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

440 0892-6638/14/0028-0440 © FASEB


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[18F]fluorodeoxyglucose positron emission tomographic Committee of the University of Shizuoka and were approved
and computed tomographic scans have identified a sig- by the Committee for the Use of Experimental Animals.
nificant amount of metabolically active BAT in human
Biochemical analyses
adults, especially in older people (7–9). Because BAT
activity is reduced in obese men, its modulation may have Between 11:00 and 12:00 AM, mice were anesthetized with
therapeutic implications for obesity and obesity-related diethyl ether, the abdominal cavity was rapidly opened, and
disorders. blood was drawn from the abdominal vena cava into syringes.
Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn- Serum and plasma samples were separated by centrifugation
glycero-3-phosphocholine) is considered to be a potent and stored at ⫺80°C before analyses. Tissues (the liver
phospholipid mediator with biological activities such as median lobe, BAT, WAT, hindlimb skeletal muscle, lung,
the stimulation of platelets and neutrophils and the spleen, and kidney) collected for RNA extraction were imme-
diately frozen in liquid nitrogen and then stored at ⫺80°C.
enhancement of vascular permeability and smooth Serum glucose concentrations were determined by the
muscle contraction (10 –12). PAF is generated in vari- hexokinase method using commercial reagents (R-Biopharm
ous inflammatory cells upon the chemical or immune AG, Darmstadt, Germany). Plasma cortisol levels were deter-
stimulation associated with inflammation and anaphy- mined using the cortisol EIA kit (Cayman Chemical Co., Ann
laxis (10 –13) but is also found in normal animal tissues Arbor, MI, USA), according to the manufacturer’s directions.
[bovine brain (14), rat uterus (15), rat stomach (16), Frozen livers (⬃0.2 g) were homogenized in 20 volumes of
0.9% NaCl containing 0.1% Triton X-100, and the concen-
and mouse osteoclasts (17)]. PAF acts by binding to a
trations of triacylglycerol, total cholesterol, and nonesterified
specific G-protein-coupled 7-transmembrane receptor fatty acid were estimated enzymatically with kits from Shino-
(10, 11). The PAF receptor (PAFR) is linked to intra- Test (Tokyo, Japan).
cellular signal transduction pathways, such as the turn-
over of phosphatidylinositol, increases in intracellular Histological analyses of adipose tissues
calcium concentrations, and the activation of kinases.
With use of PAFR-knockout (PAFR-KO) mice, an ab- Epididymal WAT and interscapular BAT were removed,
sence of PAFR was shown to protect mice from osteo- minced into small pieces (2–3 mm), fixed in 4% paraformal-
dehyde in phosphate-buffered saline (PBS) for 45 min, and
porosis after ovariectomy, and PAF also activated PAFR permeabilized with PBS containing 1% Triton X-100 at 4°C.
on osteoclasts in an autocrine/paracrine manner and Immunohistochemical analyses were performed as de-
affected osteoclastic cell function including cell sur- scribed by Nishimura et al. (19). In brief, tissues were blocked
vival, cell motility, and bone resorption activity (17). with 1% bovine serum albumin and incubated with a fluores-
However, the physiological roles of endogenous PAF in cein isothiocyanate (FITC)-conjugated CD11c antibody (1:
obesity and energy expenditure remain poorly under- 500; BD Bioscience, Franklin Lakes, NJ, USA) for 1–2 d.
stood. These tissues were counterstained for 30 min at room tem-
perature with 1 ␮M BODIPY 558/568 C12 (Invitrogen, Carls-
In this study, we found that knocking out PAFR bad, CA, USA) to visualize adipocytes and with 4=,6-diamidino-2-
caused increases in adiposity and weight gain with age. phenylindole (DAPI; 0.5 ␮g/ml; Dojin Laboratories, Kumamoto,
This finding suggests that PAF or PAFR signaling plays Japan) for 30 min at room temperature to visualize nuclei.
an important role in depressing the development of Coverslips were mounted with 50% glycerol, and images were
obesity. Thus, using KO mice, we investigated the effect acquired with a Zeiss LSM510 confocal microscope (Carl
of a deficiency in PAFR on fatty acid metabolism, Zeiss, Oberkochen, Germany).
