PGD2 production in white adipose tissue of H-PGDS TG mice was increased approximately seven-fold as compared with that in wild-type WT mice.. Serum leptin and insulin levels were increas
Trang 1caused by overproduction of prostaglandin D2 in vivo
Yasushi Fujitani1,*, Kosuke Aritake1, Yoshihide Kanaoka1,2, Tsuyoshi Goto3, Nobuyuki Takahashi3,
Ko Fujimori1,4and Teruo Kawada3
1 Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Japan
2 Department of Medicine, Harvard Medical School, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Boston, MA, USA
3 Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Japan
4 Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, Japan
Introduction
The amount of adipose tissue in the body is an
impor-tant factor in the maintenance of energy balance,
through its ability to store and release fat, and is
altered in various physiological or pathological condi-tions [1] The increased adipose tissue mass associated with obesity results from an increase in the number
Keywords
adipocytes; H-PGDS; obesity; PGD2;
transgenic mouse
Correspondence
K Fujimori, Laboratory of Biodefense and
Regulation, Osaka University of
Pharmaceutical Sciences, 4-20-1 Nasahara,
Takatsuki, Osaka 569-1094, Japan
Fax: +81 726 690 1055
Tel: +81 726 690 1055
E-mail: fujimori@gly.oups.ac.jp
*Present address
Pharmaceutical Research Division, Takeda
Pharmaceutical Co Ltd., Osaka, Japan
(Received 28 October 2009, revised 22
December 2009, accepted 4 January
2010)
doi:10.1111/j.1742-4658.2010.07565.x
Lipocalin-type prostaglandin (PG) D synthase is expressed in adipose tissues and involved in the regulation of glucose tolerance and atherosclero-sis in type 2 diabetes However, the physiological roles of PGD2 in adipo-genesis in vivo are not clear, as lipocalin-type prostaglandin D synthase can also act as a transporter for lipophilic molecules, such as retinoids We gen-erated transgenic (TG) mice overexpressing human hematopoietic PGDS (H-PGDS) and investigated the in vivo functions of PGD2 in adipogenesis PGD2 production in white adipose tissue of H-PGDS TG mice was increased approximately seven-fold as compared with that in wild-type (WT) mice With a high-fat diet, H-PGDS TG mice gained more body weight than WT mice Serum leptin and insulin levels were increased in H-PGDS TG mice, and the triglyceride level was decreased by about 50%
as compared with WT mice Furthermore, in the white adipose tissue of H-PGDS TG mice, transcription levels of peroxisome proliferator-activated receptor c, fatty acid binding protein 4 and lipoprotein lipase were increased approximately two-fold to five-fold as compared with those of
WT mice Finally, H-PGDS TG mice showed clear hypoglycemia after insulin clamp These results indicate that TG mice overexpressing H-PGDS abundantly produced PGD2in adipose tissues, resulting in pronounced adi-pogenesis and increased insulin sensitivity The present study provides the first evidence that PGD2 participates in the differentiation of adipocytes and in insulin sensitivity in vivo, and the H-PGDS TG mice could consti-tute a novel model mouse for diabetes studies
Abbreviations
15d-PGJ 2 , 15-deoxy-D12,14prostaglandin J 2 ; ACC, acetyl-CoA carboxylase; aP2, fatty acid-binding protein 4, adipocyte; BAT, brown adipose tissue; CMV, cytomegalovirus; CT, computed tomography; DEX, dexamethasone; GST, glutathione S-transferase; HF, high-fat; H-PGDS, hematopoietic prostaglandin D synthase; IBMX, 3-isobutyl-1-methylxanthine; L-PGDS, lipocalin-type prostaglandin D synthase; LPL,
lipoprotein lipase; PG, prostaglandin; PGDS, prostaglandin D synthase; PPAR, peroxisome proliferator-activated receptor; SCD, stearoyl-CoA desaturase; SEM, standard error of the mean; TG, transgenic; WAT, white adipose tissue.
