The FASEB Journal • Research Communication

Antiobese function of platelet-activating factor:

increased adiposity in platelet-activating factor
receptor-deficient mice with age
Junko Sugatani,*,†,1 Satoshi Sadamitsu,* Masahiko Yamaguchi,* Yasuhiro Yamazaki,*
Ryoko Higa,* Yoshiki Hattori,* Takahiro Uchida,* Akira Ikari,* Wataru Sugiyama,‡
Tatsuo Watanabe,†,‡ Satoshi Ishii,§ Masao Miwa,* and Takao Shimizu储
*Department of Pharmaco-Biochemistry, †Global Center of Excellence for Innovation in Human
Health Sciences, School of Pharmaceutical Sciences, and ‡School of Food and Nutritional Sciences,
University of Shizuoka, Surugaku, Shizuoka City, Japan; §Department of Immunology, Graduate
School of Medicine, Akita University, Akita City, Japan; and 储Department of Lipid Signaling, National
Center for Global Health and Medicine, Tokyo, Japan

ABSTRACT Platelet-activating factor receptor (PAFR)-
deficient mice developed a more severe obese state
characterized by higher body mass (⬃25%) and epidid-
ymal fat mass (⬃55%) with age than that of wild-type
(WT) littermates. PAFR-deficient mice did not show
changes in the expression of critical genes involved in
anabolic and catabolic metabolism in adipose, liver,
and muscle tissues between 6 and 36 wk. However, a
38ⴚ81% reduction in ␤3/␤1-adrenergic receptor (AR)
and uncoupling protein 1 (UCP1) mRNA and protein
levels was observed in the interscapular brown adipose
tissue (BAT) of PAFR-deficient mice. Whereas a single
injection of the ␤3-adrenergic agonist, CL-316,243 (25
␮g/kg) increased temperatures in the brown fat and
rectums of WT mice, this increase in temperature was
markedly suppressed in PAFR-deficient mice. Acetyl-
CoA:lyso-platelet-activating factor (PAF) acetyltrans-
ferase, which is involved in PAF biosynthesis, and the
PAF receptor were predominantly localized in BAT
macrophages, whereas brown adipocytes possessed the
enzyme and functional PAF receptors. The stimulation
of brown adipocytes by PAF induced the expression of
␤3-AR mRNA and protein (1.5- and 1.9-fold, respec-
tively), but not that of UCP1. These results indicate that
obesity in PAFR-deficient mice resulted from impaired
Abbreviations: AR, adrenergic receptor; ATGL, adipose
triglyceride lipase; BAT, brown adipose tissue; c-PAF, 1-O-
hexadecyl-2-N-methylcarbamoyl-sn-glycero-3-phosphocholine;
cPLA2␣, cytosolic phospholipase A2␣; CPT1, carnitine palmi-
toyltransferase 1; DAPI, 4=,6-diamidino-2-phenylindole; FACS,
fluorescence-activated cell sorting; FAS, fatty acid synthase;
FITC, fluorescein isothiocyanate; GR, glucocorticoid receptor;
HSL, hormone-sensitive lipase; IL, interleukin; LIPC, hepatic
lipase; LPCAT2, acyl-CoA:lysophosphatidylcholine acyltrans-
ferase 2; LPL, lipoprotein lipase; PAF, platelet-activating factor;
PAFR, PAF receptor; KO, knockout; PBS, phosphate-buffered
saline; PCR, polymerase chain reaction; PPAR␥, peroxisome
proliferator activated receptor ␥; SDS, sodium dodecyl sulfate;
UCP, uncoupling protein; WAT, white adipose tissue; WT, wild
type
BAT activity and suggest that the antiobese function of PAF
occurs through ␤3-AR/UCP1 expression in BAT.—Sugatani,
J., Sadamitsu, S., Yamaguchi, M., Yamazaki, Y., Higa, R.,
Hattori, Y., Uchida, T., Ikari, A., Sugiyama, W., Watanabe,
T., Ishii, S., Miwa, M., Shimizu, T. Antiobese function of
platelet-activating factor: increased adiposity in platelet-
activating factor receptor-deficient mice with age. FASEB J.
28, 440 – 452 (2014). www.fasebj.org
Key Words: ␤1/␤3-adrenergic receptor 䡠 uncoupling protein
1 䡠 UCP1 䡠 brown adipocytes 䡠 brown adipose tissue macro-
phages 䡠 thermogenesis
Obesity, an excess of body fat, arises from an imbal-
ance between energy intake and metabolic expenditure
(1). Adipose tissue plays key physiological roles in body
weight homeostasis. Brown adipose tissue (BAT),
composed of multilocular brown adipocytes, functions
in energy consumption, whereas white adipose
tissue (WAT), with unilocular white adipocytes, regulates
lipid storage and catabolism (1– 4). Chronic inflamma-
tion in WAT, characterized by macrophage infiltration
and the production of proinflammatory adipokines,
participates in the pathophysiology of obesity-induced
metabolic complications, such as insulin resistance and
diabetes (5). In contrast, the BAT of rodents and
newborn humans was shown to be metabolically impor-
tant in the regulation of energy expenditure by ther-
mogenesis mediated by uncoupling protein 1 (UCP1;
ref. 6). Although it had been previously thought that
BAT was rapidly lost postnatally and that adult hu-
mans lacked active BAT, several recent studies using
1
Correspondence: Department of Pharmaco-Biochemistry,
School of Pharmaceutical Sciences, University of Shizuoka,
52-1 Yada, Surugaku, Shizuoka City, Shizuoka 422– 8526,
Japan. E-mail: sugatani@u-shizuoka-ken.ac.jp
doi: 10.1096/fj.13-233262
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.

