Deficiency of the leukotriene B4 receptor, BLT-1, protects against systemic insulin resistance in diet-induced obesity.
ABSTRACT Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report that a high-fat diet selectively increases the circulating levels of CD11b(+) monocytes in wild-type mice that express leukotriene B(4) receptor, BLT-1, and that this increase is abolished in BLT-1-null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1(-/-) mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1(-/-) mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: People with diabetes can suffer from diverse complications that seriously erode quality of life. Diabetes, costing the United States more than $174 billion per year in 2007, is expected to take an increasingly large financial toll in subsequent years. Accurate projections of diabetes burden are essential to policymakers planning for future health care needs and costs. Using data on prediabetes and diabetes prevalence in the United States, forecasted incidence, and current US Census projections of mortality and migration, the authors constructed a series of dynamic models employing systems of difference equations to project the future burden of diabetes among US adults. A three-state model partitions the US population into no diabetes, undiagnosed diabetes, and diagnosed diabetes. A four-state model divides the state of "no diabetes" into high-risk (prediabetes) and low-risk (normal glucose) states. A five-state model incorporates an intervention designed to prevent or delay diabetes in adults at high risk. The authors project that annual diagnosed diabetes incidence (new cases) will increase from about 8 cases per 1,000 in 2008 to about 15 in 2050. Assuming low incidence and relatively high diabetes mortality, total diabetes prevalence (diagnosed and undiagnosed cases) is projected to increase from 14% in 2010 to 21% of the US adult population by 2050. However, if recent increases in diabetes incidence continue and diabetes mortality is relatively low, prevalence will increase to 33% by 2050. A middle-ground scenario projects a prevalence of 25% to 28% by 2050. Intervention can reduce, but not eliminate, increases in diabetes prevalence. These projected increases are largely attributable to the aging of the US population, increasing numbers of members of higher-risk minority groups in the population, and people with diabetes living longer. Effective strategies will need to be undertaken to moderate the impact of these factors on national diabetes burden. Our analysis suggests that widespread implementation of reasonably effective preventive interventions focused on high-risk subgroups of the population can considerably reduce, but not eliminate, future increases in diabetes prevalence.Population Health Metrics 10/2010; 8:29. · 2.11 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The prevalence of obesity increased in the United States between 1976-1980 and 1988-1994 and again between 1988-1994 and 1999-2000. To examine trends in obesity from 1999 through 2008 and the current prevalence of obesity and overweight for 2007-2008. Analysis of height and weight measurements from 5555 adult men and women aged 20 years or older obtained in 2007-2008 as part of the National Health and Nutrition Examination Survey (NHANES), a nationally representative sample of the US population. Data from the NHANES obtained in 2007-2008 were compared with results obtained from 1999 through 2006. Estimates of the prevalence of overweight and obesity in adults. Overweight was defined as a body mass index (BMI) of 25.0 to 29.9. Obesity was defined as a BMI of 30.0 or higher. In 2007-2008, the age-adjusted prevalence of obesity was 33.8% (95% confidence interval [CI], 31.6%-36.0%) overall, 32.2% (95% CI, 29.5%-35.0%) among men, and 35.5% (95% CI, 33.2%-37.7%) among women. The corresponding prevalence estimates for overweight and obesity combined (BMI > or = 25) were 68.0% (95% CI, 66.3%-69.8%), 72.3% (95% CI, 70.4%-74.1%), and 64.1% (95% CI, 61.3%-66.9%). Obesity prevalence varied by age group and by racial and ethnic group for both men and women. Over the 10-year period, obesity showed no significant trend among women (adjusted odds ratio [AOR] for 2007-2008 vs 1999-2000, 1.12 [95% CI, 0.89-1.32]). For men, there was a significant linear trend (AOR for 2007-2008 vs 1999-2000, 1.32 [95% CI, 1.12-1.58]); however, the 3 most recent data points did not differ significantly from each other. In 2007-2008, the prevalence of obesity was 32.2% among adult men and 35.5% among adult women. The increases in the prevalence of obesity previously observed do not appear to be continuing at the same rate over the past 10 years, particularly for women and possibly for men.JAMA The Journal of the American Medical Association 01/2010; 303(3):235-41. · 29.98 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The proper functioning of the pathways that are involved in the sensing and management of nutrients is central to metabolic homeostasis and is therefore among the most fundamental requirements for survival. Metabolic systems are integrated with pathogen-sensing and immune responses, and these pathways are evolutionarily conserved. This close functional and molecular integration of the immune and metabolic systems is emerging as a crucial homeostatic mechanism, the dysfunction of which underlies many chronic metabolic diseases, including type 2 diabetes and atherosclerosis. In this Review we provide an overview of several important networks that sense and manage nutrients and discuss how they integrate with immune and inflammatory pathways to influence the physiological and pathological metabolic states in the body.Nature Reviews Immunology 01/2009; 8(12):923-34. · 32.25 Impact Factor
of September 12, 2012.