Trang 2and size of adipocytes A major role of adipocytes is
to store large amounts of triglycerides during periods
of energy excess and to mobilize these depots during
periods of nutritional deprivation The number of
adipocytes is thought to increase as a result of
differ-entiation of adipocytes Moreover, adipocytes are
highly specialized cells that secrete various
adipocyto-kines, whose release largely reflects the amounts of
stored triglyceride Insights in the molecular
mecha-nisms underlying adipogenesis may lead to the
devel-opment of strategies for reducing the prevalence of
obesity
Adipogenesis is a complex process accompanied by
various changes in hormone sensitivity and gene
expres-sion caused by many stimuli, including lipid mediators
Prostaglandins (PGs) are involved in the regulation of
adipocyte differentiation In vitro studies have shown
that PGD2 enhances adipocyte differentiation [2], but
that PGE2and PGF2asuppress adipogenesis [3–5]
PGD synthase (PGDS) consists of two types of
pro-tein [6] One is lipocalin-type PGDS (L-PGDS), and the
other is hematopoietic PGDS (H-PGDS) H-PGDS was
originally purified from rat spleen as a cytosolic,
gluta-thione-requiring enzyme [7,8], responsible for the
bio-synthesis of PGD2 in antigen-presenting cells [9], mast
cells [10,11], megakaryocytes [12,13], and type 2 helper
T-lymphocytes [14] There have been extensive
biochem-ical and genetic analyses of H-PGDS [15], and H-PGDS
was crystallized with its specific inhibitor at 1.7 A˚
reso-lution by X-ray diffraction analysis [16] H-PGDS was
shown to be a member of the sigma-class glutathione
S-transferase (GST) family, and is also called GSTS1
[17] On the other hand, L-PGDS has been purified
from rat brain [18], and is expressed in brain, heart, and
male genital organs, as well as in adipocytes and
omen-tal adipose tissues [19–22] The different types of PGDS
have no significant homology at the amino acid level,
and have different tertiary structures for catalysis
[15,23,24] Of particular note is that L-PGDS is a
bifunctional protein, having enzymatic activity with
regard to both PGD2production and transportation of
lipophilic molecules, such as retinoids [25], biliverdin,
bilirubin [26], gangliosides [27], and amyloid b-peptides
[28], with high affinities (Kd= 20–2000 nm) We
previ-ously reported that knockdown of L-PGDS inhibited
adipocyte differentiation of 3T3-L1 cells in vitro,
thereby suggesting that L-PGDS is involved in the
regu-lation of adipocyte differentiation [2] L-PGDS
knock-out mice became glucose-intolerant and
insulin-resistant, and showed increased fat deposition in the
aorta after receiving a high-fat (HF) diet [29]
Adipo-cytes of the L-PGDS knockout mice were significantly
larger than those of wild-type (WT) mice [29] Another
recent study demonstrated that L-PGDS knockout mice did not have any significant glucose or insulin tolerance, but had increased body weight and increased atheroscle-rotic lesions in the aorta [30] Thus, the role of L-PGDS
in adipogenesis and diabetes-related phenotypes is not clear Moreover, because of the dual functions of L-PGDS, whether PGD2regulates the differentiation of adipocytes in vivo remains to be elucidated
In the present study, we have generated transgenic (TG) mice, which produce abundant PGD2 by overex-pression of human H-PGDS, and used them to investi-gate the physiological significance of PGD2 in adipogenesis in vivo The H-PGDS TG mice showed obesity, pronounced adipogenesis, and increased insu-lin sensitivity when on the HF diet
Results Generation of H-PGDS TG mice Human H-PGDS cDNA under the regulatory control
of the chicken b-actin promoter and cytomegalovirus (CMV) enhancer (Fig 1A) was microinjected into the nuclei of fertilized eggs from FVB mice We established three lines of H-PGDS TG mice, termed S41, S55, and S66 Northern blot analysis for estimation of mRNA expression of the transgene revealed higher expression
in S41 and S55 mice and lower expression in S66 mice in the liver, white adipose tissue (WAT), and brown adi-pose tissue (BAT), although H-PGDS was not expressed
in each tissue of WT mice (Fig 1B) The expression of human H-PGDS in hepatocytes and adipocytes of the H-PGDS TG mice (S55) was confirmed by immunohis-tochemistry, using a specific antibody against human H-PGDS (Fig 1C) Liver homogenates from WT and
TG mice were used for PGDS activity assays As shown
