Weisberg, S. P. et al. CCR2 modulates inflammatory and metabolic effects of high-fat feeding. J. Clin. Invest. 116, 115-124

Article (PDF Available)inJournal of Clinical Investigation 116(1):115-24 · February 2006with33 Reads
DOI: 10.1172/JCI24335 · Source: PubMed
Abstract
The C-C motif chemokine receptor-2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.
Research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006 115
CCR2 modulates inflammatory and metabolic
effects of high-fat feeding
Stuart P. Weisberg,
1
Deborah Hunter,
2
Reid Huber,
2
Jacob Lemieux,
1
Sarah Slaymaker,
3
Kris Vaddi,
2
Israel Charo,
3
Rudolph L. Leibel,
1,4
and Anthony W. Ferrante Jr.
1
1
Department of Medicine, Naomi Berrie Diabetes Center, Columbia University College of Physicians and Surgeons, New York, New York, USA.
2
Incyte Corp., Wilmington, Delaware, USA.
3
Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.
4
Department of Pediatrics,
Naomi Berrie Diabetes Center, Columbia University College of Physicians and Surgeons, New York, New York, USA.
The C-C motif chemokine receptor–2 (CCR2) regulates monocyte and macrophage recruitment and is neces-
sary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although
adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand
for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic
steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays
in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of
obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the devel-
opment of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced
macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ame-
liorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with
established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage
content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improv-
ing hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adi-
pose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue
macrophages and insulin resistance once obesity and its metabolic consequences are established.
Introduction
Obesity is associated with a complex systemic inflammatory state
that has been implicated in the development of common, medi
-
cally important complications, including atherosclerosis, hepatic
steatosis, and insulin resistance (1–4). Characteristics of obesity-
induced inflammation include elevated expression and produc
-
tion of proinflammatory molecules by adipose tissue, liver, and
skeletal muscle (5–11); increased circulating concentrations of
proinflammatory proteins, including acute-phase reactants,
procoagulant factors, cytokines, and chemokines (3, 12); and the
activation of pathways that regulate inflammation, including
JNK and NF-
kB pathways (10, 13–15). Obesity also induces the
accumulation of macrophages in adipose tissue without altering
the macrophage content of liver or muscle (8, 16). Macrophages
produce some of the proinflammatory molecules released by
adipose tissue and have been implicated in the development and
maintenance of obesity-induced adipose tissue inflammation (8,
16, 17), but the mechanisms that recruit and retain macrophages
in adipose tissue remain obscure.
Monocyte chemoattractant proteins (MCPs) and their receptors
play pivotal roles in the development of inflammatory responses
and are crucial for the recruitment of immune cells to sites of
inflammation. In obese rodents and humans, the adipose tissue
expression of at least 1 MCP, C-C motif chemokine ligand–2 (CCL2
or MCP1), is increased in proportion to adiposity. Both the adipose
tissue expression and circulating concentrations of CCL2 increase
in obesity and decrease following treatment with thiazolidinedio
-
nes (18–20). Recent studies further implicate CCL2 and its recep
-
tor, CCR2, in the regulation of adipocyte function. These studies
found that CCL2 inhibits insulin-stimulated glucose uptake as well
as the adipocyte expression of metabolically important genes (e.g.,
Glut4, Pparg, and Fabp4) in a murine adipocyte cell line (21). CCR2
is a receptor for other MCPs, including CCL8 (MCP2) and CCL7
(MCP3) (22, 23), and is necessary for the recruitment of monocytes/
macrophages in murine models of atherosclerosis, rheumatoid
arthritis, and mycobacterial infections (24).
Because CCR2 regulates monocyte and macrophage chemotaxis
and local inflammatory responses, and is implicated in regulating
glucose uptake in an insulin-responsive cell line, we hypothesized
that monocyte chemoattractant molecules acting through CCR2
might regulate obesity-induced inflammation in adipose tissue
and associated perturbations in systemic glucose homeostasis. We
studied the expression of ligands for CCR2 in lean and obese mice
and in obese mice treated with a thiazolidinedione. We examined
the effects of
Ccr2 deficiency on development of diet-induced obe-
sity, insulin sensitivity, adipose tissue histology, and gene expres-
sion and on the plasma concentrations of proteins implicated in
the inflammatory and metabolic consequences of obesity. We also
examined the metabolic phenotype of obese mice treated with a
selective, CCR2 antagonist after the onset of obesity. Congenital
absence of CCR2 had no measurable metabolic effect in lean ani
-
mals. However, in a mouse model of diet-induced obesity, CCR2
deficiency attenuated the development of obesity, adipose tissue
macrophage (ATM) accumulation, adipose tissue inflammation,
and systemic insulin resistance. In mice with preexisting obesity,
Nonstandard abbreviations used: ATM, adipose tissue macrophage; CCL, chemo-
kine, C-C motif, ligand; CCR, CCL receptor; HOMA-IR, homeostasis model assess
-
ment of insulin resistance; MCP, monocyte chemoattractant protein; PAI, plasmino-
gen activator inhibitor; SVC, stromal vascular cell.
Conflict of interest: K. Vaddi, D. Hunter, and R. Huber are employed by Incyte Corp.
Citation for this article:
J. Clin. Invest. 116:115–124 (2006).
doi:10.1172/JCI24335.
Related Commentary, page 33
research article
116 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006
short-term pharmacologic antagonism of CCR2 reduced ATM
content and improved in vivo insulin sensitivity. The mechanis
-
tic relationships among these diverse metabolic phenotypes is
unclear, but these findings suggest that CCR2 modulates meta
-
bolic and inflammatory effects induced by a high-fat diet.
Results
Adipose tissue expression of Ccr2 and its ligands is regulated by obesity. The
macrophage content of adipose tissue is increased by obesity and
reduced by administration of thiazolidinedione (8, 16, 25). To iden
-
tify potential regulators of macrophage accumulation in adipose
tissue, we studied the expression profiles of epididymal adipose tis
-
sue from lean male C57BL/6J mice maintained on a low-fat diet (5%
of calories derived from fat) for 24 weeks (body mass, 28.9 ± 2.5 g;
fasting insulin, 0.31 ± 0.6 ng/ml); C57BL/6J mice made obese and
insulin resistant on a high-fat diet (45% of calories derived from
fat) for 24 weeks (45.8 ± 4.8 g; 2.3 ± 0.5 ng/ml); and C57BL/6J mice
made obese on a high-fat diet for 22 weeks and then fed the high-
fat diet mixed with pioglitazone (60 mg/kg) for 2 additional weeks
(47.2 ± 1.8 g; 0.3 ± 0.05 ng/ml). The expression of
Ccl2 (also known
as
Mcp1) is upregulated in adipose tissue of obese rodents (18)
and humans (20, 26), but its receptor, CCR2, and the other CCR2
ligands (CCL7/MCP3 and CCL8/MCP2) have not be studied. In the
adipose tissue of obese compared with lean mice, the expression of
Ccl2 was increased 7.6-fold (P < 0.01); Ccl7 was increased 8.4-fold
(P < 0.01); and Ccl8 was increased 2.1-fold (P < 0.05). Treatment
with pioglitazone significantly decreased the expression of
Ccr2
(P < 0.001), Ccl2 (P < 0.01), and Ccl7 (P < 0.01) but not Ccl8 (Figure
1). The influence of obesity and a thiazolidinedione on the expres
-
sion of
Ccr2 and its ligands is consistent with obesity-dependent
alterations in CCR2 signaling within adipose tissue.
Ccr2
–/–
mice are partially protected from diet-induced obesity. To assess
the role of CCR2 in the development of obesity and obesity-asso
-
ciated adipose tissue inflammation and insulin resistance, we fed
either low-fat (5% calories derived from fat) or high-fat (60% of
calories derived from fat) diets to male
Ccr2-deficient (C57BL/6J
Ccr2
–/–
) or wild-type (C57BL/6J Ccr2
+/+
) mice. Previous reports of
mice fed standard chow diets had not noted any
Ccr genotype-
dependent effects on body mass (27, 28). Consistent with these
reports, lean body mass, fat mass, and total body mass were not
affected by
Ccr2 genotype in mice fed the low-fat diet for 24 weeks
(Figure 2). Unexpectedly, however, after 24 weeks on the high-fat
diet,
Ccr2
–/–
mice weighed 15% less than Ccr2
+/+
mice (39.4 ± 6.9 vs.