This information is current as
Resistance in Diet-Induced Obesity
BLT-1, Protects against Systemic Insulin
Deficiency of the Leukotriene B
R. Jala and Bodduluri Haribabu
Mathis, Madhavi Kosuri, Aruni Bhatnagar, Venkatakrishna
Matthew Spite, Jason Hellmann, Yunan Tang, Steven P.
2011; 187:1942-1949; Prepublished online 8 July
, 25 of which you can access for free at:
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The Journal of Immunology
at University of Louisville on September 12, 2012
The Journal of Immunology
Deficiency of the Leukotriene B4Receptor, BLT-1, Protects
against Systemic Insulin Resistance in Diet-Induced Obesity
Matthew Spite,*,†Jason Hellmann,* Yunan Tang,* Steven P. Mathis,‡Madhavi Kosuri,*
Aruni Bhatnagar,* Venkatakrishna R. Jala,†,‡and Bodduluri Haribabu†,‡
Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in
circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous
pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report
that a high-fat diet selectively increases the circulating levels of CD11b+monocytes in wild-type mice that express leukotriene B4
receptor, BLT-1, and that this increase is abolished in BLT-1–null mice. The accumulation of classically activated (M1) adipose
tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely
blunted in adipose tissue of obese BLT-12/2mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was
increased. Obese BLT-12/2mice were protected from systemic glucose and insulin intolerance and this was associated with
a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1
prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of
chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in
obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.
Immunology, 2011, 187: 1942–1949.
sistance in obesity is induced and sustained by chronic low-grade
inflammation (3, 4). Indeed, type 2 diabetes is associated with
increased levels of inflammatory mediators that induce insulin
resistance, and adipose tissue in obese and diabetic individuals
remains in a state of chronic, unresolved inflammation (4, 5).
More than 40% of the total adipose tissue cell content from obese
rodents and humans is composed of macrophages, compared with
10% in lean counterparts, and inflammatory adipose tissue mac-
rophages (ATMs) directly promote systemic insulin resistance (4,
6, 7). Nevertheless, the mechanisms that contribute and sustain
chronic inflammation in obesity and diabetes remain poorly un-
Leukotriene B4(LTB4) is a proinflammatory lipid mediator
generated from arachidonic acid through the sequential activities
The Journal of
besity is an emerging global epidemic and is one of the
most prominent risk factors for the development of type
2 diabetes (1, 2). Extensive studies show that insulin re-
of 5-lipoxygenase, 5-lipoxygenase–activating protein, and leuko-
triene A4hydrolase (8, 9). LTB4is rapidly generated by activated
leukocytes and has well-characterized biological actions, including
the promotion of leukocyte chemotaxis and the regulation of pro-
inflammatory cytokines (8, 10). The potent biological actions of
LTB4are mediated primarily through a high-affinity interaction
with a G protein-coupled receptor termed BLT-1 (10). Although
the LTB4/BLT-1 axis plays an important role in host defense
during acute infection, chronic activation of this pathway con-
tributes to persistent inflammation characteristic of inflammatory
pathologies, including atherosclerosis and arthritis (11–16).
Recent studies show that the expression and activity of enzymes
required for leukotriene biosynthesis, including 5-lipoxygenase
and 5-lipoxygenase–activating protein, are increased in both the
liver and adipose tissue in murine models of experimental obesity
(16–18). Moreover, LTB4levels increase in adipose tissue of both
mice and rats consuming a high-fat diet (16, 17, 19). However,
a causal role for LTB4in promoting or sustaining chronic in-
flammation and insulin resistance in obesity has not been estab-
lished. In this study, we report that deficiency of BLT-1 protects
against the development of insulin resistance in diet-induced
obesity (DIO) by regulating ATM accumulation and inflamma-
tion in insulin-sensitive tissues.
Materials and Methods
Animals and treatment
Male C57BL/6J mice were purchased from The Jackson Laboratory (Bar
Harbor, ME) and BLT-12/2mice (Ltb4r1tm1Bodd) were generated as de-
scribed (12) and backcrossed for nine generations on a C57BL/6J
background as reported before (13). At 8–11 wk of age, mice were
placed on either a 10% (kilocalories from fat) low-fat diet or 60% high-
fat diet (Research Diets) and maintained for 12 additional weeks. Body
weight was recorded weekly. Glucose and insulin tolerance tests were
performed during the 12th wk of feeding, as outlined in Fig. 1A, whereas
all other parameters were evaluated upon euthanasia. All procedures
were approved by the University of Louisville Institutional Animal Care
and Use Committee.
*Division of Cardiovascular Medicine, Diabetes and Obesity Center, University of
Louisville School of Medicine, Louisville, KY 40202;†Department of Microbiology
and Immunology, University of Louisville School of Medicine, Louisville, KY
40202; and‡James Graham Brown Cancer Center, University of Louisville School
of Medicine, Louisville, KY 40202
Received for publication January 20, 2011. Accepted for publication June 5, 2011.
This work was supported by the National Institutes of Health-sponsored Diabetes and
Obesity Center (Grant P20RR024489 to M.S. and A.B.) and by National Institutes of
Health Grant CA138623 (to H.B.).