440 0892-6638/14/0028-0440 © FASEB

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[18F]fluorodeoxyglucose positron emission tomographic
and computed tomographic scans have identified a sig-
nificant amount of metabolically active BAT in human
adults, especially in older people (7–9). Because BAT
activity is reduced in obese men, its modulation may have
therapeutic implications for obesity and obesity-related
disorders.
Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-
glycero-3-phosphocholine) is considered to be a potent
phospholipid mediator with biological activities such as
the stimulation of platelets and neutrophils and the
enhancement of vascular permeability and smooth
muscle contraction (10 –12). PAF is generated in vari-
ous inflammatory cells upon the chemical or immune
stimulation associated with inflammation and anaphy-
laxis (10 –13) but is also found in normal animal tissues
[bovine brain (14), rat uterus (15), rat stomach (16),
and mouse osteoclasts (17)]. PAF acts by binding to a
specific G-protein-coupled 7-transmembrane receptor
(10, 11). The PAF receptor (PAFR) is linked to intra-
cellular signal transduction pathways, such as the turn-
over of phosphatidylinositol, increases in intracellular
calcium concentrations, and the activation of kinases.
With use of PAFR-knockout (PAFR-KO) mice, an ab-
sence of PAFR was shown to protect mice from osteo-
porosis after ovariectomy, and PAF also activated PAFR
on osteoclasts in an autocrine/paracrine manner and
affected osteoclastic cell function including cell sur-
vival, cell motility, and bone resorption activity (17).
However, the physiological roles of endogenous PAF in
obesity and energy expenditure remain poorly under-
stood.
In this study, we found that knocking out PAFR
caused increases in adiposity and weight gain with age.
This finding suggests that PAF or PAFR signaling plays
an important role in depressing the development of
obesity. Thus, using KO mice, we investigated the effect
of a deficiency in PAFR on fatty acid metabolism,
triacylglycerol storage, and thermogenesis. We showed
that the expression of the ␤3-adrenergic receptor (AR)
and UCP1 in BAT was lower in PAFR-KO mice than in
wild-type (WT) mice. Thus, this study focused on the
fine morphology of BAT, gene expression in BAT, and
␤3-adrenergic agonist-mediated thermogenesis in BAT.
Furthermore, we investigated the molecular mecha-
nisms underlying the increased adiposity observed in
PAFR-deficient mice.
MATERIALS AND METHODS
Experimental animals
PAFR-KO mice and WT littermates (18) were maintained on
12-h light-dark cycles under controlled environmental set-
tings (23⫾1°C), with free access to water. At the age of 4 wk,
mice were fed standard chow (CE-2; Nihon Crea, Tokyo,
Japan). All experiments were conducted with 1.7- to 36-wk-old
male mice. Fighting between animals did not occur during
the experiments described. All experiments followed proto-
cols approved by the Institutional Animal Care and Use
BAT DYSFUNCTION IN PAF RECEPTOR-DEFICIENT MICE
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Committee of the University of Shizuoka and were approved
by the Committee for the Use of Experimental Animals.
Biochemical analyses
Between 11:00 and 12:00 AM, mice were anesthetized with
diethyl ether, the abdominal cavity was rapidly opened, and
blood was drawn from the abdominal vena cava into syringes.
Serum and plasma samples were separated by centrifugation
and stored at ⫺80°C before analyses. Tissues (the liver
median lobe, BAT, WAT, hindlimb skeletal muscle, lung,
spleen, and kidney) collected for RNA extraction were imme-
diately frozen in liquid nitrogen and then stored at ⫺80°C.
Serum glucose concentrations were determined by the
hexokinase method using commercial reagents (R-Biopharm
AG, Darmstadt, Germany). Plasma cortisol levels were deter-
mined using the cortisol EIA kit (Cayman Chemical Co., Ann
Arbor, MI, USA), according to the manufacturer’s directions.
Frozen livers (⬃0.2 g) were homogenized in 20 volumes of
0.9% NaCl containing 0.1% Triton X-100, and the concen-
trations of triacylglycerol, total cholesterol, and nonesterified
fatty acid were estimated enzymatically with kits from Shino-
Test (Tokyo, Japan).
Histological analyses of adipose tissues
Epididymal WAT and interscapular BAT were removed,
minced into small pieces (2–3 mm), fixed in 4% paraformal-
dehyde in phosphate-buffered saline (PBS) for 45 min, and
permeabilized with PBS containing 1% Triton X-100 at 4°C.
Immunohistochemical analyses were performed as de-
scribed by Nishimura et al. (19). In brief, tissues were blocked
with 1% bovine serum albumin and incubated with a fluores-
cein isothiocyanate (FITC)-conjugated CD11c antibody (1:
500; BD Bioscience, Franklin Lakes, NJ, USA) for 1–2 d.
These tissues were counterstained for 30 min at room tem-
perature with 1 ␮M BODIPY 558/568 C12 (Invitrogen, Carls-
bad, CA, USA) to visualize adipocytes and with 4=,6-diamidino-2-
phenylindole (DAPI; 0.5 ␮g/ml; Dojin Laboratories, Kumamoto,
Japan) for 30 min at room temperature to visualize nuclei.
Coverslips were mounted with 50% glycerol, and images were
acquired with a Zeiss LSM510 confocal microscope (Carl
Zeiss, Oberkochen, Germany).
Pieces of BAT were incubated with 0.1 ␮M MitoTracker
Red CMX Ros (Lonza Inc., Allendale, NJ. USA) for 30 min at
room temperature for staining of mitochondria. The tissues
were fixed with 4% paraformaldehyde, embedded in paraffin,
and cut into sections for hematoxylin and eosin staining.
Dishes were washed twice with PBS and fixed with 10%
buffered formalin overnight at 4°C for Oil Red O staining.
Cells were then stained for 15 min at room temperature with
a filtered Oil Red O solution (lipid assay kit; Primary Cell Co.,
Sapporo, Japan), washed 3 times with distilled water, and
visualized.
Images of 150 –200 low-power fields for the quantitative
analysis of adipocytes and crown-like structures were acquired
from 5 animals in each group, and the diameters of 5000
randomly selected cells in each group were measured using
ImageJ software (U.S. National Institutes of Health, Bethesda,
MD, USA). To quantify the crown-like structures, we obtained
150 –200 images of cells stained with BODIPY 558/568 C12,
DAPI, and CD11c antibody and determined the numbers of
crown-like structures (defined as an adipocyte surrounded by
accumulated cells and/or engulfing macrophages).
Determination of RNA levels
Total RNA was obtained from the BAT, WAT, liver, hindlimb
skeletal muscle, lung, spleen, or kidney using TRIzol reagent
441
(Invitrogen). cDNA synthesized from 75 ng of total RNA was
subjected to a quantitative real-time polymerase chain reac-
tion (PCR), as described previously (20), with a Stratagene
Mx3000P Real-Time qPCR System (Agilent Technologies
Japan, Tokyo, Japan) using SYBR Premix Ex Taq reagent
(Takara Bio Inc., Otsu, Japan) for the intercalation reaction
with SYBR Green I, according to the manufacturer’s instruc-
tions. Primer sequences (forward and reverse) used for SYBR
assays are listed in Supplemental Table S1.
Western blot analysis
Tissue or cell lysates were prepared from pooled BAT
(n⫽2–5) or brown adipocytes cultured in a 75T flask by
homogenization in 2⫻ sodium dodecyl sulfate (SDS) sample
buffer containing 4% SDS, 20% glycerol, 0.125 M Tris-HCl
buffer (pH 6.8), 1 ␮g/ml leupeptin and aprotinin, and 50
␮g/ml phenylmethylsulfonyl fluoride and were centrifuged at
16,000 g for 15 min. Protein concentrations were determined
by the Bradford assay (protein assay kit; Bio-Rad Laboratories,
Hercules, CA, USA) using bovine serum albumin. Samples
(20 ␮g) were resolved by electrophoresis on a SDS-5⫺20%
polyacrylamide gel (Atto, Tokyo, Japan) and electroblotted
onto a polyvinylidene difluoride membrane (Millipore Corp.,
Redford, MA, USA). Immunoblots were incubated with anti-
mouse ␤1-AR (1:1000, ab85037; Abcam plc, Cambridge, UK),
anti-human ␤3-AR (1:1000; Trans Genic Inc., Kobe, Japan),
anti-human UCP1 (1:10,000, ab23841; Abcam plc), and anti-
chicken ␣-tubulin (1:2000; Calbiochem, Darmstadt, Ger-
many) antibodies. Antigen-antibody complexes were detected
using the appropriate secondary antibody conjugated with