46.3 ± 4.1 g [mean ± SD];
P < 0.05). Even though the mean body
mass of the high-fat diet–fed
Ccr2
–/–
mice was lower than that of
comparably fed
Ccr2
+/+
mice, most Ccr2
–/–
mice did in fact become
obese, weighing between 40 and 50 g, with adiposity between 40%
and 50% (Figure 2A). Two-way ANOVA confirmed that both high-
fat diet (
P < 0.001) and Ccr2 genotype (P < 0.05) contributed to the
variation in body mass, though the genotype-diet interaction did
not reach significance (
P = 0.14). Hence, Ccr2 deficiency attenu-
ated but did not prevent the development of obesity in mice fed
a high-fat diet.
To better understand why
Ccr2
–/–
mice gained less weight while
ingesting the high-fat diet, we examined food intake and body
composition of individually caged
Ccr2
–/–
and Ccr2
+/+
mice fed
a high-fat diet for 6 weeks. Despite having similar body weights
(19.16 ± 1.05 g vs. 19.48 ± 0.63 g;
P > 0.05) and body compositions
at the study’s outset,
Ccr2
–/–
mice consumed fewer kilocalories per
day than
Ccr2
+/+
mice during the observation period (18.32 ± 4.08
kcal/d vs. 28.27 ± 14.25 kcal/d;
P < 0.005; Figure 2B). At the end
of the 6-week period,
Ccr2
–/–
mice weighed significantly less than
the
Ccr2
+/+
mice (26.96 ± 2.20 g vs. 31.02 ± 3.40 g, P < 0.05; Supple-
mental Figure 1; supplemental material available online with this
article; doi:10.1172/JCI24335DS1); had modestly but significantly
lower lean body mass (20.86 ± 0.69 g vs. 22.66 ± 1.57 g;
P < 0.05;
Supplemental Figure 1) and modestly but not significantly lower
fat mass (5.36 ± 1.94 g vs. 7.82 ± 3.36 g; Supplemental Figure 1)
and adiposity (20.06% ± 5.10% vs. 25.02% ± 8.36% fat). These data
suggest an unrecognized role for CCR2 in the regulation of feed
-
ing behavior in the context of a highly palatable diet.
Ccr2 genotype modulates insulin sensitivity in obesity. Decreased fat
mass in
Ccr2
–/–
mice fed a high-fat diet would be expected to reduce
their inflammatory response and to improve systemic metabolic
parameters, including insulin sensitivity. Therefore, a compari
-
Figure 1
Expression of Ccr2 and its ligands in lean mice, obese mice, and
obese mice treated with pioglitazone. Expression of Ccr2 and genes
that encode 3 of its ligands, Ccl2, Ccl7 and Ccl8, were measured
in lean mice (white bars), obese mice (black bars), and obese mice
treated with the insulin sensitizer pioglitazone (gray bars). *P < 0.05,
lean vs. obese;
P < 0.05 obese vs. obese pioglitazone-treated (n = 4).
Values are expressed as mean ± SD.
Figure 2
Body mass of Ccr2
–/–
mice. (A) C57BL/6J Ccr2
+/+
(black symbols)
and Ccr2
–/–
(gray symbols) mice were fed a low-fat (triangles) or a
high-fat diet (squares) for 24 weeks. There was no significant differ-
ence in body mass between mice of each genotype on the low-fat diet
(n = 5; P > 0.05). The mean body mass of Ccr2
–/–
mice fed a high-fat
diet was significantly lower than that of the Ccr2
+/+
mice (39.3 ± 6.9 g
vs. 46.3 ± 4.1 g; n = 10; P < 0.05). (B) Average daily food intake was
measured over a 6-week period for Ccr2
+/+
and Ccr2
–/–
mice fed a
high-fat diet. *P < 0.05 vs. Ccr2
+/+
.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006 117
son of all high-fat diet–fed Ccr2
–/–
mice with their Ccr2
+/+
coun-
terparts might overstate the true effect of
Ccr2 genotype on ATM
accumulation, adipose tissue inflammatory response, and insulin
sensitivity in obese mice. To distinguish the direct effects of CCR2
deficiency on insulin sensitivity and ATM accumulation from the
secondary effects of reduced weight and adiposity, we compared
ATM accumulation and insulin sensitivity in mice from both high-
fat diet–fed groups that weighed more than 40 g. Among these
high-fat diet–fed mice, there was no significant difference in body
mass, total fat mass, or percent body fat (Table 1).
We measured fasting blood glucose and plasma insulin concen
-
trations and calculated homeostasis model assessment of insulin
resistance (HOMA-IR) values in 4 groups of mice: lean
Ccr2
+/+
, lean
Ccr2
–/–
, obese Ccr2
+/+
, and adiposity-matched obese Ccr2
–/–
mice.
No difference was observed in the fasting blood glucose or plas
-
ma insulin concentrations between lean
Ccr2
–/–
and lean Ccr2
+/+
mice. Likewise, there was no effect of
Ccr2 genotype on insulin
sensitivity (as measured by HOMA-IR) in lean animals (Figure 3).
However, obese
Ccr2
–/–
mice had lower fasting blood glucose and
insulin concentrations compared with adiposity-matched obese
Ccr2
+/+
mice (Figure 3). Consistent with increased insulin sensi-
tivity, the HOMA-IR values of obese
Ccr2
–/–
mice were 50% lower
than those of equally obese
Ccr2
+/+
animals (P < 10
–4
; Figure 3).
Two-way ANOVA demonstrated a significant interaction between
body weight and
Ccr2 genotype in modulating fasting glucose
(P < 0.0005), insulin (P < 0.05), and HOMA-IR (P < 0.001).
Obese
Ccr2
–/–
mice were also more insulin sensitive than obese
Ccr2
+/+
animals as measured by an insulin tolerance test (Figure
4). Similarly,
Ccr2 deficiency improved glucose tolerance dur-
ing an intraperitoneal glucose tolerance test. Obese
Ccr2
–/–
mice
were less hyperglycemic compared with obese
Ccr2
+/+
animals at
45, 60, and 90 minutes following intraperitoneal injection of a
glucose bolus (Figure 4).
Ccr2 deficiency attenuates obesity-induced hepatic steatosis. Hepatic
insulin resistance in obese mice and humans is strongly associated
with hepatomegaly and hepatic steatosis (29). To determine wheth
-
er improved insulin sensitivity associated with
Ccr2 deficiency ame-
liorated hepatomegaly or reduced hepatic steatosis, we analyzed the
livers from obese
Ccr2
+/+
and Ccr2
–/–
mice. As expected, obese Ccr2
+/+
mice had a more than 10-fold higher concentration of hepatic
triglycerides (TGs) than lean
Ccr
+/+
mice (82.1 ± 19.6 vs. 7.8 ± 2.6
mg TG/g tissue;
P < 0.001). Adiposity-matched obese Ccr2
–/–
mice
Table 1
Body compositions and plasma lipids of animals examined in this study
Genotype Diet Treatment Body mass Lean mass Fat mass % Body fat Cholesterol TGs NEFAs
(g) (g) (g) (mg/dl) (mg/dl) (mmol/l)
Ccr2
+/+
Low-fat None 23.8 ± 0.88 19.4 ± 0.41 3.4 ± 0.43 14.8 ± 1.5 124 ± 59 95 ± 23 2.8 ± 0.48
Ccr2
–/–
Low-fat None 22.6 ± 0.94 18.6 ± 0.80 3.0 ± 0.22 13.9 ± 0.85 99 ± 9 96 ± 20 2.4 ± 0.34
Ccr2
+/+
High-fat None 47.4 ± 3.7 27.2 ± 2.5 21.3 ± 2.1 44.0 ± 2.8 240 ± 31 71 ± 7.4 2.6 ± 0.33
Ccr2
–/–
High-fat None 45.4 ± 5.7 24.2 ± 1.6 20.5 ± 4.1 45.4 ± 5.2 201 ± 56 81 ± 4.0
A
2.5 ± 0.15
Ccr2
+/+
High-fat INCB3344 46.3 ± 3.2 23.4 ± 1.0 19.7 ± 1.6 45.6 ± 1.8 272 ± 44 100 ± 7.1 2.4 ± 0.19
Ccr2
+/+
High-fat Vehicle 46.6 ± 2.4 23.3 ± 1.4 20.7 ± 2.2 47.0 ± 2.3 227 ± 35 111 ± 15 2.3 ± 0.37
Unselected lean and obese Ccr2
–/–
and Ccr2
+/+
mice matched for body mass as well as obese Ccr2
+/+
mice treated with CCR2 antagonist or vehicle were
studied. Body mass, lean mass, fat mass, and percent body fat did not differ between lean Ccr2
+/+
and Ccr2
–/–
animals. Lean body mass was slightly
lower in obese Ccr2
–/–
compared with obese Ccr2
+/+
mice (n = 10; P < 0.05). Daily subcutaneous injection (17 days) of obese mice with CCR2 antagonist
INCB3344 did not alter body mass, lean mass, fat mass, or percent body fat when compared with obese mice treated with vehicle. There were no signifi-
cant genotype- or treatment-dependent differences in circulating fasting concentrations of lipids, except that TG concentrations were lower in plasma from
obese Ccr2
+/+
mice compared with obese Ccr2
–/–
animals (
A
P < 0.05). Values are expressed as mean ± SD. NEFA, nonesterified fatty acid.