Address correspondence and reprint requests to Dr. Matthew Spite, University of
Louisville, Delia Baxter Building, 580 S. Preston Street, Room 404F, Louisville,
KY 40202 and address requests for material and mice to Dr. Bodduluri Haribabu,
505 S. Hancock Street, Room 324 CTR Building, Louisville, KY 40202. E-mail
addresses: Matthew.firstname.lastname@example.org (M.S.) and H0bodd01@louisville.edu (B.H.)
The online version of this article contains supplemental material.
Abbreviations used in this article: ALT, alanine aminotransferase; ATM, adipose
tissue macrophage; CLS, crown-like structures; DIO, diet-induced obesity; HOMA-
IR, homeostatic model assessment of insulin resistance; LTB4, leukotriene B4; MGL-1,
macrophage galactose-type C-type lectin 1; WT, wild-type.
at University of Louisville on September 12, 2012
Glucose/insulin tolerance tests
Glucose tolerance tests were performed following a 6 h fast by i.p. injection
of D-glucose (1 mg/g) in sterile saline. Insulin tolerance tests were per-
formed on nonfasted animals by i.p. injection of 1.5 U/kg Humulin R (Eli
Lilly, Indianapolis, IN). Blood samples were obtained from the tail and
glucose levels were measured at indicated time points using an Aviva
Accu-Chek glucometer. The homeostatic model assessment of insulin re-
sistance (HOMA-IR) score was calculated based on the formula: glucose
(mmol) 3 insulin (mU/ml)/22.5.
Immunoblotting and PCR
Tissue lysates were prepared as described in Horrillo et al. (17). Equal
amounts of protein were separated by SDS-PAGE, transblotted, and probed
for phospho-Akt (Ser473), total Akt, phospho-JNK (Thr183/Tyr185), total
JNK, phospho-IkBa, and total IkBa (Cell Signaling Technology). Western
blots were developed using ECL Plus followed by luminescence detection
using a Typhoon 9400 variable mode imager (Amersham Biosciences).
Quantification of band intensities was performed using ImageQuant TL
For quantitative RT-PCR, RNA was extracted from tissues using the
RNeasy lipid tissue kit (Qiagen), followed by cDNA synthesis. Real-time
PCR amplification was performed with SYBR Green qPCR Master Mix
(SABiosciences) using a 7900HT Fast Real-Time PCR System (Applied
Biosystems) and commercially available primers for Emr-1, IL-10, IL-6,
PPAR-g, and PPAR-a (Super Array Biosciences). The following primers
were also used (Integrated DNA Technologies): Ccl2, forward, 59-ATG-
CAGGTCCCTGTCATG-39, reverse, 59-GCTTGAGGTGGTTGTGGA-39;
Blt-1, forward, 59-TCC CTT TTT CCT CCA CTT TC-39, reverse, 59-GAA
AAG ACA CCA CCC AGA TG-39; Ym-1, forward, 59-GGGCATACCTT-
TATCCTGAG-39, reverse, 59-CCACTGAAGTCATCCATGTC-39; Arg-1,
forward, 59-ATGGAAGAGACCTTCAGCTAC-39, reverse, 59-GCTGTC-
TTCCCAAGAGTTGGG-39 (20). Relative expression was determined by
the 22DDCTmethod after internal normalization to hprt or b-actin.
Immunohistochemistry and quantification of crown-like
H&E staining was performed following standard procedures on formalin-
fixed paraffin-embedded epididymal adipose tissue and liver concurrent
sections. Crown-like structures (CLS) were identified as adipocytes com-
pletely surrounded by infiltrating cells and were quantified in five random
fields per animal. Oil Red O staining was performed on OCT-embedded
liver sections as described in Horrillo et al. (17).
Total plasma cholesterol, high-density lipoproteins, low-density lipopro-
teins, triglycerides, total protein, albumin (cholesterol CII enzymatic kit;
L-type TG-H kit; Bradford reagent, bromocresol green; Wako, Richmond,
VA), alanine aminotransferase (ALT), and aspartate aminotransferase (In-
finity, ThermoElectron) levels were measured using commercially avail-
able assay reagents as indicated. The total hepatic triglyceride content
was determined after chloroform/methanol extraction of frozen liver sam-
ples. Assays were performed using a Cobas Mira Plus 5600 Autoanalyzer
(Roche, Indianapolis, IN). Plasma insulin was measured by ELISA
Flow cytometry analysis
Analysisof cells in spleenand peripheralbloodwas carriedoutas described
in Mathis et al. (21) with minor modifications. Cells from spleen were
isolated and filtered, while 30 ml peripheral blood was used for analysis.
Cells were stained with anti-mouse CD16/32 (BD Pharmingen, San Diego,
CA) in 1% FBS in PBS. Biotinylated anti-mouse BLT-1 (3D7) was then
used for assessment of BLT-1 expression. Cells were subsequently washed
and stained. Abs used include PE-conjugated anti-B220, PerCP-Cy5.5–
conjugated anti-CD8, PE-conjugated anti-NK1.1, FITC-conjugated anti-
CD3 molecular complex, PE-conjugated anti–Siglec-F, PerCP-Cy5.5–con-
jugated anti-CD11b (BD Pharmingen, San Diego, CA), allophycocyanin-
conjugated anti-CD4, FITC-conjugated anti-GR (eBioscience, San Diego,
CA), allophycocyanin-conjugated streptavidin, and PE-conjugated anti-
gdTCR (BioLegend, San Diego, CA). Flow cytometry was carried out us-
ing a FACSCalibur flow cytometer equipped with CellQuest Pro software
(BD Biosciences) and analyzed with FlowJo v.4.3 software.