horseradish peroxidase and visualized with an enhanced
chemiluminescence system (GE Healthcare Bio-Sciences, Pis-
cataway, NJ, USA).
Acute response of thermogenesis to CL-316,243
Thermogenesis in BAT in response to a single injection of the
␤3-AR agonist CL-316,243, a disodium salt (Tocris Bioscience,
Gottingen, Germany), was assessed by measuring tempera-
ture changes in BAT and the rectum, as described by Ino-
kuma et al. (21). Male mice (6 wk old) were anesthetized with
ethyl carbamate (urethane, 250 mg/kg; Wako Pure Chemi-
cals, Tokyo, Japan), a small incision was made above the
scapula, and the interscapular brown fat pads were partially
separated from the muscle below, with the vasculature and
nerve supplies to the pads left intact. Mice were then placed
on a heat plate, and a thermistor was placed under the fat
pads. Another thermistor was inserted into the rectum, and
the plate was heated gently. After the rectal temperature had
reached a steady level (⬃37°C), CL-316,243 (25 or 50 ␮g/kg)
or saline was injected intraperitoneally, and temperature
changes were monitored for 40 min. The responses to CL-
316,243 at 50 ␮g/kg were similar to those to CL-316,243 at 25
␮g/kg, but the former dose was lethal (1 of 4 mice died) and
the latter dose was nonlethal.
Assay for acetyl-CoA:lyso-PAF acetyltransferase
Acetyl-CoA:lyso-PAF acetyltransferase activity was determined
using microsomes prepared from pooled BAT, spleens, and
lungs as described previously (22).
Extraction and quantitation of PAF in BAT
Total lipids were extracted using the method of Bligh and
Dyer (23) at 4°C in the presence of 50 mM glycine-HCl buffer
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(pH 2.8) in a 1-phase system to inactivate PAF acetylhydrolase
(24) and separated on a silica gel G plate using a solvent
system of CHCl3-CH3OH-H2O (65:35:6, vol/vol/vol). The
plate was exposed to 6-p-toluidine-2-naphthalene sulfonic
acid, and PAF, located on the thin-layer plate between the
areas corresponding to sphingomyelin and lysophosphatidyl-
choline, was scraped off and extracted using the method of
Bligh and Dyer (23). The sample obtained after thin-layer
chromatography was used to determine the PAF concentra-
tion. PAF was quantified by bioassay using rabbit platelet
aggregation (16), i.e., by comparison through platelet suspen-
sions stimulated by experimental samples with that through a
suspension stimulated by a known quantity of 1-O-hexadecyl-
2-acetyl-sn-glycero-3-phosphocholine.
Cell culture conditions and treatments
Mouse brown preadipocytes were isolated and cultured ac-
cording to a published method (25) with a few modifications.
In brief, 4-wk-old C57BL/6 mice were killed, and interscapu-
lar BATs were collected and digested with collagenase
(0.1%). The digestion mixture was filtered through a sterile
100-␮m nylon mesh and left at room temperature for 10 min
until a discrete fat layer had formed on the top. This layer was
discarded, and the lower phase was centrifuged at 700g for 10
min at room temperature. The sediments were suspended
and filtered again through a 40-␮m nylon mesh. The result-
ing stromal vascular cells were incubated in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf
serum, 17 ␮M d-pantothenic acid, 33 ␮M biotin, 100 ␮M
ascorbic acid, 1 ␮M octanoic acid, 50 nM triiodothyronine, 50
U/ml penicillin G, and 1 ␮g/ml streptomycin. After becom-
ing confluent, cells were dispersed and cultured in new
dishes. Subconfluent cells were differentiated in standard
medium supplemented with 12.5 nM dexamethasone, 100
ng/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine. Rat
brown preadipocytes (1⫻105 cells/2 ml/well; Takara Bio)
were cultured and differentiated according to the manufac-
turer’s directions. Brown adipocytes at d 2, 3, and 4 of
differentiation were treated with 1-O-hexadecyl-2-N-methycar-
bamoyl-sn-glycero-3-phosphocholine (c-PAF; Sigma-Aldrich,
St. Louis, MO, USA) at concentrations of 5 ⫻ 10⫺11 to 5 ⫻
10⫺8 M with or without 5 ⫻ 10⫺6 M WEB2086 (a gift from
Boehringer Ingelheim GmbH, Ingelheim, Germany) for an-
other 24 h.
Fluorescence-activated cell sorting (FACS) analysis of the
stromal vascular cells derived from BAT
Stromal vascular cells derived from the pooled BAT of WT
mice at 9⫺11 wk of age (n⫽12⫺18) were stained with
FITC-conjugated anti-CD31 and anti-CD45 and phycoeryth-
rin-conjugated anti-Sca-1 antibodies (BD Biosciences, San
Diego, CA, USA) for 30 min at room temperature. After
staining, cells were suspended in PBS containing 2% fetal calf
serum and 2 ␮g/ml propidium iodide. Cell sorting was
performed using a FACSAria II flow cytometer (BD Immuno-
cytometry Systems, Mountain View, CA, USA). Debris and
dead cells were excluded by forward scatter, side scatter, and
propidium iodide gating. Data were collected using FACSDiva
software (BD Biosciences). RNA was purified from sorted
cells for quantitative PCR using an RNeasy Micro Kit (Qiagen,
Hilden, Germany), according to the manufacturer’s instruc-
tions, and cDNAs were then synthesized with a QuantiTect
Reverse Transcription Kit (Qiagen), according to the manu-
facturer’s instructions.
SUGATANI ET AL.
Statistical analysis
Values are expressed as means ⫾ sem. The data were analyzed
by analysis of variance unless otherwise stated. Fisher’s pro-
tected least significant difference test was used to determine
the significance of differences among the groups. The level of
significance was set at a value of P ⬍ 0.05.
RESULTS
Increase in body weight and enhanced adiposity in
PAFR-KO mice
We monitored body weight and food intake in PAFR-
KO mice and WT littermates on a weekly basis for
a period of 36 wk. There was no significant difference
in food intake between the 2 groups fed standard chow
throughout the observation period (Table 1), but a
significantly higher body weight gain was observed in
PAFR-KO mice (Fig. 1A, B). Notably, the sizes of the
epididymal white adipose deposits corrected for total
body weight were markedly and significantly increased
in PAFR-KO mice at 12 wk of age and thereafter (Fig.
1C). This increase in body weight was largely due to a
significant elevation in fat mass. In contrast, there was
no significant difference in the weights of the other
major organs, such as the liver (Fig. 1D).
Moreover, we examined liver and serum lipid levels.
No significant differences were observed in serum and
liver levels of triacylglycerols, total cholesterol, or
nonesterified fatty acids between the two groups, ex-
cept that the triacylglycerol content in the liver was
significantly higher in 36-wk-old KO mice (Table 1). In
addition, we observed higher fasting serum glucose
levels in PAFR-KO mice at 36 wk of age (but this was not
significant, P⫽0.2) than in WT mice.
We observed marked changes in the morphology of
obese adipose tissue: adiposity in mice at ⬎24 wk of age
was accompanied by increases in both the number and
size of adipocytes and elevated levels of mRNAs for
several macrophage markers (F4/80, CD11c, and
CD68), including the expression of macrophage-spe-
cific proteins within crown-like structures in adipose
tissue, although there was no significant difference in
macrophage infiltration in the WAT of PAFR-KO and
WT mice at ⬍18 wk of age (Figs. 1E and 2). In contrast,
there was no significant change in the WAT gene
expression of adipocytokines, such as adiponectin and
leptin, or anti-inflammatory cytokine interleukin
(IL)-10 between PAFR-KO and WT mice, whereas the
expression of tumor necrosis factor ␣, IL-6, and chemo-
kine (C-C motif) ligand 3 (CCL3) mRNAs was in-
creased in epididymal fat in mice at ⬎24 wk of age
(Fig. 2A).
No change in the expression of critical genes
involved in anabolic and catabolic metabolism in the
adipose, liver, and muscle tissues of PAFR-KO mice
We further examined the effect of the functional PAFR
deficiency on the gene regulation of the lipogenic
enzyme [fatty acid synthase (FAS)], the mitochondrial
enzyme [carnitine palmitoyltransferase 1 (CPT1a and
CPT1b)] involved in fatty acid oxidation, and the lipo-
lytic enzymes [lipoprotein lipase (LPL), adipose triglyc-
eride lipase (ATGL), hormone-sensitive lipase (HSL),
and hepatic lipase (LIPC)] in the major metabolic