Figure 3
Insulin sensitivity in obese Ccr2
–/–
and obese Ccr2
+/+
mice.
Fasting plasma insulin (A) and blood glucose concentra-
tions (B) were measured in lean Ccr2
+/+
(black bars) and
Ccr2
–/–
(gray bars) mice and mice of both genotypes made
obese following 20 weeks of high-fat diet feeding. There
were no significant genotype-dependent differences in fast-
ing glucose or insulin concentrations in lean animals. How-
ever, fasting glucose and insulin concentrations were lower
in obese Ccr2
–/–
compared with obese Ccr2
+/+
mice despite
similar degrees of adiposity (insulin: P < 0.005; glucose:
P < 10
–4
). (C) HOMA-IR values (expressed as IU-mg/dl) were
significantly lower (P < 10
–4
) among obese Ccr2
–/–
than obese
Ccr2
+/+
mice. (D) A plot of HOMA-IR values against body
mass among all Ccr2
–/–
(gray squares) and Ccr2
+/+
(black
circles) mice reveals that the relationship between insulin
sensitivity and body mass differs between mice dependent
upon Ccr2 genotype. **P < 0.01 compared with wild type.
Values are expressed as mean ± SD.
research article
118 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006
had 50% lower hepatic TG content than obese Ccr2
+/+
animals
(43.9 ± 23.6 vs. 82.1 ± 19.6 mg TGs/g tissue;
P < 0.001). Similarly,
livers of obese
Ccr2
+/+
mice weighed 50% more than those of compa-
rably obese
Ccr2
–/–
animals (2.4 ± 0.5 g vs. 1.6 ± 0.6 g; P < 0.001).
Ccr2 deficiency decreases macrophage content of adipose tissue. In ear-
lier mouse studies, ATM content was strongly correlated with adi
-
posity (8, 16). To determine whether the accumulation of ATMs
during the development of obesity is dependent upon CCR2,
we compared adipocyte size and ATM content in diet-induced
obese
Ccr2
–/–
and Ccr2
+/+
mice that were matched for adiposity.
We measured macrophage content and adipocyte morphology by
immunohistochemistry in epididymal and subcutaneous adipose
tissue depots obtained from obese
Ccr2
–/–
and Ccr2
+/+
mice. In epi-
didymal adipose tissue, adipocyte morphology was similar in both
genotypes. However, adipocyte size (median cross-sectional area)
was greater in the
Ccr2
–/–
mice (median, 4,457 ± 650 mm
2
; mean,
5,005 ± 621
mm
2
) compared with the obese Ccr2
+/+
controls (medi-
an, 3,588 ± 561
mm
2
; mean, 4,345 ± 460 mm
2
; P < 0.05 for median).
As a percentage of cells, adipose tissue from obese
Ccr2
+/+
mice
contained more ATMs (25% ± 5.6%) than that from lean
Ccr2
+/+
animals (7.2% ± 4.9%). However, despite having adipocytes that
were larger and total adiposity that was equal, the ATM content of
epididymal adipose tissue in obese
Ccr2
–/–
(16.3% ± 3%) mice was
significantly less than the ATM content of adipose tissue in obese
Ccr2
+/+
mice (25% ± 5.6%; P < 0.005) (Figure 5). A similar
reduction in macrophage content was also detected in
subcutaneous adipose tissue depots (Figure 5)
Flow cytometry can also be used to isolate and quan
-
tify the fraction of ATMs that comprises stromal vascu
-
lar cells (SVCs) of adipose tissue. However, a portion of
ATMs is not isolated in the SVCs in standard protocols
because of increased buoyancy of lipid-laden ATMs
from obese animals and adherence of ATMs to adi
-
pocytes (8, 16). Nevertheless, characterization of SVC
populations by flow cytometry has also shown a con
-
sistent increase in the fraction of ATMs in adipose tis
-
sue from obese compared with lean mice (8, 16). Flow
cytometry, therefore, provides an alternative means of
determining the relative abundance of ATMs. Using
flow cytometry, we quantified the macrophage popula
-
tions (F4/80
+
, Cd11b
+
) in SVCs of peri-renal adipose tis-
sue from lean
Ccr2
+/+
mice and obese Ccr2
–/–
and Ccr2
+/+
mice. Consistent with our immunohistochemical anal
-
ysis, we found that the proportion of macrophages in
SVCs isolated from obese wild-type mice (17.6% ± 5%
of SVCs) was significantly greater than the propor
-
tion in the SVCs isolated from lean
Ccr2
+/+
mice (8.1% ± 2.9%) and
obese
Ccr2
–/–
mice (9.4% ± 1.7%; P < 0.001 vs. obese Ccr2
+/+
). Thus,
in obese animals, intraabdominal and subcutaneous adipose tis
-
sue depots have fewer macrophages in
Ccr2-deficient mice than
in adiposity-matched wild-type animals. However,
Ccr2 deficiency
does not normalize ATM content to that observed in lean animals,
indicating that macrophage accumulation is modulated by CCR2-
independent factors as well.
Ccr2 deficiency attenuates obesity-induced changes in adipose tissue gene
expression. The development of obesity and insulin resistance is
associated with stereotypical changes in adipose tissue expression
of inflammatory and metabolic genes. Increased expression of
genes that encode proinflammatory and macrophage-associated
proteins is a hallmark of adipose tissue from obese animals and
humans (30). To assess the role of
Ccr2 in mediating the transcrip-
tional inflammatory response to obesity, we compared the expres
-
sion of genes encoding proinflammatory and macrophage marker
proteins in epididymal adipose tissue from lean and obese
Ccr2
+/+
Figure 5
CCR2 deficiency lowers ATM content in obese mice. The frac-
tion of F4/80-expressing macrophages was determined by
immunohistochemical analysis of epididymal adipose tissue from
obese Ccr2
+/+
(A) and Ccr2
–/–
(B) mice with the macrophage-specific
marker F4/80 (EMR1). (C) The fraction of ATMs (F4/80-stained cells/
total cells) in periepididymal adipose tissue of obese Ccr2
+/+
mice
(white bar) was significantly greater than the fraction of ATMs in lean
Ccr2
+/+
mice (black bar) (P < 10
4
) and obese Ccr2
–/–
mice (gray bar)
(P < 0.005). (D) The average fraction of ATMs was also greater in
subcutaneous adipose tissue of obese Ccr2
+/+
mice (black bar) com-
pared with obese Ccr2
–/–
mice (gray bar). Values are expressed as
mean ± SD. **P < 0.01 compared with obese Ccr2
+/+
mice.
Figure 4
Glucose homeostasis in obese Ccr2
–/–
and Ccr2
+/+
mice. (A) The response of fast-
ed obese Ccr2
+/+
(black circles) and Ccr2
–/–
(gray squares) mice following a single
intraperitoneal injection of insulin (1.5 U/kg) was monitored by serially measuring
blood glucose concentrations. The percent reduction in glucose concentration in
obese Ccr2
–/–
mice was significantly greater than that in obese Ccr2
+/+
mice at
75, 90, and 130 minutes (*P < 0.05). (B) Intraperitoneal injection of a bolus of
glucose to fasted obese Ccr2
+/+
(black circles) and Ccr2
–/–
(gray squares) mice
lead to similar peak glucose concentrations but lower glucose concentrations at
45, 60, and 90 minutes. *P < 0.05 compared with wild type. Values are expressed
as mean ± SD.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006 119
and Ccr2
–/–
mice. As expected, obese Ccr2
+/+
mice compared with
lean
Ccr2
+/+
mice showed increased expression of genes encoding
proinflammatory molecules including
Tnfa (TNF-α) and Serpine1
(plasminogen activator inhibitor–1 [PAI-1]) and the macrophage
markers
Emr1 (F4/80) and Cd68 (CD68). Consistent with the histo-
logical data, expression of the macrophage markers
Emr1 and Cd68
was reduced in obese
Ccr2
–/–
mice compared with obese Ccr2
+/+
mice. By 2-way ANOVA, the variation in the expression of these
genes was attributable to a significant interaction between diet
and
Ccr2 genotype (Figure 6; ANOVA interaction P < 0.05). Paral-
leling the decrease in macrophage-specific gene expression,
Tnfa
expression was reduced in obese
Ccr2
–/–
mice (Figure 6; ANOVA
interaction
P < 0.005). No significant differences in the expression
of these genes were observed between lean
Ccr2
–/–
and Ccr2
+/+
mice
(Figure 6), nor were differences observed in
Serpine1, Ccl2, or Ccl7
expression between obese
Ccr2
–/–
and Ccr2
+/+
mice (data not shown).