Adipose tissue stromal vascular cells were isolated as in Lumeng et al.
(22). Stromal vascular cell pellets were incubated with Fc Block prior to
staining with fluorescent-conjugated primary Abs or isotype controls. Abs
used included Alexa Fluor 647 anti–Mgl-1 (CD301a; AbD Serotec), FITC
anti-CD11c (BD Biosciences), and PE anti-F4/80 (BioLegend). Flow cy-
tometry was carried out using a BD LSRII flow cytometer equipped with
FACSDiva v.6.0. Analysis was performed with FlowJo v.7.6 software.
ulates peripheral blood monocyte expan-
sion in DIO. A, Feeding protocol. Starting
at 8 wk age, WTand BLT-1–deficient mice
were fed a low-fat (10%) or high-fat
(60%) diet for 12 wk. Glucose tolerance
tests (GTT) and insulin tolerance tests
(ITT) were performed during the 12th
week of the feeding protocol. B, Surface
expression of BLT-1 on peripheral blood
monocytes and neutrophils (PMN) in WT
mice fed a low-fat or high-fat diet (n =
5/group). C, Weight gain of WT and BLT-
1–deficient mice on a high-fat diet for
12 wk (n = 8–12/group). D, Peripheral
blood monocytes in WT and BLT-1–
deficient mice (n = 5/group). E, Whole
blood leukocyte differentials from WT
and BLT-1–deficient mice (n = 5/group).
F, BLT-1 mRNA expression in liver and
skeletal muscle (n = 6/group). Data are
presented as means 6 SEM. *p , 0.05
by Student t test (F), one-way ANOVA
(D), or two-way ANOVA (C).
Expression of BLT-1 reg-
The Journal of Immunology1943
at University of Louisville on September 12, 2012
Data are expressed as means 6 SEM. Multiple group comparisons were
made using one-way or two-way ANOVA, followed by Bonferroni post-
tests. Direct comparisons were made using an unpaired Student t test. A p
value ,0.05 was considered significant.
BLT-1 regulates peripheral blood monocyte expansion in
High-fat feeding increases circulating levels of peripheral blood
monocytes that infiltrate fat depots and other dysfunctional tissues
(6, 23, 24). Because LTB4increases in adipose tissue in obesity
(16, 17, 19), we asked whether activation of BLT-1 regulates
monocyte recruitment in obesity. In mice fed a high-fat diet for
12 wk (see protocol in Fig. 1A), BLT-1 expression increased on
CD11b+circulating monocytes (Fig. 1B). Importantly, this in-
crease was selective for monocytes, as BLT-1 expression on poly-
morphonuclear neutrophils (PMNs) did not change with high-fat
feeding. To determine whether BLT-1 is required for monocyte
expansion in obesity, mice deficient in BLT-1 were placed on
either a low-fat or high-fat diet. Interestingly, BLT-1–deficient
mice gained weight to a similar extent as did their wild-type (WT)
counterparts when fed a high-fat diet (Fig. 1C). Note that the BLT-
1–deficient mice weighed less than their WT counterparts at the
initiation of the study (23.59 6 0.37 versus 26.76 6 0.41 g, re-
spectively), and thus the BLT-1–deficient mice weighed less than
the WT mice on a high-fat diet throughout the study protocol
(Supplemental Fig. 1A). However, at the end of the feeding period,
there were no significant differences in body weight between WT
and BLT-1–deficient mice, indicating that the BLT-1–deficient
mice had accelerated weight gain toward the end of the study
protocol (Supplemental Fig. 1B). Consistent with previous reports,
high-fat feeding led to an increase in the number of circulating
CD11b+monocytes in WT mice (Fig. 1D) (23). Significantly, this
increase was completely abolished in BLT-1–deficient mice (Fig.
1D). Notably, this regulation was specific to monocytes, as other
peripheral blood leukocyte populations, including GR-1+PMNs,
B cells, NK cells, and both CD4+and CD8+T cells, were not
affected by either high-fat feeding or BLT-1 deficiency (Fig. 1E).
Moreover, leukocyte populations in the spleen were also un-
affected by high-fat feeding or BLT-1 deficiency (Supplemental
We next evaluated changes in the expression of BLT-1 in liver,
skeletal muscle, and adipose tissue of lean and obese mice. As
shown in Fig. 1F, BLT-1 was expressed in the liver and skeletal
muscle of lean mice, although the expression of BLT-1 in adipose
tissue was not evident. In mice fed a high-fat diet, a decrease in
the expression of BLT-1 was observed in both liver and skeletal
Deficiency of BLT-1 improves glucose and insulin tolerance in
We next evaluated whether BLT-1 deficiency modulates metabolic
derangements associated with DIO. High-fat feeding markedly
increased fasting plasma glucose levels in WT mice (Fig. 2A).