TABLE 1. Food intake, liver lipids, serum lipids, and serum glucose levels of male PAFR-KO and WT mice

12 wk 24 wk 36 wk

Parameter WT KO WT KO WT KO

Food intake (g/wk) 23.67 ⫾ 0.52 24.46 ⫾ 0.53 27.76 ⫾ 0.50 26.04 ⫾ 0.48 26.52 ⫾ 1.00 24.22 ⫾ 1.36
Liver weight (g) 1.04 ⫾ 0.05 1.10 ⫾ 0.02 1.44 ⫾ 0.10 1.55 ⫾ 0.08 1.40 ⫾ 0.04 1.70 ⫾ 0.15***
Liver triacylglycerol 12.27 ⫾ 0.70 19.30 ⫾ 1.25 26.88 ⫾ 8.23 24.36 ⫾ 5.04 32.89 ⫾ 2.27 50.39 ⫾ 10.00*
(mg/g liver)
Liver total cholesterol 3.657 ⫾ 0.169 4.388 ⫾ 0.226 4.609 ⫾ 0.418 4.11 ⫾ 0.548 4.556 ⫾ 0.220 5.339 ⫾ 0.425
(mg/g liver)
Liver nonesterified 8.77 ⫾ 0.52 10.52 ⫾ 0.34 11.63 ⫾ 0.85 10.68 ⫾ 1.02 12.07 ⫾ 0.42 13.16 ⫾ 0.66
fatty acid (␮Eq/g
liver)
Fasting serum 0.362 ⫾ 0.051 0.333 ⫾ 0.045 0.373 ⫾ 0.038 0.312 ⫾ 0.031 0.403 ⫾ 0.028 0.384 ⫾ 0.021
triacylglycerol
(mg/ml)a
Fasting serum total 0.862 ⫾ 0.028 0.702 ⫾ 0.043* 0.829 ⫾ 0.028 0.723 ⫾ 0.059 0.787 ⫾ 0.040 0.811 ⫾ 0.050
cholesterol
(mg/ml)a
Fasting serum 1.367 ⫾ 0.095 1.549 ⫾ 0.080 1.101 ⫾ 0.085 1.203 ⫾ 0.101 1.231 ⫾ 0.083 1.191 ⫾ 0.053
nonesterified fatty
acid (␮Eq/ml)a
Fasting serum glucose 1.285 ⫾ 0.104 0.968 ⫾ 0.060* 1.584 ⫾ 0.082 1.398 ⫾ 0.044 1.594 ⫾ 0.139 1.805 ⫾ 0.140
(␮g/ml)a

Values are means ⫾ sem of 6 –10 determinations/group. aAnalysis of serum triacylglycerol, total cholesterol, nonesterified fatty acid and
glucose of animals after overnight food withdrawal. *P ⬍ 0.05, ***P ⬍ 0.001 vs. WT animals.

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Figure 1. Increased body weights and fat weights of PAFR-KO mice. A) Photograph showing the increased body size of PAFR-KO
mice at 18 wk of age. B) Body weight development in PAFR-KO mice vs. WT mice over 36 wk. C, D) Tissue weights of the
epididymal fat pads (C) and livers (D) of PAFR-KO and WT mice. Tissue weights were normalized against body weights. E) Size
of adipocytes in epididymal adipose tissue (n⫽100 low-power fields in each group) of male PAFR-KO and WT mice at 18, 24,
and 36 wk of age. Results are means ⫾ sem (n⫽6⫺15). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 for PAFR-KO mice vs. WT mice.

tissues. PAFR-KO mice at 12 wk of age, which exhibited
fat accumulation, expressed levels of LPL, ATGL, HSL,
and LIPC similar to those of their WT littermates
(Fig. 3) in the BAT, liver, and skeletal muscle. Of note,
PAFR-KO mice expressed elevated mRNA levels of LPL,
ATGL, and HSL in the WAT. Furthermore, we ob-
served that the expression of FAS mRNA in the BAT,
WAT, liver, and skeletal muscle was not significantly
different between PAFR-KO and WT mice over 36 wk,
except for the WAT of 12-wk-old mice and the skeletal
muscle of 24-wk-old mice (Fig. 4A). In addition, no
significant differences were observed in the expression
of CPT1a mRNA in the BAT, WAT, and liver and
CPT1b mRNA in the skeletal muscle between PAFR-KO
and WT mice over 36 wk, except for the BAT of
1.7-wk-old mice, WAT of 6-wk-old mice, and BAT, WAT,
and liver of 24-wk-old mice (Fig. 4B).
Decreases in ␤1/␤3-AR and UCP1 in the BAT of
PAFR-KO mice
We next examined the expression of ␤1-, ␤2-, and
␤3-ARs in the BAT, WAT, liver, and skeletal muscle,
which were involved in the sympathetic regulation of
lipid metabolism. There were no significant differences
in the expression of these ARs in the WAT, liver, and
skeletal muscle of PAFR-KO and WT mice at 6 wk of age
before the onset of obesity (Fig. 4C). However, the
expression of ␤1- and ␤3-ARs was significantly lower and
the expression of ␤2-AR was significantly higher in the
BAT of PAFR-KO mice than in that of WT littermates.
Furthermore, we examined the gene expression levels
of the uncoupling proteins (UCP1–3). The levels of
UCP1 and UCP3 mRNA in the BAT and UCP2 mRNA
in the WAT were significantly lower in PAFR-KO mice
than in WT littermates (Fig. 4D).
We then examined changes in the protein expression
levels of ␤1- and ␤3-ARs and UCP1 in the BAT of
PAFR-KO and WT mice with age. Western blot analysis
revealed that the relative levels of ␤3-AR expression vs.
␣-tubulin expression in the BAT of PAFR-KO mice were
significantly lower than those of WT littermates at 6, 12,
18, and 24 wk, whereas there was no significant differ-
ence in the BAT weight corrected for the body weight
(Fig. 5A, C, E). The levels of ␤1-AR expression in the
BAT of PAFR-KO mice were also lower than those of
WT littermates (Fig. 5A, B). Furthermore, Western blot
analysis revealed significantly lower levels of UCP1
protein in the BAT of PAFR-KO mice than in WT
littermates at 6⫺24 wk of age (Fig. 5A, D).