Pieces of BAT were incubated with 0.1 ␮M MitoTracker
triacylglycerol storage, and thermogenesis. We showed
Red CMX Ros (Lonza Inc., Allendale, NJ. USA) for 30 min at
that the expression of the ␤3-adrenergic receptor (AR) room temperature for staining of mitochondria. The tissues
and UCP1 in BAT was lower in PAFR-KO mice than in were fixed with 4% paraformaldehyde, embedded in paraffin,
wild-type (WT) mice. Thus, this study focused on the and cut into sections for hematoxylin and eosin staining.
fine morphology of BAT, gene expression in BAT, and Dishes were washed twice with PBS and fixed with 10%
␤3-adrenergic agonist-mediated thermogenesis in BAT. buffered formalin overnight at 4°C for Oil Red O staining.
Furthermore, we investigated the molecular mecha- Cells were then stained for 15 min at room temperature with
a filtered Oil Red O solution (lipid assay kit; Primary Cell Co.,
nisms underlying the increased adiposity observed in Sapporo, Japan), washed 3 times with distilled water, and
PAFR-deficient mice. 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
MATERIALS AND METHODS randomly selected cells in each group were measured using
ImageJ software (U.S. National Institutes of Health, Bethesda,
Experimental animals MD, USA). To quantify the crown-like structures, we obtained
150 –200 images of cells stained with BODIPY 558/568 C12,
PAFR-KO mice and WT littermates (18) were maintained on DAPI, and CD11c antibody and determined the numbers of
12-h light-dark cycles under controlled environmental set- crown-like structures (defined as an adipocyte surrounded by
tings (23⫾1°C), with free access to water. At the age of 4 wk, accumulated cells and/or engulfing macrophages).
mice were fed standard chow (CE-2; Nihon Crea, Tokyo,
Japan). All experiments were conducted with 1.7- to 36-wk-old Determination of RNA levels
male mice. Fighting between animals did not occur during
the experiments described. All experiments followed proto- Total RNA was obtained from the BAT, WAT, liver, hindlimb
cols approved by the Institutional Animal Care and Use skeletal muscle, lung, spleen, or kidney using TRIzol reagent

BAT DYSFUNCTION IN PAF RECEPTOR-DEFICIENT MICE 441


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(Invitrogen). cDNA synthesized from 75 ng of total RNA was (pH 2.8) in a 1-phase system to inactivate PAF acetylhydrolase
subjected to a quantitative real-time polymerase chain reac- (24) and separated on a silica gel G plate using a solvent
tion (PCR), as described previously (20), with a Stratagene system of CHCl3-CH3OH-H2O (65:35:6, vol/vol/vol). The
Mx3000P Real-Time qPCR System (Agilent Technologies plate was exposed to 6-p-toluidine-2-naphthalene sulfonic
Japan, Tokyo, Japan) using SYBR Premix Ex Taq reagent acid, and PAF, located on the thin-layer plate between the
(Takara Bio Inc., Otsu, Japan) for the intercalation reaction areas corresponding to sphingomyelin and lysophosphatidyl-
with SYBR Green I, according to the manufacturer’s instruc- choline, was scraped off and extracted using the method of
tions. Primer sequences (forward and reverse) used for SYBR Bligh and Dyer (23). The sample obtained after thin-layer
assays are listed in Supplemental Table S1. chromatography was used to determine the PAF concentra-
tion. PAF was quantified by bioassay using rabbit platelet
Western blot analysis aggregation (16), i.e., by comparison through platelet suspen-
sions stimulated by experimental samples with that through a
Tissue or cell lysates were prepared from pooled BAT suspension stimulated by a known quantity of 1-O-hexadecyl-
(n⫽2–5) or brown adipocytes cultured in a 75T flask by 2-acetyl-sn-glycero-3-phosphocholine.
homogenization in 2⫻ sodium dodecyl sulfate (SDS) sample
buffer containing 4% SDS, 20% glycerol, 0.125 M Tris-HCl
Cell culture conditions and treatments
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 Mouse brown preadipocytes were isolated and cultured ac-
by the Bradford assay (protein assay kit; Bio-Rad Laboratories, cording to a published method (25) with a few modifications.