Similar to the effects of BLT-1 deficiency on weight gain, no
differences in plasma glucose were observed with high-fat feeding
compared with WT mice. DIO increased plasma insulin in WT
mice, whereas insulin was not significantly elevated in BLT-12/2
mice (Fig. 2B). Importantly, BLT-12/2mice were less insulin re-
sistant than were obese WT mice, as determined by the homeo-
stasis model assessment of insulin resistance (HOMA-IR; Fig.
tects against systemic insulin resistance
in DIO. A, Fasting blood glucose. B,
Fasting plasma insulin levels. C, Cal-
culated HOMA-IR score. D and E,
Glucose tolerance tests. F, Area under
the curve measurement of glucose tol-
erance tests in D and E. G and H, Insulin
tolerance tests. I, Area under the curve
measurement of insulin tolerances tests
in G and H. Data are presented as
means 6 SEM. *p , 0.05 by one-way
(A–C, F, I) or two-way (D, E, G, H)
ANOVA; n = 8–12/group. A.U.C., area
under the curve.
Deficiency of BLT-1 pro-
1944BLT-1 AND OBESITY-INDUCED INSULIN RESISTANCE
at University of Louisville on September 12, 2012
2C). To evaluate how BLT-1 deficiency affects glucose homeo-
stasis, we performed glucose and insulin tolerance tests in WTand
BLT-12/2mice. DIO induced pronounced glucose intolerance in
WT mice (Fig. 2D). Similarly, obese BLT-12/2mice were glucose
intolerant (Fig. 2E), although area under the curve measurements
indicated that the magnitude of glucose intolerance was less in the
BLT-12/2mice than their WT counterparts (Fig. 2F). Importantly,
high-fat feeding decreased insulin-stimulated glucose disposal
in WT mice, whereas BLT-12/2mice were completely protected
from this insulin intolerance (Fig. 2G–I). Deficiency of BLT-1 did
not modulate changes in total plasma cholesterol, high-density
lipoproteins, low-density lipoproteins, plasma triglycerides, or
total protein levels in DIO (Supplemental Fig. 2). Modest changes
in the heart/body weight ratios were observed between obese WT
and BLT-12/2mice (Supplemental Fig. 2). Serum creatinine and
lactate dehydrogenase levels were not significantly increased with
high-fat feeding in WT mice (Supplemental Fig. 2). Taken to-
gether, these results indicate that deficiency of BLT-1 protects
from glucose and insulin intolerance in obesity.
Adipose tissue macrophage accumulation and inflammation
are decreased in obese BLT-12/2mice
The accumulation of classically activated (M1) macrophages in
adipose tissue is a critical underlying component linking adipose
tissue expansion with systemic insulin resistance (6, 7, 25). We
next asked whether deficiency of BLT-1 affects the accumulation
of M1 ATMs. As shown in Fig. 3A, epididymal fat pad weights
were increased in both WT and BLT-12/2mice on a high-fat diet,
consistent with the observed increase in total body mass (see
above). Notably, the magnitude of epididymal fat pad expansion
in obese BLT-12/2mice was significantly higher than in obese
WT mice. However, despite this increase, expression of pan-
macrophage marker Emr-1 (F4/80) was significantly decreased
in BLT-12/2mice relative to WT mice (Fig. 3B). Additionally, the
expression of monocyte chemoattractant Ccl2 was also decreased
in BLT-12/2mice (Fig. 3C). Interestingly, the expression of
PPARa, which is a nuclear receptor for LTB4that regulates the
expression of b-oxidation genes, was significantly elevated in
obese BLT-1–deficient mice (Fig. 3D) (26). Histological analysis
of adipose tissue from obese WT mice showed an increase in the
formation of CLS compared with their low fat-fed counterparts
(Fig. 3E) (7). The formation of CLS in obese BLT-12/2mice was
not significantly increased compared with their low fat-fed
counterparts. Because M1 ATMs contribute to insulin resistance
by producing inflammatory cytokines that block insulin action
(25, 27), we next evaluated the expression of IL-6 in adipose
tissue. Notably, IL-6 was drastically reduced in BLT-12/2mice
(Fig. 3F). To further elucidate how adipose tissue inflammation is
affected by BLT-1 deficiency, we evaluated the activation of JNK,
which has been shown to play a causal role in obesity-induced
insulin resistance (28). Indeed, phosphorylation of JNK was sig-
nificantly decreased in adipose tissue of BLT-12/2mice compared
with obese WT mice (Fig. 3G). Nevertheless, as reported pre-
viously (29), high-fat feeding for 12 wk did not induce insulin
tion and macrophage accumulation are
decreased in BLT-1–deficient mice on
a high-fat diet. A, Ratio of epididymal
fat pad weight to total body weight. B,
Quantification of mRNA expression of
macrophage gene Emr-1 in adipose tissue
isolated from WT or BLT-1–deficient
mice. C and D, Quantification of Ccl2
and PPARa in adipose tissue. E, Repre-
sentative histological analysis (H&E
stain) of epididymal adipose tissue from
WT and BLT–1 deficient mice (original
magnification 310); right panel, quan-
tification of crown-like structures per
field. F, Adipose tissue mRNA expres-
sion of IL-6 in WT and BLT-1–deficient
mice fed a high-fat diet. G, Phosphory-
lation of JNK in adipose tissue of WT
and BLT-1–deficient mice fed a high-fat
diet. Data are presented as means 6
SEM. *p , 0.05 by one-way ANOVA
(A, E) or Student t test (B–D, F, G); n =
Adipose tissue inflamma-
The Journal of Immunology1945