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Figure 2. Infiltration of macrophages into obese adipose tissue and the expression of inflammatory and immune cell markers
in obese adipose tissue of PAFR-KO mice. A) Real-time PCR analysis of the macrophage membrane protein and cytokine
expression in the epididymal WAT of male PAFR-KO and WT mice over 36 wk. Relative mRNA expression was normalized to
that in control WT mice at 6 wk of age. Results are means ⫾ sem (n⫽6⫺15). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 for PAFR-KO
mice vs. WT mice. B) Immunohistochemical identification of macrophages (CD11c, red) in the epididymal WAT of male
PAFR-KO and WT mice at 36 wk of age. Adipocytes were counterstained with BODIPY (blue) and the nuclei with DAPI (green).
Scale bars ⫽ 100 ␮m. C) Numbers of crown-like structures (indicated by white arrows in panel B) in epididymal WAT
(n⫽150 –200 low-power fields in each group).

In addition, we detected elevated levels of thermo-
genic fat cell-selective gene UCP1 mRNA in epididymal
WAT of WT mice at 24 and 36 wk of age [4.15⫾1.10-
fold (P⫽0.054) and 6.09⫾2.68-fold (P⫽0.024) in the
control mice at 6 wk, respectively; Fig. 6A]. However,
PAFR-KO mice at 24 and 36 wk expressed lower levels
of UCP1 mRNA in WAT (1.36⫾0.16- and 2.68⫾0.61-
fold in the control mice at 6 wk, respectively; Fig. 6A).
The UCP1-immunopositive cells, which had a unilocu-
lar morphology typical of white adipocytes, were in-
creased in epididymal WAT of WT mice at 24 and 36
wk, compared with that of PAFR-KO mice (Fig. 6B).
Because not only classic brown adipocytes derived from
a myf-5 cellular lineage but also beige adipocytes
(UCP1-positive cells that emerge in white fat from a
non-myf-5 lineage) have been reported to respond to
cAMP stimulation with high UCP1 expression (26), our
present data suggest that PAF or PAFR signaling also
contributes to the expression of UCP1 in the beige
adipocytes within white adipose tissues.
Furthermore, to investigate whether elevations in plasma
glucocorticoid levels contributed to the reduction in

Figure 3. Lipase gene expression in BAT, WAT, liver, and skeletal muscle of PAFR-KO and WT mice. Real-time PCR analysis of
LPL (A), ATGL (B), HSL (C), and LIPC (D) mRNA expression in BAT, WAT, liver, and skeletal muscle of PAFR-KO and WT
mice at 12 wk of age. Relative mRNA expression was normalized against 18S rRNA. Results are expressed as the means ⫾ sem
(n⫽6⫺9). *P ⬍ 0.05, ***P ⬍ 0.001 for PAFR-KO mice vs. WT mice.

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Figure 4. Analysis of FAS, CPT1, ␤-AR, and UCP
mRNA expression in BAT, WAT, liver, and
skeletal muscle of PAFR-KO and WT mice over
36 wk. A, B) Real-time PCR analysis of FAS (A)
and CPT1a (BAT, WAT, and liver) and CPT1b
[skeletal muscle (SM)] (B) gene expression in
BAT, WAT, liver, and skeletal muscle of
PAFR-KO and WT mice over 36 wk. Relative
mRNA expression was normalized to that of
control WT mice at 6 wk of age. Results are
means ⫾ sem (n⫽6⫺15). *P ⬍ 0.05, **P ⬍ 0.01,
***P ⬍ 0.001 for PAFR-KO mice vs. WT mice. C,
D) Real-time PCR analysis of ␤1-AR, ␤2-AR, and
␤3-AR (C) and UCP1, UCP2, and UCP3 (D) gene
expression in BAT, WAT, liver, and skeletal
muscle of PAFR-KO and WT mice at 6 wk of
age. Relative mRNA expression was normalized
against 18S rRNA. Results are means ⫾ sem
(n⫽6). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 for
PAFR-KO mice vs. WT mice.

␤3-AR and UCP1 expression in the BAT of PAFR-KO mice
(27–29), we determined peripheral plasma cortisol levels
in WT and PAFR-KO mice. Unexpectedly, plasma cortisol
levels in PAFR-KO mice were significantly lower than
those in WT mice. The levels of glucocorticoid receptor
(GR) mRNA in the BAT of PAFR-KO mice was slightly
lower than those in the BAT of WT littermates (P⫽0.07;
Fig. 7), indicating that the decrease in ␤3-AR expression
did not result from elevations in plasma cortisol levels or
BAT GR mRNA expression.
Reduced thermogenesis in PAFR-KO mice
We subsequently examined the physiological response
of PAFR-KO mice to the ␤3-AR agonist, CL-316,243. We
monitored temperature changes in the interscapular
BAT and rectums of PAFR-KO and WT mice at 6 wk of
age after a single injection of CL-316,243. The basal
temperatures of the BAT and rectums were 35.4 ⫾ 0.2
and 37.1 ⫾ 0.2°C in PAFR-KO mice and 35.3 ⫾ 0.2 and
36.8 ⫾ 0.2°C in WT mice, respectively, which were not
significantly different between the 2 groups, whereas
the BAT temperature was slightly lower than the rectal
temperature in both genotypes of mice. The responses
in PAFR-KO mice 14 min after the CL-316,243 (25
␮g/kg) injection and thereafter were significantly less
than those in WT mice (Fig. 8). The injection of saline
did not change the basal temperature of the BAT or
rectums in either type of mice.
To further evaluate whether the dysfunction in BAT
contributed to the impaired thermogenic activity in
PAFR-KO mice, we examined the morphology and
expression of several genes involved in mitochondrial
biogenesis in BAT. Although adipocyte cell size in BAT
(interscapular fat) was higher in PAFR-KO mice than in
WT mice (Fig. 5F), PAFR-KO mice showed levels of
mitochondrial density similar to those of WT litter-
mates, as determined by staining with MitoTracker
(Fig. 5G), and the gene expression levels of Ndufc1,
Sdha, Cybb, and Cox5a involved in mitochondrial respi-
ratory complex I, complex II⫺III, and complex IV,
were also similar except for those of 6-wk-old mice
(Supplemental Fig. S1). The levels of Ndufc1 and Sdha
mRNA were slightly lower in PAFR-KO mice than in WT
mice at 6 wk of age and reached a plateau in the mutant
mice at 12 wk similar to that in WT mice, which
indicates the delayed formation of the mitochondrial
respiratory complex in PAFR-KO mice.