Hercules, CA, USA) using bovine serum albumin. Samples In brief, 4-wk-old C57BL/6 mice were killed, and interscapu-
(20 ␮g) were resolved by electrophoresis on a SDS-5⫺20% lar BATs were collected and digested with collagenase
polyacrylamide gel (Atto, Tokyo, Japan) and electroblotted (0.1%). The digestion mixture was filtered through a sterile
onto a polyvinylidene difluoride membrane (Millipore Corp., 100-␮m nylon mesh and left at room temperature for 10 min
Redford, MA, USA). Immunoblots were incubated with anti- until a discrete fat layer had formed on the top. This layer was
mouse ␤1-AR (1:1000, ab85037; Abcam plc, Cambridge, UK), discarded, and the lower phase was centrifuged at 700g for 10
anti-human ␤3-AR (1:1000; Trans Genic Inc., Kobe, Japan), min at room temperature. The sediments were suspended
anti-human UCP1 (1:10,000, ab23841; Abcam plc), and anti- and filtered again through a 40-␮m nylon mesh. The result-
chicken ␣-tubulin (1:2000; Calbiochem, Darmstadt, Ger- ing stromal vascular cells were incubated in Dulbecco’s
many) antibodies. Antigen-antibody complexes were detected modified Eagle’s medium supplemented with 10% fetal calf
using the appropriate secondary antibody conjugated with serum, 17 ␮M d-pantothenic acid, 33 ␮M biotin, 100 ␮M
horseradish peroxidase and visualized with an enhanced ascorbic acid, 1 ␮M octanoic acid, 50 nM triiodothyronine, 50
chemiluminescence system (GE Healthcare Bio-Sciences, Pis- U/ml penicillin G, and 1 ␮g/ml streptomycin. After becom-
cataway, NJ, USA). ing confluent, cells were dispersed and cultured in new
dishes. Subconfluent cells were differentiated in standard
Acute response of thermogenesis to CL-316,243 medium supplemented with 12.5 nM dexamethasone, 100
ng/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine. Rat
Thermogenesis in BAT in response to a single injection of the brown preadipocytes (1⫻105 cells/2 ml/well; Takara Bio)
␤3-AR agonist CL-316,243, a disodium salt (Tocris Bioscience, were cultured and differentiated according to the manufac-
Gottingen, Germany), was assessed by measuring tempera- turer’s directions. Brown adipocytes at d 2, 3, and 4 of
ture changes in BAT and the rectum, as described by Ino- differentiation were treated with 1-O-hexadecyl-2-N-methycar-
kuma et al. (21). Male mice (6 wk old) were anesthetized with bamoyl-sn-glycero-3-phosphocholine (c-PAF; Sigma-Aldrich,
ethyl carbamate (urethane, 250 mg/kg; Wako Pure Chemi- St. Louis, MO, USA) at concentrations of 5 ⫻ 10⫺11 to 5 ⫻
cals, Tokyo, Japan), a small incision was made above the 10⫺8 M with or without 5 ⫻ 10⫺6 M WEB2086 (a gift from
scapula, and the interscapular brown fat pads were partially Boehringer Ingelheim GmbH, Ingelheim, Germany) for an-
separated from the muscle below, with the vasculature and other 24 h.
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 Fluorescence-activated cell sorting (FACS) analysis of the
the plate was heated gently. After the rectal temperature had stromal vascular cells derived from BAT
reached a steady level (⬃37°C), CL-316,243 (25 or 50 ␮g/kg)
or saline was injected intraperitoneally, and temperature Stromal vascular cells derived from the pooled BAT of WT
changes were monitored for 40 min. The responses to CL- mice at 9⫺11 wk of age (n⫽12⫺18) were stained with
316,243 at 50 ␮g/kg were similar to those to CL-316,243 at 25 FITC-conjugated anti-CD31 and anti-CD45 and phycoeryth-
␮g/kg, but the former dose was lethal (1 of 4 mice died) and rin-conjugated anti-Sca-1 antibodies (BD Biosciences, San
the latter dose was nonlethal. Diego, CA, USA) for 30 min at room temperature. After
staining, cells were suspended in PBS containing 2% fetal calf
Assay for acetyl-CoA:lyso-PAF acetyltransferase 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
Acetyl-CoA:lyso-PAF acetyltransferase activity was determined
dead cells were excluded by forward scatter, side scatter, and
using microsomes prepared from pooled BAT, spleens, and
propidium iodide gating. Data were collected using FACSDiva
lungs as described previously (22).
software (BD Biosciences). RNA was purified from sorted
cells for quantitative PCR using an RNeasy Micro Kit (Qiagen,
Extraction and quantitation of PAF in BAT Hilden, Germany), according to the manufacturer’s instruc-
tions, and cDNAs were then synthesized with a QuantiTect
Total lipids were extracted using the method of Bligh and Reverse Transcription Kit (Qiagen), according to the manu-
Dyer (23) at 4°C in the presence of 50 mM glycine-HCl buffer facturer’s instructions.