at University of Louisville on September 12, 2012
resistance in the adipose tissue, as assessed by insulin-stimulated
phosphorylation of Akt (data not shown). These results indicate
that BLT-12/2mice are protected from infiltration of inflammatory
monocytes into adipose tissue and consequently from adipose
Deficiency of BLT-1 alters adipose tissue macrophage
Adipose tissue from lean mice contains resident M2 macrophages
that serve a homeostatic role. It is now widely accepted that
macrophages that infiltrate the expanded adipose tissue in obesity
are derived from circulating monocytes and assume an M1 phe-
notype (22, 30). Because BLT-1 deficiency prevented the increase
in CD11b+monocytes in DIO and also decreased ATM accumu-
lation, we questioned whether the phenotype of ATMs was af-
fected by BLT-1 deficiency. For this, we isolated stromal vascular
cells from adipose tissue obtained from WT or BLT-12/2mice
and assessed the surface expression of CD11c and macrophage
galactose-type C-type lectin 1 (MGL-1), markers of M1 and M2
macrophages, respectively, on the total macrophage (F4/80+)
population (22, 30). High-fat feeding markedly increased the
population of ATMs expressing CD11c and lacking MGL-1 (Fig.
4A). Importantly, this increase was prevented in BLT-12/2mice.
Moreover, the amount of ATMs expressing MGL-1 and lacking
CD11c (M2) was significantly increased in BLT-12/2mice com-
pared with WT mice. Consistent with a recent report, we also
identified a CD11c+MGL-1+population of ATMs, which was
present in both WT and BLT-12/2mice, but this population was
significantly increased only in the BLT-12/2mice (30). Repre-
sentative dot plots of the F4/80+ATMs are shown in Fig. 4B. We
next measured the expression of characteristic M2 genes to rig-
orously assess how ATM phenotype is affected by BLT-1 de-
ficiency. The expression of M2 genes, Ym-1 and PPARg, was
significantly increased in BLT-12/2mice compared with obese
WT mice, whereas the expression of other M2 genes, such as IL-
10 and Arg-1, was not affected (Fig. 4C). Collectively, these
results suggest that BLT-1 deficiency prevents accumulation of
M1 ATMs in obesity and thus alters the balance between inflamma-
tory M1 and M2 ATMs.
Obesity-induced hepatic steatosis and inflammation are
alleviated by BLT-1 deficiency
Macrophage-mediated adipose tissue inflammation is sufficient to
promote insulin resistance in other insulin-sensitive tissues, such
as the liver and skeletal muscle (31–33). In particular, insulin re-
sistance in the liver can be driven by fatty acid release from ad-
ipose tissue, resulting in hepatic triglyceride accumulation, and it
can also arise from increased production of inflammatory cyto-
kines from the adipose tissue (31, 32). Thus, we asked whether
a deficiency of BLT-1 would alleviate insulin resistance and he-
patic steatosis in DIO. Obesity caused profound liver damage in
WT mice, as evidenced by increased plasma ALT levels (Fig. 5A).
Surprisingly, this increase was completely abolished in BLT-12/2
mice. The liver/body weight ratio was significantly decreased in
BLT-12/2mice compared with obese WT mice (Supplemental
Fig. 3A). Moreover, the high-density lipoprotein/low-density li-
poprotein ratio was significantly decreased in obese WT mice, but
this decrease was prevented in BLT-12/2mice (Supplemental Fig.
3B). Histological analysis of the liver showed an apparent de-
crease in fat accumulation in BLT-12/2mice (Fig. 5B). Accord-
ingly, the total triglyceride content of the liver was significantly
decreased in BLT-12/2mice (Fig. 5C). Fat staining with Oil Red
O showed a reduction in total fat content of the liver in BLT-12/2
mice compared with obese WT mice (Supplemental Fig. 3C). In
contrast to the adipose tissue, PPARa transcripts were not mod-
ulated by BLT-1 deficiency in the liver of high fat-fed mice
(Supplemental Fig. 3D). Because steatosis drives hepatic inflam-
mation, we next evaluated whether the inflammatory NF-kB
pathway was affected by BLT-1 deficiency. Indeed, increased
phosphorylation of IkBa, an upstream mediator of NF-kB acti-
vation, was observed with DIO in WT mice, whereas this increase
was abolished in BLT-12/2mice (Fig. 5D). Increased inflam-
matory signaling through this pathway directly contributes to in-
sulin resistance, so we next evaluated how insulin signaling was
affected in the liver of BLT-12/2mice. As expected with this
duration of high-fat feeding, the liver was markedly insulin re-
sistant in WT mice, as assessed by reduced insulin-stimulated
types. A, Quantification of isolated adipose tissue macrophage populations
in WT and BLT-1–deficient mice. B, Representative flow cytometry dot
plots of F4/80+adipose tissue macrophages from WT or BLT-1–deficient
mice. C, Adipose tissue mRNA expression of characteristic alternatively
activated adipose tissue macrophage genes. Data are presented as means 6
SEM. *p , 0.05 by one-way ANOVA (A) or Student t test (C); n = 6–9/
BLT-1 deficiency alters adipose tissue macrophage pheno-
1946BLT-1 AND OBESITY-INDUCED INSULIN RESISTANCE
at University of Louisville on September 12, 2012
phosphorylation of Akt. Importantly, insulin signaling was largely
preserved in the liver of obese BLT-12/2mice (Fig. 5E).