In contrast to the alterations in
Tnfa expression in adipose tissue,
we were consistently unable to detect circulating TNF-
α protein
(<3.2 pg/ml) in mice of either genotype or level of adiposity (data
not shown). These data suggest that the local proinflammatory
changes that occur in adipose tissue during the development of
obesity are, in part, CCR2 dependent.
Concomitant with an increase in inflammatory gene expres
-
sion, obesity leads to decreased expression of genes required for
mitochondrial function, TG metabolism, and adipocyte differen
-
tiation (8, 31–33). Recent studies suggested that CCL2 directly
effects similar changes in adipocyte gene expression (21). We mea
-
sured the expression of 4 genes downregulated in obesity and by
Ccl2 treatment of adipocytes: a nuclear encoded mitochondrial
gene (
Gpam) (8, 32), a lipase (Lipe) (8, 31), a fatty acid binding pro-
tein (
Fabp4) (32), and a transcription factor required for adipocyte
differentiation (
Pparg) (32). In lean animals there was no effect of
Ccr2 genotype on the expression of these genes, but in obese mice
the expression of these genes was increased in
Ccr2
–/–
compared
with
Ccr2
+/+
mice (Figure 7).
The effects of
Ccr2 genotype on the expression of this select
group of inflammatory and metabolic genes suggested that obe
-
sity-induced alterations in adipose tissue gene expression are, in
part, CCR2 dependent. To more fully define the alterations in
gene expression associated with
Ccr2 deficiency in adipose tis-
sue of obese mice, we used oligonucleotide microarrays to com
-
pare the expression profiles of epididymal adipose tissue from
Ccr2
–/–
and Ccr2
+/+
mice with dietary obesity. Using an algorithm
that identifies predefined functional classes (based on the Gene
Ontology [GO] Group gene classification scheme; http://geneon
-
tology.org) of coregulated genes, we found that 71 classes of genes
were significantly downregulated (
P < 0.001, corrected for mul-
tiple testing) (34). Forty-one of the downregulated groups repre
-
sent inflammatory functional classes and include classes of genes
involved in the regulation of immune responses (GO:0050778),
interleukin receptor activity (GO:0004907), and NF-
kB signaling
(GO:0043122) (Supplemental Table 1). These data suggest that
the inflammatory profile of adipose tissue in obese mice
is broad-
ly attenuated by deficiency of CCR2. We also found that 52 classes
of genes were significantly upregulated in
Ccr2
–/–
compared with
Ccr2
+/+
obese mice. Consistent with the relative normalization in
metabolic gene expression and with the increase in expression of
Gpam, Lipe, and Glut4, 23 of the upregulated functional classes in
obese
Ccr2
–/–
adipose tissue are involved in intermediary metabo-
lism, most notably mitochondria and peroxisome function, and
lipid metabolism (Supplemental Table 2).
Ccr2-dependent regulation of expression and circulating concentra-
tions of adipose tissue–derived molecules. The microarray dataset also
revealed that several individual genes with higher levels of expres
-
sion in
Ccr2
–/–
than in Ccr2
+/+
obese mice encode hormones pro-
duced by adipocytes including leptin (
Lep) (P < 0.05), resistin
(Retn) (P < 0.05), and adiponectin (Acdc) (P < 0.05). Perturbations
of each of these hormones has been implicated in the develop
-
ment of insulin resistance. Leptin deficiency leads to a syndrome
of severe obesity and insulin resistance; elevation of circulating
resistin is implicated in obesity-induced insulin resistance; and,
conversely, decreases in the antiinflammatory and insulin-sensi
-
tizing protein adiponectin (encoded by
Acdc), lead to the develop-
ment of obesity-induced insulin resistance and hepatic steatosis
(35, 36). Despite alterations in adipose tissue gene expression,
we did not detect any differences in circulating concentrations
Figure 6
Expression of genes involved in inflammation. Quantitative RT-PCR
was used to measure the expression in periepididymal adipose tissue of
genes involved in macrophage function and inflammation (Tnfa, Cd68,
Emr1, and Serpine1). Obese Ccr2
–/–
(light gray bars) and obese Ccr2
+/+
(dark gray bars) mice with body mass greater than 40 g were studied,
as were lean Ccr2
+/+
mice (black bars) and lean Ccr2
–/–
mice (white
bars). *P < 0.05, obese mice on high-fat chow compared with mice of
the same genotype on low-fat chow;
§
P < 0.05, obese Ccr2
+/+
compared
with obese Ccr2
–/–
mice. Values are expressed as mean ± SEM.
Figure 7
Expression of genes involved in adipocyte function. Quantitative
RT-PCR was used to measure the expression in periepididymal adi-
pose tissue of genes involved in adipocyte function (Fabp4, Gpam,
Lipe, Pparg, and Acdc). Obese Ccr2
–/–
(light gray bars) and obese
Ccr2
+/+
(dark gray bars) mice with body mass greater than 40 g were
studied, as were lean Ccr2
+/+
mice (black bars) and lean Ccr2
–/–
mice
(white bars). *P < 0.05, mice with dietary obesity compared with lean
mice of the same genotype;
§
P < 0.05, obese Ccr2
+/+
compared with
obese Ccr2
–/–
mice.
#
NS. Values are expressed as mean ± SEM.
research article
120 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006
of leptin or resistin between obese Ccr2
–/–
and Ccr2
+/+
mice (Sup-
plemental Table 3). We confirmed adipose tissue gene expres
-
sion of adiponectin by quantitative PCR and found, as expected,
decreased expression of adiponectin in obese compared with lean
Ccr2
+/+
mice. No difference in adipose tissue Acdc expression was
detected between lean
Ccr2
+/+
and Ccr2
–/–
animals. However, con-
sistent with their increased systemic insulin sensitivity, adiponec
-
tin gene expression was higher in obese
Ccr2
–/–
mice compared
with obese
Ccr2
+/+
animals (Figure 7). Furthermore, the amount
of circulating adiponectin in plasma, while similar in lean mice of
both genotypes, was significantly higher in obese mice deficient
in
Ccr2 than in wild-type obese animals (Figure 8).
IL-6 and PAI-1 are circulating proteins that are produced by
multiple organs and tissues including adipose tissue, liver, leu
-
kocytes, and endothelial cells. Both have been implicated in obe
-
sity-induced insulin resistance, and the adipose tissue expression
of both is increased in obesity (18, 26, 37–40). We did not detect
Ccr2-dependent modulation in adipose tissue expression of IL-6
or PAI-1. Consistent with the gene expression data, there were no
CCR2-dependent alterations in the circulating concentration of
IL-6. In contrast, we detected lower circulating concentrations of
PAI-1 in lean and obese
Ccr2
–/–
mice when compared with weight-
matched
Ccr2
+/+
mice (Supplemental Table 3). These data reveal
that CCR2 modulates circulating PAI-1 by a mechanism that is
not primarily dependent upon adipose tissue expression or the
development of obesity.
Effects of Ccr2 genotype on plasma lipid concentrations. In rodents and
humans, obesity is associated with a dyslipidemia characterized by
elevated serum concentrations of cholesterol (41, 42). As expected,
fasting total plasma cholesterol concentrations were higher in
obese compared with lean
Ccr2
+/+
mice. In obese Ccr2
–/–
mice, the
increase was similar, and there were no genotype-dependent dif
-
ferences between fasting cholesterols of adiposity-matched mice
(Table 1). Fasting plasma concentrations of TGs and nonesteri
-
fied fatty acids were not different between lean and obese animals.
No genotype-dependent effects on plasma nonesterified fatty acid
(NEFA) concentrations were detected. TG concentrations were
modestly higher in obese
Ccr2
–/–
mice compared with obese Ccr2
+/+
mice (Table 1), though still lower than TG concentrations in lean
mice. In contrast to other metabolic phenotypes examined,
Ccr2
deficiency did not significantly reverse the lipid abnormalities
associated with obesity.