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Figure 5. Changes in ␤1/␤3-AR and UCP1 protein expression in BAT of PAFR-KO and WT mice. A) Protein levels in pooled BAT
(n⫽2⫺5) of PAFR-KO and WT mice were determined by Western blotting with the indicated antibodies. B, C, D) Relative
protein expressions of ␤1-AR (B), ␤3-AR (C), and UCP1 (D) were normalized to those of control WT mice at 6 wk of age. E) BAT
weights of PAFR-KO and WT mice. Tissue weights were normalized against body weights. F, G) Histological sections of BAT of
PAFR-KO and WT mice at 6 wk of age stained with hematoxylin and eosin (H.E.; F) and with MitoTracker (red) and DAPI (blue;
G). Scale bar ⫽ 50 ␮M. Results are means ⫾ sem (n⫽3⫺4). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 for PAFR-KO mice vs. WT mice;
#
P ⬍ 0.05, ##P ⬍ 0.01 for WT mice at 12, 18, and 24 wk vs. control WT mice at 6 wk.

Induction of ␤3-AR mRNA and protein expression in
brown adipocytes by PAF
Because ␤3-AR is a potent stimulator of thermogenesis in
the BAT of humans and animals, including rodents, we
examined the effect of PAFR stimulation on the in vitro
expression of ␤3-AR in mouse and rat brown adipocytes.
c-PAF, a nonhydrolyzable agonist of PAFR, was used for
PAFR stimulation of the cultured brown adipocytes, be-
cause serum PAF acetylhydrolase immediately hydrolyzes
PAF (30). The expression of ␤3-AR mRNA increased with
the process of differentiation and maturation after treat-
ment of brown preadipocytes with dexamethasone and
insulin, whereas that of UCP1 mRNA was not found until
d 5 (Supplemental Fig. S2). The administration of c-PAF
to mouse brown adipocytes differentiated at d 5 at a
concentration of 5 ⫻ 10⫺9 M resulted in a significant
elevation in the expression of ␤3-AR mRNA and protein
(1.5- and 1.9-fold, respectively; Fig. 9A, B), whereas ␤3-AR
mRNA levels were significantly increased after treatment
with c-PAF at 5 ⫻ 10⫺10 M in rat brown adipocytes
differentiated at d 5 (1.5-fold; Supplemental Fig. S2A).
Adipogenic differentiation was confirmed by peroxisome
proliferator-activated receptor ␥ (PPAR␥) mRNA expres-
sion and Oil Red O staining (Fig. 9C, D). WEB2086, an
antagonist of PAFR, significantly prevented the induction
of ␤3-AR mRNA and protein expression by c-PAF. In
contrast, c-PAF at 5 ⫻ 10⫺10 to 5 ⫻ 10⫺8 M could not
induce the expression of UCP1 mRNA (Fig. 9A and
Supplemental Fig. 2B) and protein (Fig. 9B) in mouse
and rat brown adipocytes differentiated at d 3⫺5, which
suggests that PAF did not directly influence the induction
of UCP1.
Occurrence of PAF in the BAT of WT mice
The PAF content in BAT was 0.230 ⫾ 0.058 pmol/g tissue
(n⫽4), with the results presented as means ⫾ sem for
combined samples from 10⫺14 mice at 6 wk of age in the
number of experiments indicated (Fig. 10). It was
confirmed that the substance activating washed rabbit
platelets was PAF, because the PAF antagonist WEB2086
completely inhibited its aggregation activity on platelets
(Fig. 10).
Expression of acyl-CoA:lysophosphatidylcholine
acyltransferase 2 (LPCAT2), cytosolic phospholipase
A2␣ (cPLA2␣), and PAFR mRNA in the BAT, BAT
macrophages, and brown adipocytes of WT mice
To evaluate the potential for PAF biosynthesis in BAT,
stromal vascular cells derived from BAT, and brown
adipocytes, we measured the gene expression of
LPCAT2 (which catalyzes the final reaction for PAF bio-

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Figure 6. Induction of UCP1 expression in epididymal WAT
of PAFR-KO and WT mice. A) Real-time PCR analysis of UCP1
gene expression in epididymal WAT of PAFR-KO and WT
mice over 36 wk. Solid and open bars represent the mRNA
levels of UCP1 in WAT of WT and PAFR-KO mice, respec-
tively. Relative mRNA expression is normalized to that in the
control WT mice at 6 wk of age. Results are means ⫾ sem
(n⫽6⫺15). *P ⬍ 0.05, **P ⬍ 0.01 for WT mice at 24 and 36
wk vs. control WT mice at 6 wk; #P ⬍ 0.05 for PAFR-KO mice
vs. WT mice. B) Immunohistochemical identification of
brown-like adipocytes (arrow; UCP1, red) in epididymal WAT
of PAFR-KO and WT mice at 36 wk of age. Adipocytes were
counterstained with BODIPY (blue) and the nuclei with DAPI
(green). Scale bar ⫽ 50 ␮m.

synthesis during the remodeling pathway of glycero-

phospholipids; ref. 31) and cPLA2␣ (which is an im-
portant PLA2 for lyso-PAF production in the remodel-
ing pathway; ref. 32). The activities of LPCAT2 in BAT
were significantly higher but were less than those in the
lung and spleen (Fig. 11A). The gene expression levels
of LPCAT2 and cPLA2␣ were also significantly higher in
BAT and brown adipocytes but were less than those in
the lung and spleen (Fig. 11B, C). Furthermore, we
examined the gene expression of PAFR in the major
metabolic tissues such as the adipose tissue, liver, and
skeletal muscle in addition to the lung, spleen, and
kidney of WT mice. The level of PAFR mRNA in the
BAT of WT mice was similar to that in the lung and
spleen and higher than that in the liver, skeletal
Figure 7. Plasma cortisol and BAT GR mRNA levels in
PAFR-KO and WT mice. Plasma cortisol levels (A) and BAT
GR mRNA levels (B) were determined in PAFR-KO and WT
mice at 12 wk of age. Relative mRNA expression was normal-
ized against 18S rRNA. Results are means ⫾ sem (n⫽6⫺11).
**P ⬍ 0.01 for PAFR-KO mice vs. control WT mice.
Figure 8. Acute response of BAT thermogenesis to CL-
316,243. PAFR-KO and WT mice were anesthetized, and
temperature changes in the interscapular BAT (A) and
rectums (B) were monitored after the injection of CL-316,243
(25 ␮g/kg) or saline. Results are means ⫾ sem (n⫽3⫺5).
Difference between values for PAFR-KO and WT mice was
significant (P ⬍ 0.05) at 14 min and thereafter.
muscle, kidney, and mouse brown adipocytes (Fig.
11D). Similar levels of PAFR mRNA were observed in
the BAT of WT mice at 6⫺36 wk of age, indicating that
they were not dependent on age, whereas LPCAT2 and
cPLA2␣ mRNA levels increased and decreased with
age, respectively (Fig. 12). In contrast, the levels of
LPCAT2 and cPLA2␣ mRNAs in BAT of PAFR-KO mice
were increased over 24 wk of age and were significantly
higher than those of WT mice (Fig. 12A, B).
We then established the gene expression patterns of
LPCAT2 and PAFR in stromal vascular cells derived
from the BAT of WT mice. Sca-1⫹/CD31⫺/CD45⫺
stem cells, which were isolated by removing CD45⫹
hematopoietic cells and CD31⫹ endothelial cells, ex-
pressed levels of LPCAT2 mRNA (9.6⫾3.6/18S rRNA⫻
104) similar to those in brown adipocytes (6.6⫾0.4/18S
rRNA⫻104). Among the stromal vascular cells, LPCAT2
mRNA was predominantly localized in Sca-1⫺/CD31⫹/
CD45⫹ sorted cells (130⫾42/18S rRNA⫻104), which
markedly expressed the CD68 gene (BAT macro-
phages; Fig. 11). The PAFR gene was also predomi-
nantly localized in Sca-1⫺/CD31⫹/CD45⫹ sorted cells