Deficiency of BLT-1 preserves insulin signaling in the skeletal
muscle in DIO
In addition to the liver, insulin resistance in skeletal muscle is
in pronounced insulin resistance in skeletal muscle, as evidenced
by a relative lack of insulin-stimulated Akt phosphorylation in high
fat-fed mice (Supplemental Fig. 4). Although the level of insulin-
stimulated phosphorylation of Akt in BLT-12/2mice fed a low-fat
diet was lower in magnitude than in lean WT mice, obese BLT-1–
deficient mice were markedly protected from obesity-induced loss
in insulin signaling (Supplemental Fig. 4).
The LTB4/BLT-1 axis is an important proinflammatory pathway
involved in host defense. During the acute inflammatory response,
LTB4is one of the first leukocyte chemoattractants generated and
it promotes leukocyte migration in response to invading pathogens
and tissue injury (8, 12, 14). Indeed, BLT-1–deficient mice have
defects in leukocyte infiltration in acute peritonitis and show
a decrease in leukocyte-mediated bacterial clearance (12, 14, 34).
Conversely, increased activation of BLT-1 is associated with
chronic inflammatory diseases, and deficiency of BLT-1 confers
protection against the development of arthritis and atherosclerosis.
Although BLT-1 contributes to the progression of chronic in-
flammation and recent studies show that LTB4is increased in
obesity, a direct causative role of this pathway in DIO and insulin
resistance had not been assessed before (16, 17, 19). The results
of the current study document a previously unrecognized role of
BLT-1 in promoting monocyte recruitment, inflammation, and
insulin resistance in obesity.
Obesity promotes the mobilization of monocytes from the bone
marrow in part by activating the chemokine receptor CCR2 (23,
24, 35). Global deficiency of Ccr2 or its ligand, Ccl2 (MCP-1), in
mice results in a failure of monocyte mobilization and is associ-
ated with protection from monocyte infiltration into adipose tissue
and insulin resistance (24, 35). The current study demonstrates
that BLT-1 is also required for obesity-induced increases in pe-
ripheral monocytes and subsequent ATM accumulation. Inter-
estingly, BLT-1 deficiency also led to a decrease in the adipose
tissue expression of Ccl2, suggesting that activation of the
BLT-1 pathway may be important for subsequent production of
chemokine-driven amplification loops in obesity. Indeed, activa-
tion of BLT-1 by LTB4induces production of CCL2 in monocytes
and, conversely, CCL2 stimulates the production of LTB4to es-
tablish a positive feed-forward cycle (13, 36). Recently, BLT-1
was shown to be required for the initiation of cytokine and che-
mokine gradients in K/BxN-induced arthritis (11). Thus, in light
1–deficient mice. B, Histological analysis (H&E staining) of liver tissues from WT or BLT-1–deficient mice (original magnification 310). C, Hepatic
triglyceride levels in WTor BLT-1–deficient mice. D, Immunoblot of phospho-IkBa in the liver of WTand BLT-1–deficient mice, with quantification shown
(lower panel). E, Insulin-stimulated Akt phosphorylation in liver of WT and BLT-1–deficient mice fed a low-fat (LF) or high-fat (HF) diet. Lower panel,
Quantification of Akt phosphorylation in high fat-fed WTand BLT-1–deficient mice. Data are presented as means 6 SEM. *p , 0.05 by one-way ANOVA;
n = 6–9/group.
Hepatic steatosis, inflammation, and insulin resistance are diminished in BLT-1–deficient mice in DIO. A, Plasma ALT levels in WTand BLT-
The Journal of Immunology1947
at University of Louisville on September 12, 2012
of these previous findings, our current data indicate that activation
of BLT-1 may be an important early contributor to the increase in
circulating monocytes and monocyte infiltration into tissues in
obesity. One potential caveat of our study is that the WT and
BLT-1–deficient mice used were not littermates. However, the
BLT-12/2mice have been backcrossed for more than nine gen-
erations onto a C57BL/6 background, and we routinely house the
WT mice obtained from the vendor for a 2- to 3-wk period for
acclimatization in the same facility before they enter the study.