Short-term treatment with a CCR2 antagonist reduces ATM content
and improves insulin sensitivity in obese mice. CCR2 has been iden-
tified as a potential therapeutic target in several inflammatory
disorders, including rheumatoid arthritis and multiple sclerosis.
INCB3344 (Incyte Corp.) was developed as a selective small mole
-
cule antagonist of CCR2. Previous studies characterized the phar
-
macokinetic and pharmacodynamic profile of this molecule and
demonstrated that with a single daily subcutaneous dose (100
mg/kg), a significant inhibition (>50%) of murine CCR2 receptor
activity is achieved in vivo (11). To test whether short-term treat
-
ment with a CCR2 antagonist reverses any of the obesity-induced
effects that are prevented in CCR2-deficient animals, we treated
C57BL/6J mice made obese by feeding a high-fat diet with daily
subcutaneous injections of INCB3344 (100 mg/kg) for 17 days.
At the end of the treatment period, body mass and composition
were not significantly different between INCB3344- and vehicle-
treated animals (Table 1).
Following 2 weeks of treatment with INCB3344, we assessed
glucose homeostasis in the INCB3344-treated and vehicle-treated
obese animals. After a 6-hour fast, animals receiving INCB3344
were significantly less hyperglycemic than animals receiving injec
-
tions of vehicle (143 ± 29 mg/dl vs. 181 ± 31 mg/dl;
P < 0.05; Figure
9). After an overnight fast (14 hours), blood glucose concentra
-
tions in the 2 groups of animals were similar. However, fasting
plasma insulin concentrations were approximately 33% lower
in INCB3344-treated compared with control animals (
P < 0.05);
hence, insulin sensitivity as measured by HOMA-IR was improved
(8.3 ± 2.5 vs. 13.5 ± 4.4 IR units;
P < 0.05; Figure 9). Consistent with
improved insulin sensitivity, an intraperitoneal injection of insulin
lowered blood glucose concentrations more in INCB3344-treated
than in vehicle-injected control obese mice (Figure 9). Similar to
the effects of
Ccr2 deficiency, INCB3344 treatment also improved
glucose homeostasis following a glucose tolerance test (Figure 9).
Treatment of obese mice with INCB3344 increased adipose tis
-
sue expression of adiponectin by 29.8% (
P < 0.05), although the
increase in circulating adiponectin concentration did not reach
statistical significance (
P > 0.05).
We also examined the effect of CCR2 inhibition with INCB3344
on the ATM content of periepididymal adipose tissue in wild-
type mice with established obesity. Immunohistochemical anal
-
ysis showed a modest but significant decrease in the fraction
of F4/80-expressing ATMs in obese mice that had been treated
with INCB3344 (21.8% vs. 15.7%, vehicle- vs. INCB3344-treated;
P < 0.05). These data implicate CCR2-dependent pathways in the
maintenance of the macrophage population in obese adipose tis
-
sue and imply a turnover sufficiently rapid to permit detectable
differences after 2–3 weeks of CCR2 antagonism. In contrast to the
ability of short-term treatment with a CCR2 antagonist to improve
insulin sensitivity and lower macrophage content in adipose tis
-
sue, the effects of a short-term treatment regimen with INCB3344
on proinflammatory adipose tissue gene expression were less con
-
sistent. Short-term INCB3344 treatment consistently and signifi
-
cantly lowered
Ccr2 expression by 65% (P < 0.01; n = 12). However,
Figure 8
Plasma adiponectin in lean and obese Ccr2
–/–
and Ccr2
+/+
mice. Pro-
teins in 1 ml of plasma drawn from lean (white bars) and adiposity-
matched obese (black bars) Ccr2
–/–
and Ccr2
+/+
mice were denatured,
reduced, and separated using SDS-PAGE. We performed immunob-
lotting for adiponectin with an antibody specific for adiponectin. Values
are expressed as mean ± SD. *P < 0.05.
research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006 121
in contrast, a more modest (20–30%) reduction in expression of
other inflammatory genes, including
Tnfa, Csf1r, and Emr1, was
not significant in all cohorts tested (data not shown). Similarly,
short-term antagonist treatment did not have a measurable effect
on hepatomegaly (data not shown). Thus, the development of obe
-
sity and the obesity-induced expression of several key inflamma
-
tory genes were not reversed with 2-week antagonism of CCR2.
Discussion
CCR2 signaling plays pivotal roles in immune responses and ath
-
erosclerosis. Its role in metabolic disorders has not been previ
-
ously appreciated. However, findings presented here demonstrate
a complex and important role for CCR2 in both the development
and maintenance of obesity and obesity-associated phenotypes. In
lean animals, no effect of
Ccr2 genotype on metabolic traits could
be detected. In contrast, when mice were fed a high-fat diet,
Ccr2
genotype modulated feeding behavior, the development of obesity,
and the development of obesity-associated adipose tissue inflam
-
mation, ATM accumulation, adiponectin expression, hepatic ste
-
atosis, and insulin sensitivity.
Once obesity is established, short-term inhibition of CCR2
signaling with a specific antagonist attenuates obesity-induced
macrophage accumulation in adipose tissue and insulin resis
-
tance. However, adipose tissue proinflammatory gene expression
was not as consistently reduced. The expression of some genes,
e.g.,
Ccr2 and Acdc, were modulated similarly by genetic deficiency
and pharmacological antagonism of CCR2, but others, includ
-
ing the pro-typical inflammatory cytokines, were not consistently
decreased in antagonist-treated mice. Similarly, circulating adipo
-
nectin and hepatomegaly were not significantly altered by drug
treatment. That the effects of the antagonist were more modest
than the developmental effects seen with genetic deficiency may
reflect a developmental requirement for CCR2 in certain obe
-
sity-induced phenotypes and/or relate to the short duration of
impaired or absent CCR2 action.
The requirement for CCR2 in the hyperphagic response to a high-
fat diet was unexpected. Feeding behavior is integrated and regulat
-
ed by centers in the central nervous system, in particular, by nuclei
within the hypothalamus and brain stem. Populations of neurons,
astrocytes, and glial cells express CCR2, and CCL2 binding within
the hypothalamus and brain stem have been reported, although
possible noninflammatory roles of CCR2 within the central ner
-
vous system have not been characterized. Thus, CCR2 may play a
role in the ability of feeding centers to respond fully to a highly
fat-laden palatable diet. Alternatively, CCR2-dependent regulation
of feeding behavior may occur through the action of CCR2 on cells
in the periphery, which in turn release molecules that act upon
neurons in the central nervous system. Identifying specific hypo
-
thalamic and brainstem neuronal and non-neuronal populations
that express CCR2, and determining whether central or peripheral
INCB3344 can attenuate the development of diet-induced obesity
before its onset, will be important initial steps in identifying mech
-
anisms by which CCR2 modulates feeding behavior.
We had hypothesized that ATM content is CCR2 dependent.
Indeed, the fraction of ATMs in adipose tissue was 35% lower in
obese
Ccr2
–/–
compared with obese Ccr2
+/+
mice. The ability of
a CCR2 antagonist to lower ATM content by 25% in mice with
established obesity after 2 weeks of treatment suggests that the
effects of genetic deficiency in
Ccr2 are not simply attributable to
developmental defects in hematopoiesis or chemotaxis. The data
also indicate that the turnover of ATMs in obesity is relatively
high (half-life in adipose tissue of
1 month). Identifying the rel-
evant ligand(s) for CCR2-dependent accumulation of ATMs will
require further study, although the expression patterns of CCL2
and CCL7 make them strong candidates. The similarity in adi
-
pose tissue expression of CCL2 and CCL7 suggests an additional
level of complexity and potential redundancy that will compli
-
cate studies. Furthermore, neither pharmacologic modulation
nor genetic deficiency of CCR2 normalized ATM content, indi
-
cating that factors other than CCR2 participate in macrophage
recruitment to adipose tissue.
Recent data suggest that the expression of proinflammatory
molecules by adipose tissue is in part due to the expression of
these molecules by non-adipocytes, including macrophages (8,
16, 25, 43–45).