442 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org SUGATANI ET AL.
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Statistical analysis addition, we observed higher fasting serum glucose
levels in PAFR-KO mice at 36 wk of age (but this was not
Values are expressed as means ⫾ sem. The data were analyzed significant, P⫽0.2) than in WT mice.
by analysis of variance unless otherwise stated. Fisher’s pro- We observed marked changes in the morphology of
tected least significant difference test was used to determine
the significance of differences among the groups. The level of
obese adipose tissue: adiposity in mice at ⬎24 wk of age
significance was set at a value of P ⬍ 0.05. 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-
RESULTS cific proteins within crown-like structures in adipose
tissue, although there was no significant difference in
Increase in body weight and enhanced adiposity in macrophage infiltration in the WAT of PAFR-KO and
PAFR-KO mice WT mice at ⬍18 wk of age (Figs. 1E and 2). In contrast,
there was no significant change in the WAT gene
We monitored body weight and food intake in PAFR- expression of adipocytokines, such as adiponectin and
KO mice and WT littermates on a weekly basis for leptin, or anti-inflammatory cytokine interleukin
a period of 36 wk. There was no significant difference (IL)-10 between PAFR-KO and WT mice, whereas the
in food intake between the 2 groups fed standard chow expression of tumor necrosis factor ␣, IL-6, and chemo-
throughout the observation period (Table 1), but a kine (C-C motif) ligand 3 (CCL3) mRNAs was in-
significantly higher body weight gain was observed in creased in epididymal fat in mice at ⬎24 wk of age
PAFR-KO mice (Fig. 1A, B). Notably, the sizes of the (Fig. 2A).
epididymal white adipose deposits corrected for total
body weight were markedly and significantly increased No change in the expression of critical genes
in PAFR-KO mice at 12 wk of age and thereafter (Fig. involved in anabolic and catabolic metabolism in the
1C). This increase in body weight was largely due to a adipose, liver, and muscle tissues of PAFR-KO mice
significant elevation in fat mass. In contrast, there was
no significant difference in the weights of the other We further examined the effect of the functional PAFR
major organs, such as the liver (Fig. 1D). deficiency on the gene regulation of the lipogenic
Moreover, we examined liver and serum lipid levels. enzyme [fatty acid synthase (FAS)], the mitochondrial
No significant differences were observed in serum and enzyme [carnitine palmitoyltransferase 1 (CPT1a and
liver levels of triacylglycerols, total cholesterol, or CPT1b)] involved in fatty acid oxidation, and the lipo-
nonesterified fatty acids between the two groups, ex- lytic enzymes [lipoprotein lipase (LPL), adipose triglyc-
cept that the triacylglycerol content in the liver was eride lipase (ATGL), hormone-sensitive lipase (HSL),
significantly higher in 36-wk-old KO mice (Table 1). In 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 in the expression of these ARs in the WAT, liver, and
fat accumulation, expressed levels of LPL, ATGL, HSL, skeletal muscle of PAFR-KO and WT mice at 6 wk of age
and LIPC similar to those of their WT littermates before the onset of obesity (Fig. 4C). However, the
(Fig. 3) in the BAT, liver, and skeletal muscle. Of note, expression of ␤1- and ␤3-ARs was significantly lower and
PAFR-KO mice expressed elevated mRNA levels of LPL, the expression of ␤2-AR was significantly higher in the
ATGL, and HSL in the WAT. Furthermore, we ob- BAT of PAFR-KO mice than in that of WT littermates.
served that the expression of FAS mRNA in the BAT, Furthermore, we examined the gene expression levels
WAT, liver, and skeletal muscle was not significantly of the uncoupling proteins (UCP1–3). The levels of
different between PAFR-KO and WT mice over 36 wk, UCP1 and UCP3 mRNA in the BAT and UCP2 mRNA
except for the WAT of 12-wk-old mice and the skeletal in the WAT were significantly lower in PAFR-KO mice
muscle of 24-wk-old mice (Fig. 4A). In addition, no than in WT littermates (Fig. 4D).
significant differences were observed in the expression We then examined changes in the protein expression
of CPT1a mRNA in the BAT, WAT, and liver and levels of ␤1- and ␤3-ARs and UCP1 in the BAT of
CPT1b mRNA in the skeletal muscle between PAFR-KO PAFR-KO and WT mice with age. Western blot analysis
and WT mice over 36 wk, except for the BAT of revealed that the relative levels of ␤3-AR expression vs.