Notably, the high-fat feeding protocol itself extends over 12 wk,
during which time the mice were housed under identical conditions,
thus minimizing the contribution of differences in housing to the
Consistent with our findings that BLT-1 expression increases
on peripheral blood monocytes in obesity, BLT-1 is also highly
expressed on classically activated “inflammatory” human mono-
cytes (CD14+CD162) (37, 38). Significantly, the expression of
BLT-1 is lower on CD14+CD16+“resident” monocytes, and this
dynamic expression pattern parallels that of CCR2 (37). Our
findings also demonstrate that DIO decreases the expression of
BLT-1 in tissues. Previous studies have documented that BLT-1
expression decreases on leukocytes after they infiltrate arthritic
joints and are exposed to ligand LTB4(11, 39). Moreover, the
expression of both BLT-1 and CCR2 is negatively regulated by
proinflammatory cytokines, including IFN-g and TNF-a (37, 40).
This well-documented negative feedback system likely explains
why BLT-1 expression decreased in both skeletal muscle and liver
of obese mice and was not readily detected in adipose tissue.
Although our studies suggest a key role of BLT-1 in mediating
tissue recruitment of monocytes in obesity, we cannot rule out that
BLT-1 might also be an important factor in perpetuating in-
flammatory signaling at the tissue level. As noted, recent reports
demonstrate that LTB4levels are increased in adipose tissue in
obesity, and our studies show a profound decrease in the activation
of inflammatory signaling in both adipose tissue and liver of BLT-
1–deficient mice. Thus, it is likely that the LTB4/BLT-1 pathway
plays an important role in both recruitment and local activation of
monocytes/macrophages in obesity.
Macrophage polarization toward a classically activated (M1)
or alternatively activated (M2) state depends largely on soluble
factors, such as cytokines and lipid mediators (38, 41). Adipose
tissue expansion in DIO is associated with the infiltration of M1
macrophages that localize to CLS surrounding apoptotic adipo-
cytes (42). A high ratio of inflammatory M1 ATMs (F4/80+
CD11c+MGL-12) to resident M2 ATMs (F4/80+CD11c2MGL-1+)
is reflective of both adipose tissue as well as systemic insulin
resistance (7, 22, 30). In comparison with WT controls, ATMs
were drastically decreased in obese BLT-12/2mice and this was
associated with a higher M2/M1 ratio. This observation was
confirmed by the identification of higher M2 transcripts, includ-
ing Ym-1 and PPARg, in BLT-12/2mice. Because PPARg controls
alternative activation in macrophages and the expression of this
nuclear receptor is associated with increased insulin sensitivity
(43), it is likely that PPARg may be in part responsible for
the higher proportion of M2 macrophages in adipose tissue of
BLT-12/2mice. Additional studies are required to fully elucidate
The central role of ATM-mediated inflammation to systemic
insulin resistance is evidenced by studies showing that adipocyte-
specific expression of Ccr2 promotes adipose tissue macrophage
accumulation and is sufficient to induce insulin resistance in other
tissues such as the liver (33). In our studies, BLT-1–deficient mice
were largely protected from ATM accumulation, and insulin sig-
naling was preserved in the liver as well as in the skeletal muscle.
These data suggest that BLT-1 regulates infiltration of macro-
phages into the adipose tissue and agrees with the paradigm that
adipose tissue inflammation drives insulin resistance and in-
flammation in other insulin-sensitive tissues (31–33). Consistent
with this scenario, triglyceride accumulation in the liver was
largely blunted in BLT-1–deficient mice, which is in agreement
with the well-documented phenomenon that lipolysis in adipose
tissue promotes hepatic triglyceride accumulation and subsequent
insulin resistance (32). Note that we chose a duration of high-fat
feeding that is sufficient to drive adipose tissue inflammation with
associated insulin resistance in the liver and skeletal muscle, but
precedes the development of insulin resistance in the adipose
tissue itself, which requires nearly 14 wk of high-fat feeding to
Activation of both the JNK and NF-kB pathways directly pro-
motes insulin resistance through serine phosphorylation of the
insulin receptor substrate-1 and deficiencies of jnk1 and Ikkb
protect mice from obesity-induced insulin resistance (28, 44). Our
study also shows that activation of these inflammatory signaling
pathways is decreased in the adipose tissue and the liver of BLT-
12/2mice. Importantly, the expression of IL-6, which directly
promotes insulin resistance in the liver and adipocytes (27), was
also decreased in adipose tissue of BLT-12/2mice. These pro-
tective effects of BLT-1 deficiency are in agreement with the
known role of BLT-1–dependent signaling in the activation of JNK
and NF-kB by LTB4in macrophages (15, 16, 45). The activation
of these signaling pathways has been shown to underlie the BLT-
1–dependent induction of both CCL2 and IL-6 by LTB4(46).
Collectively, these studies lend new insight into the mechanisms
by which chronic inflammation contributes to insulin resistance
and implicate BLT-1 as a key regulator of macrophage accumula-
this pathway points toward promising new avenues for the thera-
peutic management of obesity and type 2 diabetes.
We thank David Young and Laura Wheat for expert technical assistance.
The authors have no financial conflicts of interest.
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