Ccr2 deficiency attenuates the development of a
proinflammatory expression profile by adipose tissue of obese
animals, consistent with ATMs being a source of obesity-induced
inflammation. However, short-term treatment with a CCR2 antag
-
onist decreased the fraction of macrophages in adipose tissue and
improved insulin sensitivity without a similarly broad impact
on proinflammatory expression profile in mice with established
obesity. This suggests that if the increased production of proin
-
Figure 9
Insulin sensitivity in CCR2 antagonist–treated mice. (A) Hyperglyce-
mia after a daytime fast (6 hours) was reduced in high-fat diet–fed
obese mice that received the selective CCR2 antagonist INCB3344
for 14 days (white bars) compared with high-fat dietfed obese
mice that received vehicle injections (black bars; n = 7 per group;
*P < 0.05). (B) Following an overnight fast (14 hours), blood glucose
concentrations of INCB3344 and vehicle-treated obese animals were
not significantly different. However, fasting insulin concentrations and
HOMA-IR values (expressed as IU-mg/dl) were significantly lower in
the INCB3344-treated animals (n = 7 per group; *P < 0.05). (C) Fol-
lowing an intraperitoneal injection of glucose, obese mice treated with
INCB3344 (9 days of daily injections; open circles) were significantly
less hyperglycemic (n = 8 per group; **P < 0.01 at 10, 20, 30, and 60
minutes) than those treated with vehicle (filled squares). (D) Following
an intraperitoneal injection of insulin (1.5 U/kg) the percentage reduc-
tion in blood glucose concentration was greater in obese mice treated
with INCB3344 (14 days of daily injections) than in vehicle-treated
controls (n = 7; *P < 0.05 at 30, 45, 75, 90, and 130 minutes). Values
are expressed as mean ± SD.
research article
122 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006
flammatory molecules in obesity were only the result of a quan-
titative increase in ATM number, then their expression should be
uniformly reduced in both the CCR2-deficient and antagonist-
treated obese mice. Therefore, the data from obese
Ccr2
–/–
mice
suggest that expression of macrophage-derived proinflammatory
molecules is also dependent on the maturity or activation state
of the ATMs, and not solely on reduction in ATM content per se.
Furthermore, differences in reduction in proinflammatory gene
expression observed in obese
Ccr2
–/–
and antagonist-treated mice
are likely in part attributable to adipocyte-derived proinflammato
-
ry gene expression being differentially affected by complete genetic
deficiency and pharmacological antagonism of CCR2.
Adiponectin is an adipocyte-derived hormone that positively reg
-
ulates insulin sensitivity in liver and muscle. The enhanced insulin
sensitivity of
Ccr2
–/–
compared with Ccr2
+/+
obese mice is consistent
with higher expression of adiponectin and higher circulating con
-
centrations of adiponectin observed in
obese Ccr2
–/–
mice. These
findings suggest the existence of a CCR2-dependent mechanism
for obesity-induced downregulation of adiponectin. Modulation
of adiponectin expression may occur indirectly through interac
-
tions of adipocytes with CCR2-expressing ATMs and the modu
-
lation of obesity-induced inflammatory gene expression. Inflam
-
matory cytokines, including TNF-
α, are known to downregulate
adiponectin expression by adipocytes, and therefore, the lower
inflammatory expression profile of adipose tissue from obese
Ccr2
–/–
mice may contribute to the maintenance of adiponectin
expression. Alternatively or in addition, the expression of CCR2 by
adipocytes raises the possibility that
Ccr2-dependent downregula-
tion of adiponectin may occur via a direct effect of CCR2 on adi
-
pocyte gene expression.
The improved insulin sensitivity of obese
Ccr2
–/–
mice is not
entirely attributable to elevated adiponectin expression. Enhanced
insulin sensitivity of obese mice treated with a CCR2 antagonist,
in which adiponectin expression is similarly elevated but total
circulating adiponectin is not significantly different, implicates
an additional CCR2-dependent but adiponectin-independent
mechanism of insulin resistance. The identity and location of the
CCR2-expressing cell(s) required for this effect are not apparent.
While the correlation of ATM content with systemic insulin resis
-
tance is intriguing, we cannot determine whether ATM content
and insulin sensitivity are causally related. Defining the functional
relationship between these 2 phenotypes requires development of
tools that will permit specific manipulation of ATMs as opposed
to other hematopoietic or macrophage populations (e.g., ATM-
specific promoter for Cre-based genetic manipulations).
The adiponectin-independent mechanism(s) of CCR2 regula
-
tion of insulin sensitivity may occur through direct action on insu
-
lin-responsive cell types. The expression and function of CCR2 in
nonhematopoietic cells has not been well characterized. It has
been reported that CCR2 is expressed by adipocytes, neurons,
endothelial cells, and myocyte precursors. To our knowledge, there
is no evidence that mature myocytes or hepatocytes express CCR2.
However, we detected increased CCR2 transcript expression in liv
-
ers of obese mice in the absence of any increase in the number of
Kupffer cells (hepatic macrophages) (ref. 8 and S.P. Weisberg and
A.W. Ferrante Jr., unpublished observations). Thus, CCR2 in the
liver, either in Kupffer cells or nonhematopoietic cells, may modu
-
late the insulin responsiveness of hepatocytes. In contrast, we have
not detected any obesity-associated changes in CCR2 expression in
whole skeletal muscle.
Here we have shown that CCR2 plays important roles in the
pathogenesis of obesity and obesity-associated phenotypes and
identify CCR2 as a possible therapeutic target in efforts to treat
obesity and its complications.
Methods
Animals and animal care. Ccr2
–/–
were previously generated and have been
backcrossed 10 times to the C57BL/6J background (46). C57BL/6J mice
used in the INCB3344 studies were obtained from the Jackson Laboratory
at 6–8 weeks of age. All mice were housed in Plexiglas ventilated cages
(1–3 animals/cage) within a pathogen-free barrier facility that maintained
a 12-hour light/12-hour dark cycle. Mice had free access to autoclaved
water and irradiated pellet food. In all studies, including studies of Ccr2
genotype effects, and INCB3344 treatment, obesity was induced by a high-
fat diet that derived approximately 60% of calories from lipids (D12492;
Research Diets Inc.) for 24 weeks beginning at age 6 weeks. Prior to age 6
weeks, mice were fed a standard pellet diet, in which 5% of calories were
provided as fat (PicoLab Rodent 20; LabDiet). Mice on a low-fat diet
received the standard chow pellet throughout life.
Mice were sacrificed by CO
2
asphyxiation during the second and third
hour of the light cycle. Animals were weighed, and adipose tissues (epi-
didymal, subcutaneous, and perirenal) and liver were removed. Tissues
analyzed by FACS were processed immediately. Other samples were fro-
zen in liquid nitrogen and stored at –80°C prior to RNA extraction and
immunohistochemical analysis.
Mice treated with INCB3344 were lightly anesthetized with inhaled
2% isoflurane and injected subcutaneously with INCB3344 (100 mg/ml)
emulsified in 10% carboxymethylcellulose as vehicle. Control animals were
similarly anesthetized but subcutaneously injected with vehicle alone. Body
composition was determined by dual x-ray absorbance spectroscopy using a
PIXImus DEXA scanner specifically designed for mice (Lunar Corp.). Cali-
bration was performed before each set of measurements and data obtained
according to manufacturer’s protocols. All procedures were approved by
Columbia University’s Institutional Animal Care and Use Committee.
CCR2 antagonist. INCB3344 was synthesized by the medicinal chemistry
department of Incyte Corp. The specificity of this compound for CCR2
and its pharmakinetics of INCB3344 have been described elsewhere (11).
Metabolic testing. Insulin tolerance testing was carried out in animals
that were fasted for 6 hours beginning at approximately 10 am. After an
intraperitoneal bolus injection of recombinant human regular insulin (1.5
U/kg) (Novolin R; Novo Nordisk Inc.), blood glucose concentrations were
measured using a Glucometer ELITE XL blood glucose meter (Bayer Corp.)
before and 15, 30, 45, 75, 90, and 130 minutes after injection. Glucose toler-
ance tests were performed after an overnight (14-hour) fast. Blood glucose
concentrations were measured before and 15, 30, 45, 60, 90, 120 minutes
after an intraperitoneal injection of dextrose dissolved in water (1 g/kg).
Microarray gene expression. Total RNA was extracted from the perigonadal
(epididymal or parametrial) adipose tissue of individual mice using a com-
mercially available acid-phenol reagent (TRIzol; Invitrogen Inc.). RNA
concentration was assessed by absorbance spectroscopy and RNA integrity
confirmed by nondenaturing agarose gel electrophoresis. Twenty micro-
grams of RNA from each sample were further purified from contaminating
organics and non-RNA species using a silica resin (RNEasy; QIAGEN) pro-
tocol according to the manufacturer’s instructions. Total RNA from single
animals was individually converted into biotinylated, fragmented cRNA
using protocols recommended by the microarray manufacturer (Affyme-
trix). cRNA samples derived from single animals were hybridized in recom-
mended buffer to microarrays (Affymetrix Murine Genome Array U430
2.0) at 45°C for 16 hours. The samples were stained and washed according
to the manufacturer’s protocol on a Fluidics Station 400 (Affymetrix) and
research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006 123
scanned on a GeneArray Scanner (Affymetrix). Primary data extraction was
performed with Microarray Suite 5.0 (Affymetrix), and signal normaliza-
tion across samples was carried out using all probe sets with a mean expres-
sion value of 500.