1.7-wk-old mice, WAT of 6-wk-old mice, and BAT, WAT, ␣-tubulin expression in the BAT of PAFR-KO mice were
and liver of 24-wk-old mice (Fig. 4B). significantly lower than those of WT littermates at 6, 12,
18, and 24 wk, whereas there was no significant differ-
Decreases in ␤1/␤3-AR and UCP1 in the BAT of ence in the BAT weight corrected for the body weight
PAFR-KO mice (Fig. 5A, C, E). The levels of ␤1-AR expression in the
BAT of PAFR-KO mice were also lower than those of
We next examined the expression of ␤1-, ␤2-, and WT littermates (Fig. 5A, B). Furthermore, Western blot
␤3-ARs in the BAT, WAT, liver, and skeletal muscle, analysis revealed significantly lower levels of UCP1
which were involved in the sympathetic regulation of protein in the BAT of PAFR-KO mice than in WT
lipid metabolism. There were no significant differences littermates at 6⫺24 wk of age (Fig. 5A, D).

444 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org SUGATANI ET AL.
Downloaded from www.fasebj.org to IP 206.83.48.110. The FASEB Journal Vol.28, No.1 , pp:440-452, September, 2016
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- wk, compared with that of PAFR-KO mice (Fig. 6B).
genic fat cell-selective gene UCP1 mRNA in epididymal Because not only classic brown adipocytes derived from
WAT of WT mice at 24 and 36 wk of age [4.15⫾1.10- a myf-5 cellular lineage but also beige adipocytes
fold (P⫽0.054) and 6.09⫾2.68-fold (P⫽0.024) in the (UCP1-positive cells that emerge in white fat from a
control mice at 6 wk, respectively; Fig. 6A]. However, non-myf-5 lineage) have been reported to respond to
PAFR-KO mice at 24 and 36 wk expressed lower levels cAMP stimulation with high UCP1 expression (26), our
of UCP1 mRNA in WAT (1.36⫾0.16- and 2.68⫾0.61- present data suggest that PAF or PAFR signaling also
fold in the control mice at 6 wk, respectively; Fig. 6A). contributes to the expression of UCP1 in the beige
The UCP1-immunopositive cells, which had a unilocu- adipocytes within white adipose tissues.
lar morphology typical of white adipocytes, were in- Furthermore, to investigate whether elevations in plasma
creased in epididymal WAT of WT mice at 24 and 36 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 in PAFR-KO mice 14 min after the CL-316,243 (25
(27–29), we determined peripheral plasma cortisol levels ␮g/kg) injection and thereafter were significantly less
in WT and PAFR-KO mice. Unexpectedly, plasma cortisol than those in WT mice (Fig. 8). The injection of saline
levels in PAFR-KO mice were significantly lower than did not change the basal temperature of the BAT or
those in WT mice. The levels of glucocorticoid receptor rectums in either type of mice.