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 Figure 9. ␤3-AR expression in mouse brown adipocytes induced by
c-PAF. A, B) Mouse brown adipocytes at d 4 of differentiation
triggered by dexamethasone and insulin were treated with c-PAF at
the indicated concentrations for 24 h. A) Relative mRNA levels of
␤3-AR and UCP1 were expressed by taking the control values
obtained from preadipocytes as 1; mRNA levels of ␤3-AR and UCP1
normalized against 18S rRNA (⫻105) in preadipocytes were 229 ⫾
24 and 1.00 ⫾ 0.10, respectively. B) Relative protein levels of ␤3-AR
were normalized to that of control brown adipocytes at d 5. C)
Relative mRNA levels of PPAR␥ at d 0, 4, 5, 6, and 7 were expressed
by taking the control values obtained from preadipocytes as 1.
Results are means ⫾ sem (n⫽3⫺4). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍
0.001 for c-PAF-treated cells vs. control cells; #P ⬍ 0.05, ###P ⬍
0.001 for c-PAF-treated cells with WEB2086 vs. c-PAF-treated cells
without WEB2086. D) Mouse brown preadipocytes and adipocytes at d 5 of differentiation were stained with Oil Red O to
assess lipid accumulation.

(237⫾42/18S rRNA⫻104), whereas low PAFR mRNA
levels were expressed in Sca-1⫹/CD31⫺/CD45⫺ stem
cells (0.2⫾0.2/18S rRNA⫻104) and brown adipocytes
(3.6⫾0.7/18S rRNA⫻104; Fig. 11).
DISCUSSION
This study indicated that body weight with age and
adiposity was higher in PAFR-KO mice than in WT
Figure 10. Platelet aggregation induced by BAT PAF. PAF in
the 25-mg sample of BAT was used. Trace a: platelets
activated by BAT PAF. Trace b: Platelets preincubated with
2.5 ⫻ 10⫺6 M WEB2086 and activated by BAT PAF. Traces
c: Platelets activated by 1.25 ⫻ 10⫺11, 2.5 ⫻ 10⫺11, and 5 ⫻
10⫺11 M PAF.
littermates (Fig. 1). The adiposity was accompanied by
increases in both the number and size of adipocytes
with age, but mRNA levels for several macrophage
markers (F4/80, CD11b, CD11c, and CD68) were
prominently elevated only at 24 wk, including the
expression of M1 macrophage-specific protein CD11c
within crown-like structures in adipose tissue, sugges-
tive of macrophage infiltration of WAT (Figs. 1 and 2).
These results indicate that macrophage infiltration of
WAT participated in the inflammatory pathway and
made a limited contribution to body weight increases in
PAFR-deficient mice with age. Whereas PAFR-KO mice
exhibited metabolic disorders such as mild hyperglyce-
mia and fatty liver at 36 wk, their serum levels of total
cholesterol and triacylglycerols and gene expression
levels of adiponectin and leptin in the WAT were not
significantly changed (Table 1 and Fig. 2). PAF exhibits
biological activities such as enhanced vascular permea-
bility, the regulation of blood pressure, and induction
of smooth muscle contraction and edema (11, 12) and
is also involved in bone metabolism (17). However, the
role of PAF and its receptor in the development and
maintenance of adiposity remains to be resolved. It is of
interest to elucidate the molecular mechanisms under-
lying PAFR-mediated antiobesity exclusively.
Obesity is caused by an imbalance in energy intake
and expenditure (1). Because there was no significant
difference in food intake between PAFR-KO and WT
mice over 36 wk in the present study, we determined
the effect of the PAFR deficiency on energy expendi-
ture. ATGL, HSL, and LPL mRNA levels were higher in
the WAT of obese 12-wk-old PAFR-KO mice than in the
WAT of the WT mice (Fig. 3), indicating that the PAFR

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Figure 11. Lyso-PAF acetyltransferase activity (A); LPCAT2 (B), cPLA2␣ (C), and PAFR (D) gene expression in BAT; and CD68
(F), LPCAT2 (G), and PAFR (H) gene expression in each fraction (R1, R2, R3, and R4) of the stromal vascular cells derived from
BAT (E–H). A) Lyso-PAF acetyltransferase activity in microsomal fractions prepared from pooled BAT, spleens, and lungs of WT
mice at 18 wk of age. Results are means ⫾ sem (n⫽3⫺4). B–H) Real-time PCR analysis of each gene expression in the lung,
spleen, BAT, kidney, liver, and skeletal muscle of WT mice at 6 wk of age and mouse brown adipocytes at d 5 of differentiation
(B–D), and each fraction of the stromal vascular cells derived from BAT of WT mice at 9⫺11 wk of age (E–H). R1, R2, R3, and
R4 cell fractions indicate Sca-1⫹/CD31⫺/CD45⫺, Sca-1⫹/CD31⫹/CD45⫹, Sca-1⫺/CD31⫹/CD45⫹, and Sca-1⫺/CD31⫺/CD45⫺
cells, respectively, in the stromal vascular cells. Relative mRNA expression was normalized against 18S rRNA. Results are
means ⫾ sem (n⫽3⫺5).

deficiency did not cause a reduction in lipase in
adipose tissues. In contrast, we demonstrated that
PAFR-KO mice exhibited significantly elevated mRNA
levels of the fatty acid-synthesizing enzyme FAS in the
WAT at 12 wk and significantly decreased mRNA levels
of the ␤-oxidation-related enzyme CPT1a in the WAT at
6 wk (Fig. 4). These findings appeared to contribute, at
least in part, to the development of adiposity at 12 wk
and thereafter, although it remains to be determined
whether the increase in FAS mRNA and decrease in
CPT1a mRNA, leading to lipid accumulation and a
reduction in lipid consumption, respectively, result
from a lack of PAFR.
The activation of energy expenditure in the WAT
and BAT is mediated by the sympathetic nervous system
and is particularly reliant on ␤-AR signaling (33, 34). In
the BAT of PAFR-deficient mice, the expression of not
only ␤3-AR but also ␤1-AR was decreased (Figs. 4 and 5),
whereas ␤1-AR could elicit cAMP-mediated responses to
promote UCP1 expression and activate BAT in ␤3-AR