We examined gene expression only for those genes deemed “present” by
the Microarray Suite software. To calculate the probability that genes were
up- or downregulated in Ccr2
–/–
compared with wild-type animals, we used
Microsoft Excel (Microsoft Corp.) to calculate Student’s t test scores for
each “present” gene. To identify functionally related groups of genes with
similar expression patterns, we analyzed the list of Student’s t test scores
using Gene Ontology Class Scoring software (34, 47).
Immunohistochemistry. Adipose tissue samples were fixed for 12–16 hours
at room temperature in zinc-formalin fixative (Anatech Ltd.) and embed-
ded in paraffin. Five-micrometer sections, cut at 50-mm intervals, were
mounted on charged glass slides, deparaffinized in xylene, and stained
for expression of F4/80 as described by Cecchini et al. with an anti-F4/80
monoclonal antibody (CALTAG Laboratories) (48). For each individual
mouse tissue block, 15–20 different ×20 fields from each of 4 different
sections were analyzed. The total number of nuclei and the number of
nuclei of F4/80-expressing cells were counted for each field using a soft-
ware algorithm designed for the image analysis software Image-Pro Plus
1.0 (Media Cybernetics Inc.). The fraction of F4/80-expressing cells for
each sample was calculated as the sum of the number of nuclei of F4/80
expressing cells divided by the total number of nuclei in sections of a
sample. Average adipocyte cross-sectional area was determined for each
adipocyte in each field analyzed using image analysis software (SPOT ver-
sion 3.3; Diagnostic Instruments Inc.).
Isolation of adipose tissue SMCs. Adipose tissue was isolated from mice
immediately after CO
2
asphyxiation. Tissues were handled using sterile
techniques and minced into fine (<10-mg) pieces. Minced samples were
placed in HEPES-buffered DMEM (Invitrogen Corp.) supplemented with
10 mg/ml fatty acid–poor BSA (FAP-BSA; Sigma-Aldrich) and centrifuged
at 1000 g for 10 minutes at room temperature to pellet erythrocytes and
other blood cells. An LPS-depleted collagenase cocktail (Liberase 3; Roche
Diagnostics Corp.) at a concentration of 0.03 mg/ml and 50 U/ml DNAse I
(Sigma-Aldrich) were added to the tissue filtrate and the samples incubated
at 37°C on an orbital shaker (215 Hz) for 45–60 minutes. Once digestion
was complete, samples were passed through a sterile 250-mm nylon mesh
(Sefar Inc.). The suspension was centrifuged at 500 g for 10 minutes. The
pelleted cells were collected as the SVCs, and the floating cells were collect-
ed as the adipocyte-enriched fraction. The SVCs were resuspended in eryth-
rocyte lysis buffer and incubated at room temperature for 5 minutes. The
erythrocyte-depleted SVCs were centrifuged at 500 g for 5 minutes and the
pellet resuspended in FACS buffer (PBS, 0.2%, FAP-BSA, 5 mM EDTA).
Immunophenotyping and flow cytometry. SVCs isolated from adipose tissue
samples were cooled on ice and counted using a hemocytometer. Cell sur-
vival rates ranged from 70% to 90% as determined by trypan blue exclusion.
After counting, cells were centrifuged at 500 g for 5 minutes and resus-
pended in FACS buffer at a concentration of 7 × 10
6
cells/ml. Cells were
incubated in the dark at 4°C on a bidirectional shaker for 30 minutes in
FcBlock (20 mg/ml; BD Biosciences — Pharmingen), then for an additional
50 minutes with fluorophore-conjugated primary antibodies or isotype
control antibodies. Antibodies employed in these studies included: CD11b-
PE (2 mg/ml) and F4/80-APC (5 mg/ml) from (CALTAG Laboratories). Fol-
lowing incubation with primary antibodies, 1 ml FACS buffer was added to
the cells. Cells were centrifuged at 500 g for 5 minutes and resuspended in 1
ml of FACS buffer. The wash was repeated 2 times. Cells were analyzed on a
FACSCalibur and analysis was performed using CellQuest software (BD).
Quantitative real-time PCR. Total RNA was extracted from frozen adipose
tissue (100 mg) using a commercially available acid-phenol reagent (TRIzol;
Invitrogen Corp.). First-strand cDNA was synthesized using Superscript
III reverse transcriptase and random hexamer primers as described in the
manufacturer’s protocol (Invitrogen Corp.). Samples of cDNA were dilut-
ed 1:25 in nuclease-free water (QIAGEN). Samples from each cDNA pool
were diluted 1:10, 1:30, 1:90, and 1:270 in order to create a standard curve
for calculation of relative gene expression levels. PCR amplification mix-
tures (20 ml) contained 10 ml ×2 PCR SYBR Green I Quantitect Master Mix
(QIAGEN), 0.4 ml of 25-mM reverse and forward primer mix, and 11.6 ml
diluted cDNA template. Real-time quantitative PCR was carried out using
the DNA Engine Opticon (MJ Research) instruments with the following
cycling parameters — polymerase activation: 15 minutes, 95°C; amplifica-
tion for 40 cycles: 15 seconds, 94°C; 20 seconds, 58°C; 20 seconds, 72°C.
After amplification, melting curve analysis was performed as described in
the manufacturers’ protocol (QIAGEN). Relative expression values were
calculated based on the standard curve.
The expression rates of 2 macrophage-specific genes (Emr1, Cd68) that
correlated with body mass in our microarray studies, an adipocyte-spe-
cific gene (Acdc), lipid metabolism genes (Lipe, Gpam, Fabp4), and proin-
flammatory genes (Tnfa, Ccr2, Ccl2/Mcp1, Ccl7/Mcp3, Serpine1) were deter-
mined by quantitative RT-PCR.
To normalize expression data, we used multiple internal control
genes as described by Vandesompele et al. (49). The internal control
genes encoding HMG-14 (Hmg14) and ribosomal protein S3 (Rps3) were
selected from our murine microarray data set for having high expres-
sion and little sample-to-sample variability. For each transcript assayed,
intron-spanning primers were designed using publicly available genomic
contig sequences obtained through Entrez Gene (http://www.ncbi.nlm.
nih.gov/entrez/query.fcgi?db=gene), the public domain primer design
software Primer3 (http://frodo.wi.mit.edu/primer3/primer3_code.html),
and the DNA analysis software Vector NTI Suite Version 7 (Informax
Inc.). Primer sequences were as follows: Rps3 forward, 5-ATCAGAGA-
GTTGACCGCAGTTG-3; Rps3 reverse, 5-AATGAACCGAAGCACAC-
CATAG-3; Emr1 forward, 5-CTTTGGCTATGGGCTTCCAGTC-3;
Emr1 reverse, 5-GCAAGGAGGACAGAGTTTATCGTG-3; Cd68 for-
ward, 5-CTTCCCACAGGCAGCACAG-3; Cd68 reverse, 5-AATGAT-
GAGAGGCAGCAAGAGG-3; Tnfa forward, 5-CCAGACCCTCACTA-
GATCA-3; Tnfa reverse, 5-CACTTGGTGGTTTGCTACGAC-3; Acdc
forward, 5-GCTCCTGCTTTGGTCCCTCCAC-3; Acdc reverse, 5-
GCCCTTCAGCTCCTGTCATTCC-3; Hmg14 forward, 5-GCAGAAAAT-
GGAGAGACGGAAAACC-3; Hmg14 reverse, 5-AAGGGAGGCGGGAC-
CACTGAC-3; Ccl2 forward, 5-AGGTCCCTGTCATGCTTCTGG-3;
Ccl2 reverse, 5-CTGCTGCTGGTGATCCTCTTG-3; Pparg forward, 5-
GCCCTTTGGTGACTTTATGGAG-3; Pparg reverse, 5-GCAGCAGGTT-
GTCTTGGATG-3; Lipe forward, 5-ACGAGCCCTACCTCAAGAACTG-
3; Lipe reverse, 5-ATCTGGCACCCTCACTCCATAG-3; Fabp4 forward,
5-AAGAAGTGGGAGTGGGCTTTG-3; Fabp4 reverse, 5-CTGTC-
GTCTGCGGTGATTTC-3; Serpine1 forward, 5-TCCTCATCCTGCCTA-
AGTTCTC-3; Serpine1 reverse, 5-GTGCCGCTCTCGTTTACCTC-3;
Gpam forward, 5-TCCAGAAGGTGAAAAGGAAAGC-3; Gpam reverse,
5-GGCAAAAGAGGATGAAGGTGAG-3.