(GR) mRNA in the BAT of PAFR-KO mice was slightly To further evaluate whether the dysfunction in BAT
lower than those in the BAT of WT littermates (P⫽0.07; contributed to the impaired thermogenic activity in
Fig. 7), indicating that the decrease in ␤3-AR expression PAFR-KO mice, we examined the morphology and
did not result from elevations in plasma cortisol levels or expression of several genes involved in mitochondrial
BAT GR mRNA expression. biogenesis in BAT. Although adipocyte cell size in BAT
(interscapular fat) was higher in PAFR-KO mice than in
Reduced thermogenesis in PAFR-KO mice WT mice (Fig. 5F), PAFR-KO mice showed levels of
mitochondrial density similar to those of WT litter-
We subsequently examined the physiological response mates, as determined by staining with MitoTracker
of PAFR-KO mice to the ␤3-AR agonist, CL-316,243. We (Fig. 5G), and the gene expression levels of Ndufc1,
monitored temperature changes in the interscapular Sdha, Cybb, and Cox5a involved in mitochondrial respi-
BAT and rectums of PAFR-KO and WT mice at 6 wk of ratory complex I, complex II⫺III, and complex IV,
age after a single injection of CL-316,243. The basal were also similar except for those of 6-wk-old mice
temperatures of the BAT and rectums were 35.4 ⫾ 0.2 (Supplemental Fig. S1). The levels of Ndufc1 and Sdha
and 37.1 ⫾ 0.2°C in PAFR-KO mice and 35.3 ⫾ 0.2 and mRNA were slightly lower in PAFR-KO mice than in WT
36.8 ⫾ 0.2°C in WT mice, respectively, which were not mice at 6 wk of age and reached a plateau in the mutant
significantly different between the 2 groups, whereas mice at 12 wk similar to that in WT mice, which
the BAT temperature was slightly lower than the rectal indicates the delayed formation of the mitochondrial
temperature in both genotypes of mice. The responses 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 contrast, c-PAF at 5 ⫻ 10⫺10 to 5 ⫻ 10⫺8 M could not
brown adipocytes by PAF induce the expression of UCP1 mRNA (Fig. 9A and
Supplemental Fig. 2B) and protein (Fig. 9B) in mouse
Because ␤3-AR is a potent stimulator of thermogenesis in and rat brown adipocytes differentiated at d 3⫺5, which
the BAT of humans and animals, including rodents, we suggests that PAF did not directly influence the induction
examined the effect of PAFR stimulation on the in vitro of UCP1.
expression of ␤3-AR in mouse and rat brown adipocytes.
c-PAF, a nonhydrolyzable agonist of PAFR, was used for Occurrence of PAF in the BAT of WT mice
PAFR stimulation of the cultured brown adipocytes, be-
cause serum PAF acetylhydrolase immediately hydrolyzes The PAF content in BAT was 0.230 ⫾ 0.058 pmol/g tissue
PAF (30). The expression of ␤3-AR mRNA increased with (n⫽4), with the results presented as means ⫾ sem for
the process of differentiation and maturation after treat- combined samples from 10⫺14 mice at 6 wk of age in the
ment of brown preadipocytes with dexamethasone and number of experiments indicated (Fig. 10). It was
insulin, whereas that of UCP1 mRNA was not found until confirmed that the substance activating washed rabbit
d 5 (Supplemental Fig. S2). The administration of c-PAF platelets was PAF, because the PAF antagonist WEB2086
to mouse brown adipocytes differentiated at d 5 at a completely inhibited its aggregation activity on platelets
concentration of 5 ⫻ 10⫺9 M resulted in a significant (Fig. 10).
elevation in the expression of ␤3-AR mRNA and protein
(1.5- and 1.9-fold, respectively; Fig. 9A, B), whereas ␤3-AR Expression of acyl-CoA:lysophosphatidylcholine
mRNA levels were significantly increased after treatment acyltransferase 2 (LPCAT2), cytosolic phospholipase
with c-PAF at 5 ⫻ 10⫺10 M in rat brown adipocytes A2␣ (cPLA2␣), and PAFR mRNA in the BAT, BAT
differentiated at d 5 (1.5-fold; Supplemental Fig. S2A). macrophages, and brown adipocytes of WT mice
Adipogenic differentiation was confirmed by peroxisome
proliferator-activated receptor ␥ (PPAR␥) mRNA expres- To evaluate the potential for PAF biosynthesis in BAT,
sion and Oil Red O staining (Fig. 9C, D). WEB2086, an stromal vascular cells derived from BAT, and brown
antagonist of PAFR, significantly prevented the induction adipocytes, we measured the gene expression of
of ␤3-AR mRNA and protein expression by c-PAF. In 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 Figure 8. Acute response of BAT thermogenesis to CL-
316,243. PAFR-KO and WT mice were anesthetized, and
were significantly higher but were less than those in the
temperature changes in the interscapular BAT (A) and
lung and spleen (Fig. 11A). The gene expression levels rectums (B) were monitored after the injection of CL-316,243
of LPCAT2 and cPLA2␣ were also significantly higher in (25 ␮g/kg) or saline. Results are means ⫾ sem (n⫽3⫺5).