Figure 12. LPCAT2, cPLA2␣, and PAFR gene expression in interscapular BAT of PAFR-KO and WT mice over 36 wk. Real-time
PCR analysis was used to qualify relative mRNA abundance of LPCAT2 (A), cPLA2␣ (B), and PAFR (C) in the interscapular BAT
of PAFR-KO and WT mice. Relative mRNA expression was normalized to that of control WT mice at 6 wk of age. Results are
means ⫾ sem (n⫽6⫺15). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 for WT mice at 12, 18, 24, and 36 wk of age vs. WT mice at 6
wk of age; #P ⬍ 0.05, ##P ⬍ 0.01, ###P ⬍ 0.001 for PAFR-KO mice at 6, 12, 18, 24, and 36 wk of age vs. WT mice at 6 wk of age;
⫹⫹⫹
P ⬍ 0.001 for PAFR-KO mice vs. WT mice at the same age.

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knockout mice because of adaptations compensating
for the ␤3-AR deficiency (35, 36). Although ␤1- and
␤3-AR mRNA and protein have been reported to be
down-regulated by glucocorticoids through a glucocor-
ticoid receptor-mediated mechanism (27–29), plasma
glucocorticoid and BAT GR mRNA levels were not
increased in PAFR-KO mice (Fig. 7). The reduced
expression of ␤1- and ␤3-AR in the BAT of PAFR-KO
mice did not appear to be mediated by GR.
Recent studies have suggested that the metabolically
active BAT of human adults is more abundant than
previously thought and could be a target for the
treatment of obesity (7–9, 37). Whereas thermogenesis
in response to alterations in energy intake is largely
attributed to the activation of the thermogenic organ
BAT, ␤3-AR stimulation does not cause BAT thermo-
genesis in mice lacking UCP1 (21). Thus, ␤3-AR-medi-
ated thermogenesis depends on UCP1, which uncou-
ples mitochondrial oxidative phosphorylation and
generates heat by dissipating the proton gradient. The
present study showed that ␤3-AR and UCP1 were found
predominantly in BAT (Figs. 4 and 5). The levels of
UCP1 mRNA and protein in BAT were lower in
PAFR-KO mice than in WT mice, which may have
resulted from the suppressed production, but not deg-
radation, of UCP1 (Figs. 4D and 5D). Because of the
BAT dysfunction, which resulted from the reduced
expression of ␤3/␤1-AR and UCP1, PAFR-KO mice
appeared to exhibit less thermogenesis with ␤3-AR
stimulation (Fig. 8). In fact, lipid vacuoles were en-
larged in the BAT of PAFR-KO mice, indicating that the
accumulation of lipids in BAT was enhanced (Fig. 5F).
Because the appearance of UCP1 with age was also
suppressed in the WAT of PAFR-deficient mice (Fig. 6),
PAFR-KO mice appeared to be more susceptible to
diet-induced obesity than WT mice because of the
reduced ability to use UCP1-mediated thermogenesis.
The present study showed that PAFR mRNA levels
were high not only in the BAT of control mice but also
in mouse brown adipocytes (Fig. 11D). This prompted
us to investigate the role of PAF in the expression of
␤-ARs in mouse brown adipocytes (Fig. 9). Our findings
demonstrated that c-PAF induced the expression of
␤3-AR mRNA and protein but not of UCP1 mRNA and
protein in mouse brown adipocytes. These effects were
receptor-mediated because they were not observed in
WEB2086-treated cells. In immortal brown adipocytes
of p53-KO mice, UCP1 mRNA was not detected, but was
markedly activated after ␤3-AR stimulation by addition
of norepinephrine or CL316,243 (25). Norepinephrine
is supplied to BAT in vivo under physiological stimula-
tion but not to cultured cells in vitro. Therefore, c-PAF
did not directly influence the expression of UCP1
mRNA in mouse adipocytes, which suggests that the
decreased levels of UCP1 mRNA and protein in the
BAT of PAFR-KO mice resulted in a reduction in
PAF-induced ␤3-AR expression. PAFR signaling may
play a role in adipocyte development and differentia-
tion through ␤3-AR, which is linked to thermogenesis.
We then investigated whether PAF was biosynthesized
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in BAT. Brown adipocytes had low, but significant,
levels of LPCAT2 mRNA, whereas BAT macrophages
had high levels of its mRNA (Figs. 11 and 12). In
addition, BAT had a significant amount of PAF (Fig.
10). Thus, PAF appears to be produced by lyso-PAF
acetyltransferase activated in BAT macrophages in re-
sponse to extracellular stimuli or in brown adipocytes
in an autocrine/paracrine manner.
Exposure of mice to cold temperatures rapidly pro-
motes the alternative activation of adipose tissue mac-
rophages, which secrete catecholamines to induce ther-
mogenic gene expression in BAT (38). Because the
PAFR gene was predominantly localized in BAT macro-
phages, in addition to ␤3-AR expression in brown
adipocytes by PAF, the absence of BAT macrophages
activated by PAF in PAFR-KO mice may have sup-
pressed the secretion of catecholamines and caused
BAT dysfunction. We are currently in the process of
investigating the precise mechanism of BAT macro-
phage activation associated with thermogenesis through
PAF.
In summary, our findings in the PAFR-KO genetic
model revealed that PAFR deficiency caused BAT dys-
function, which induced the development of obesity
due to the impaired thermogenic activity of BAT. In
other words, our findings suggest that PAF/PAFR pos-
sesses an antiobese function that occurred through
␤3-AR/UCP1 expression in BAT. These findings were
consistent with the fact that the body weight of trans-
genic mice overexpressing PAFR at 14⫺17 wk was
⬃20% lighter than that of their control littermates
(39). Future studies on how PAF/PAFR signaling con-
trols UCP1 levels through ␤3-AR production in the BAT
of animals and humans may reveal new therapeutic
targets to treat metabolic disorders associated with
obesity. We are in the process of investigating the
mechanism of the antiobese action through PAF or
PAFR signaling using conditional gene-KO mice.
The authors gratefully acknowledge Masaki Imanishi,
Kengo Yasuda, and Kouki Hiramoto for excellent technical
assistance. This work was supported in part by the global
Center of Excellence program and grants-in-aid for scientific
research (21590170 and 22590068) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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Received for publication July 1, 2013.
Accepted for publication September 24, 2013.
SUGATANI ET AL.
Antiobese function of platelet-activating factor: increased
adiposity in platelet-activating factor receptor-deficient mice with
age
Junko Sugatani, Satoshi Sadamitsu, Masahiko Yamaguchi, et al.

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