Protein assays. For the experiments involving INCB3344, insulin levels were
determined using an ultrasensitive insulin ELISA (Mercodia AB). In experi-
ments comparing Ccr2
–/–
and Ccr2
+/+
mice, we performed a multiplexed
protein assay for IL-6, MCP1, PAI-1, insulin, and leptin (LincoPlex Mouse
Adipokine Panel; Linco Research Inc.). Western blots for adiponectin were
performed with 1 ml plasma from lean and adiposity-matched obese Ccr2
–/–
and Ccr2
+/+
mice. Plasma proteins were denatured, reduced, and separated
on a 16% Tris-glycine gel (Invitrogen Corp.) using SDS-PAGE. Proteins were
transferred to nitrocellulose (Invitrogen Corp.). We performed immunob-
lotting for adiponectin and detected the bands using ECL.
research article
124 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 1 January 2006
Statistics. Pairwise comparisons of quantitative phenotypes between mice
of different genotypes (e.g., obese Ccr2
–/–
vs. obese Ccr2
+/+
) or diets (e.g., lean
low-fat diet–fed Ccr2
+/+
vs. obese high-fat diet–fed Ccr2
+/+
) were assessed by
2-tailed Student’s t test assuming unequal variance. To assess the interac-
tion between genotype and diet for quantitative phenotypes, 2-way ANOVA
was performed. Analyses were performed using Prism 3.0 (GraphPad Soft-
ware Inc.). P < 0.05 was considered statistically significant.
Acknowledgments
We thank Philip Scherer for his gift of the anti-adiponectin
antibody used for Western analysis. This work was supported by
grants from the NIH (DK59960 and DK066525 to A.W. Ferran
-
te Jr.) and the Columbia Diabetes and Endocrinology Research
Center (DK063608).
Received for publication December 27, 2004, and accepted in
revised form September 12, 2005.
Address correspondence to: Anthony W. Ferrante Jr., Naomi Ber
-
rie Diabetes Center, Room 238B, 1150 St. Nicholas Avenue, New
York, New York 10032, USA. Phone: (212) 851-5322; Fax: (212)
851-5331; E-mail: awf7@columbia.edu.
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    • "Adipocytes are reported to exhibit immune cell function with the ability to independently stimulate low-grade inflammation via secretion of MCP-1 [29], while endothelial cells are also reported to increase MCP-1 secretion subsequent to damage [31]. However, these effects are primarily observed in conditions of obesity [32,33] and hypercholesterolemia [34,35]; which, rather than influencing transient changes in the inflammatory response, appear to result in chronically elevated baseline values for MCP-1 and/or CCR2. The cross-over design utilized in the present study negates the potential effect of chronically elevated baseline MCP-1 levels or CCR2 expression. "
    [Show abstract] [Hide abstract] ABSTRACT: The recruitment and infiltration of classical monocytes into damaged muscle is critical for optimal tissue remodeling. This study examined the effects of an amino acid supplement on classical monocyte recruitment following an acute bout of lower body resistance exercise. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm) ingested supplement (SUPP) or placebo (PL) immediately post-exercise in a randomized, cross-over design. Blood samples were obtained at baseline (BL), immediately (IP), 30-min (30P), 1-h (1H), 2-h (2H), and 5-h (5H) post-exercise to assess plasma concentrations of monocyte chemoattractant protein 1 (MCP-1), myoglobin, cortisol and insulin concentrations; and expressions of C-C chemokine receptor-2 (CCR2), and macrophage-1 antigen (CD11b) on classical monocytes. Magnitude-based inferences were used to provide inferences on the true effects of SUPP compared to PL. Changes in myoglobin, cortisol, and insulin concentrations were similar between treatments. Compared to PL, plasma MCP-1 was "very likely greater" (98.1% likelihood effect) in SUPP at 2H. CCR2 expression was "likely greater" at IP (84.9% likelihood effect), "likely greater" at 1H (87.7% likelihood effect), "very likely greater" at 2H (97.0% likelihood effect), and "likely greater" at 5H (90.1% likelihood effect) in SUPP, compared to PL. Ingestion of SUPP did not influence CD11b expression. Ingestion of an amino acid supplement immediately post-exercise appears to help maintain plasma MCP-1 concentrations and augment CCR2 expression in resistance trained men.
    Full-text · Article · Jul 2016
    • "Therefore, further studies are needed to confirm that CCL2 levels may serve as a potential biomarker to monitor the effects of treatment on childhood obesity. Several reports revealed that mice with targeted deletions in the genes for Mcp-1/Ccl2 and Ccr2 have decreases in macrophage content and inflammation in adipose tissue and are protected from high-fat diet-induced insulin resistance [30, 31]. Therefore, the interaction between CCL2 and its receptor CCR2 is considered to be pivotal in obesity-induced insulin resistance [32]. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent studies revealed that obesity is a low-grade, chronic inflammatory state that is accompanied by the enhanced production of multiple chemokines. In particular, metabolic syndrome (MS) and visceral adipose tissue (VAT) accumulation are significantly associated with certain chemokines in adults. However, little is known regarding this association in obese children. The aim of this study was to investigate the relationship between circulating chemokine levels and both MS and VAT accumulation in obese children. Forty-four obese schoolchildren (26 boys) with a percentage of overweight (POW) exceeding 20 were evaluated. The median age was 11.4 years (range: 6.8-16.5 years). Blood samples were drawn after overnight fasting, and serum chemokine levels (CCL2, CCL5 and CXCL10) were quantitated. Visceral fat area (VFA) determinations were conducted using computed tomography. The results showed that the median BMI Z-score, POW, waist circumference and VFA of the subjects were 2.24 SD, 49.8%, 88.3 cm and 80.8 cm2, respectively. Eighteen were diagnosed with MS. CCL2 was significantly increased in MS subjects compared with non-MS subjects (p<0.05). CXCL10 was positively correlated with VFA (r=0.425, p<0.01). There were no significant correlations between age and chemokine levels. We showed that CCL2 levels were elevated in MS and CXCL10 levels were associated with VFA in obese children. Our results suggest that CCL2 and CXCL10 play important roles in the progression of obesity-related metabolic complications in children.
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    • "These cytokines are produced primarily from macrophages, which increase to account for 40% of total cells in AT [8, 12]. Our current mechanistic understanding of obesity-associated AT inflammation and insulin resistance is based primarily on animal studies of artificial metabolic extremes (high-fat diet challenges, lipid infusion, or genetic intervention) or human studies of severely obese subjects [13][14][15]. These studies provide important insights, but their results must be validated by human studies that are designed to examine the non-extreme forms of obesity-induced metabolic disease that dominate the clinical landscape. "
    [Show abstract] [Hide abstract] ABSTRACT: The roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in obesity-associated insulin resistance have been explored in both animal and human studies. However, our current understanding of obesity-associated insulin resistance relies on studies of artificial metabolic extremes. The purpose of this study was to explore the roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in human patients with modest obesity and early metabolic dysfunction. We obtained omental adipose tissue and fasting blood samples from 51 females undergoing gynecologic surgery. We investigated serum concentrations of proinflammatory cytokines and adipokines as well as the mRNA expression of proinflammatory and macrophage phenotype markers in visceral adipose tissue using ELISA and quantitative RT-PCR. We measured adipose tissue inflammation and macrophage infiltration using immunohistochemical analysis. Serum levels of adiponectin and leptin were significantly correlated with HOMA-IR and body mass index. The levels of expression of MCP-1 and TNF-α in visceral adipose tissue were also higher in the obese group (body mass index ≥ 25). The expression of mRNA MCP-1 in visceral adipose tissue was positively correlated with body mass index (r = 0.428, p = 0.037) but not with HOMA-IR, whereas TNF-α in visceral adipose tissue was correlated with HOMA-IR (r = 0.462, p = 0.035) but not with body mass index. There was no obvious change in macrophage phenotype or macrophage infiltration in patients with modest obesity or early metabolic dysfunction. Expression of mRNA CD163/CD68 was significantly related to mitochondrial-associated genes and serum inflammatory cytokine levels of resistin and leptin. These results suggest that changes in the production of inflammatory biomolecules precede increased immune cell infiltration and induction of a macrophage phenotype switch in visceral adipose tissue. Furthermore, serum resistin and leptin have specific roles in the regulation of adipose tissue macrophages in patients with modest obesity or early metabolic dysfunction.
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