BAT and brown adipocytes but were less than those in Difference between values for PAFR-KO and WT mice was
the lung and spleen (Fig. 11B, C). Furthermore, we significant (P ⬍ 0.05) at 14 min and thereafter.
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 muscle, kidney, and mouse brown adipocytes (Fig.
kidney of WT mice. The level of PAFR mRNA in the 11D). Similar levels of PAFR mRNA were observed in
BAT of WT mice was similar to that in the lung and the BAT of WT mice at 6⫺36 wk of age, indicating that
spleen and higher than that in the liver, skeletal 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
Figure 7. Plasma cortisol and BAT GR mRNA levels in mRNA was predominantly localized in Sca-1⫺/CD31⫹/
PAFR-KO and WT mice. Plasma cortisol levels (A) and BAT
GR mRNA levels (B) were determined in PAFR-KO and WT
CD45⫹ sorted cells (130⫾42/18S rRNA⫻104), which
mice at 12 wk of age. Relative mRNA expression was normal- markedly expressed the CD68 gene (BAT macro-
ized against 18S rRNA. Results are means ⫾ sem (n⫽6⫺11). phages; Fig. 11). The PAFR gene was also predomi-
**P ⬍ 0.01 for PAFR-KO mice vs. control WT mice. 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 littermates (Fig. 1). The adiposity was accompanied by
levels were expressed in Sca-1⫹/CD31⫺/CD45⫺ stem increases in both the number and size of adipocytes
cells (0.2⫾0.2/18S rRNA⫻104) and brown adipocytes with age, but mRNA levels for several macrophage
(3.6⫾0.7/18S rRNA⫻104; Fig. 11). markers (F4/80, CD11b, CD11c, and CD68) were
prominently elevated only at 24 wk, including the
expression of M1 macrophage-specific protein CD11c
DISCUSSION within crown-like structures in adipose tissue, sugges-
tive of macrophage infiltration of WAT (Figs. 1 and 2).
This study indicated that body weight with age and These results indicate that macrophage infiltration of
adiposity was higher in PAFR-KO mice than in WT 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
Figure 10. Platelet aggregation induced by BAT PAF. PAF in mice over 36 wk in the present study, we determined
the 25-mg sample of BAT was used. Trace a: platelets
activated by BAT PAF. Trace b: Platelets preincubated with
the effect of the PAFR deficiency on energy expendi-
2.5 ⫻ 10⫺6 M WEB2086 and activated by BAT PAF. Traces ture. ATGL, HSL, and LPL mRNA levels were higher in
c: Platelets activated by 1.25 ⫻ 10⫺11, 2.5 ⫻ 10⫺11, and 5 ⫻ the WAT of obese 12-wk-old PAFR-KO mice than in the
10⫺11 M PAF. 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 CPT1a mRNA, leading to lipid accumulation and a
adipose tissues. In contrast, we demonstrated that reduction in lipid consumption, respectively, result
PAFR-KO mice exhibited significantly elevated mRNA from a lack of PAFR.
levels of the fatty acid-synthesizing enzyme FAS in the The activation of energy expenditure in the WAT
WAT at 12 wk and significantly decreased mRNA levels and BAT is mediated by the sympathetic nervous system
of the ␤-oxidation-related enzyme CPT1a in the WAT at and is particularly reliant on ␤-AR signaling (33, 34). In
6 wk (Fig. 4). These findings appeared to contribute, at the BAT of PAFR-deficient mice, the expression of not
least in part, to the development of adiposity at 12 wk only ␤3-AR but also ␤1-AR was decreased (Figs. 4 and 5),
and thereafter, although it remains to be determined whereas ␤1-AR could elicit cAMP-mediated responses to
whether the increase in FAS mRNA and decrease in 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.

450 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org SUGATANI ET AL.
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knockout mice because of adaptations compensating in BAT. Brown adipocytes had low, but significant,
for the ␤3-AR deficiency (35, 36). Although ␤1- and levels of LPCAT2 mRNA, whereas BAT macrophages
␤3-AR mRNA and protein have been reported to be had high levels of its mRNA (Figs. 11 and 12). In
down-regulated by glucocorticoids through a glucocor- addition, BAT had a significant amount of PAF (Fig.
ticoid receptor-mediated mechanism (27–29), plasma 10). Thus, PAF appears to be produced by lyso-PAF
glucocorticoid and BAT GR mRNA levels were not acetyltransferase activated in BAT macrophages in re-
increased in PAFR-KO mice (Fig. 7). The reduced sponse to extracellular stimuli or in brown adipocytes
expression of ␤1- and ␤3-AR in the BAT of PAFR-KO in an autocrine/paracrine manner.
mice did not appear to be mediated by GR. Exposure of mice to cold temperatures rapidly pro-
Recent studies have suggested that the metabolically motes the alternative activation of adipose tissue mac-
active BAT of human adults is more abundant than rophages, which secrete catecholamines to induce ther-
previously thought and could be a target for the mogenic gene expression in BAT (38). Because the
treatment of obesity (7–9, 37). Whereas thermogenesis PAFR gene was predominantly localized in BAT macro-
in response to alterations in energy intake is largely phages, in addition to ␤3-AR expression in brown
attributed to the activation of the thermogenic organ adipocytes by PAF, the absence of BAT macrophages
BAT, ␤3-AR stimulation does not cause BAT thermo- activated by PAF in PAFR-KO mice may have sup-
genesis in mice lacking UCP1 (21). Thus, ␤3-AR-medi- pressed the secretion of catecholamines and caused
ated thermogenesis depends on UCP1, which uncou- BAT dysfunction. We are currently in the process of
ples mitochondrial oxidative phosphorylation and investigating the precise mechanism of BAT macro-
generates heat by dissipating the proton gradient. The phage activation associated with thermogenesis through
present study showed that ␤3-AR and UCP1 were found PAF.
predominantly in BAT (Figs. 4 and 5). The levels of In summary, our findings in the PAFR-KO genetic
UCP1 mRNA and protein in BAT were lower in model revealed that PAFR deficiency caused BAT dys-
PAFR-KO mice than in WT mice, which may have function, which induced the development of obesity
resulted from the suppressed production, but not deg- due to the impaired thermogenic activity of BAT. In
radation, of UCP1 (Figs. 4D and 5D). Because of the other words, our findings suggest that PAF/PAFR pos-
BAT dysfunction, which resulted from the reduced sesses an antiobese function that occurred through
expression of ␤3/␤1-AR and UCP1, PAFR-KO mice ␤3-AR/UCP1 expression in BAT. These findings were
appeared to exhibit less thermogenesis with ␤3-AR consistent with the fact that the body weight of trans-
stimulation (Fig. 8). In fact, lipid vacuoles were en- genic mice overexpressing PAFR at 14⫺17 wk was
larged in the BAT of PAFR-KO mice, indicating that the ⬃20% lighter than that of their control littermates
accumulation of lipids in BAT was enhanced (Fig. 5F). (39). Future studies on how PAF/PAFR signaling con-
Because the appearance of UCP1 with age was also trols UCP1 levels through ␤3-AR production in the BAT
suppressed in the WAT of PAFR-deficient mice (Fig. 6), of animals and humans may reveal new therapeutic
PAFR-KO mice appeared to be more susceptible to targets to treat metabolic disorders associated with
diet-induced obesity than WT mice because of the obesity. We are in the process of investigating the
reduced ability to use UCP1-mediated thermogenesis. mechanism of the antiobese action through PAF or
The present study showed that PAFR mRNA levels PAFR signaling using conditional gene-KO mice.
were high not only in the BAT of control mice but also
The authors gratefully acknowledge Masaki Imanishi,
in mouse brown adipocytes (Fig. 11D). This prompted
Kengo Yasuda, and Kouki Hiramoto for excellent technical
us to investigate the role of PAF in the expression of assistance. This work was supported in part by the global
␤-ARs in mouse brown adipocytes (Fig. 9). Our findings Center of Excellence program and grants-in-aid for scientific
demonstrated that c-PAF induced the expression of research (21590170 and 22590068) from the Ministry of
␤3-AR mRNA and protein but not of UCP1 mRNA and Education, Culture, Sports, Science and Technology, Japan.
protein in mouse brown adipocytes. These effects were
receptor-mediated because they were not observed in
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452 Vol. 28 January 2014 The FASEB Journal 䡠 www.fasebj.org SUGATANI ET AL.
Downloaded from www.fasebj.org to IP 206.83.48.110. The FASEB Journal Vol.28, No.1 , pp:440-452, September, 2016
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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