in Fig 1D, the tissue homogenates of TG mice showed higher levels of PGD2 production than those of WT mice (approximately 18-fold, 25-fold and five-fold in S41, S55 and S66 mice, respectively) These results indi-cate that the H-PGDS TG mice overexpress human H-PGDS transcripts, proteins and activities in various tissues In further experiments, we decided to use S41 and S55 mice as TG mice, because these mice showed more abundant mRNA expression and enzymatic activity of human H-PGDS
HF diet study
In order to examine the effects of PGDS overexpres-sion on adipogenesis, WT and TG mice were fed a normal or HF diet for 6 weeks after delactation (Fig 2A) TG mice showed normal growth and no
Trang 3significant differences in spontaneous locomotor
activ-ity, rectal temperature and amount of food intake
under either normal or HF diet conditions in
compari-son with WT mice (data not shown) The body weights
of WT and TG mice were almost the same at the start
of this experiment (21.2 ± 0.3 g, 20.5 ± 0.7 g and
20.2 ± 0.5 g for WT, S41 and S55 mice, respectively)
The body weights of both WT and TG mice increased
in a similar manner under normal diet conditions
(Fig 2B) In contrast, under HF diet conditions, the
body weights of both S41 and S55 mice increased
more, with statistically significant differences from WT
mice (Fig 2B) Next, we measured tissue weights of
the liver, WATs (epididymal and perirenal fat) and
BAT under HF diet condition WAT weights of TG
mice were significantly increased, by 20–30%, as
com-pared with those of WT mice The BAT mass of TG
mice was larger than that of WT mice On the other
hand, liver weights showed no difference between WT
and TG mice, under either normal or HF diet
condi-tions (Fig 2C) These results indicate that the
overex-pression of H-PGDS causes the increase in adipose
tissue mass under HF diet conditions
Body distribution of adipose tissues as
determined by computed tomography (CT) analysis
To further assess the effect of H-PGDS overexpression
on the increase in adipose tissues, the weights of
subcutaneous and visceral adipose tissues, as well as of muscle, of WT and TG (S55) mice were analyzed with a micro-CT scanner under HF diet conditions Visceral and subcutaneous adipose tissue weights of TG mice were significantly increased after 1 week of the HF diet
in comparison with those of WT mice (Fig 2D) The weights of visceral and subcutaneous adipose tissues of
TG mice were approximately 1.5-fold and 1.4-fold, respectively, of those of WT mice after 6 weeks of the
HF diet (Fig 2D) In contrast, the weight of muscle with organ, but without fats, showed no significant dif-ference between WT and TG mice (Fig 2D) These results confirm that both subcutaneous and visceral adi-pose tissues were increased in TG mice by the HF diet
mRNA expression of adipogenic genes in WAT of
TG mice
We measured the amounts of PGD2 in WAT after
6 weeks of the HF diet WAT of TG (S55) mice con-tained significantly more PGD2 (approximately seven-fold) than that of WT mice (Fig 3A) To examine the effects of the increased PGD2level on peroxisome pro-liferator-activated receptor (PPAR) c activation, we performed quantitative RT-PCR to measure the mRNA expression levels of adipogenic genes, including several PPARc-target genes, the transcription of which is enhanced in adipogenesis [31,32] The expres-sion levels of PPARc, fatty acid-binding protein 4,
Human H-PGDS cDNA Chicken β-actin
enhancer
Liver WAT BAT
β-globin PolyA
5
10
Liver WAT
0
5
–1 ·mg
promoter
Intron
CMV
promoter
A
B
Fig 1 Generation of human H-PGDS TG mice (A) Schematic representation of human H-PGDS transfer vector The SalI–NotI fragment was microinjected into fertilized eggs of FVB mice (B) Northern blot analysis of transgene expression in the liver, WAT, and BAT Ten micrograms of total RNA was subjected to agarose gel electrophoresis, blotted onto a nylon mem-brane, and hybridized with the 32 P-labeled full-length cDNA for human H-PGDS (C) Immunohistochemical analysis of transgene expression in the liver and WAT Paraffin sections of liver and WAT from WT and TG mice (S55) were stained with antibody against human H-PGDS Bars: 100 lm (D) PGDS activity in liver of WT and S41, S55 and S66 TG mice.
Trang 4adipocyte (aP2), lipoprotein lipase (LPL),
stearoyl-CoA desaturase (SCD), CD36 and acetyl-stearoyl-CoA
carbox-ylase (ACC) in WAT of TG mice were significantly
upregulated by approximately 2.5-fold, three-fold,
five-fold, 8.6-five-fold, 1.2-fold and 22-five-fold, respectively, in
comparison with those in WT mice (Fig 3B) These
results indicate that mRNA expression of PPARc
tar-get genes is increased in WAT of TG mice, suggesting
that PPARc might be activated more in WAT of TG
mice than in WAT of WT mice
Serum levels of triglyceride, glucose, leptin and
insulin, and insulin sensitivity, in TG mice
After 6 weeks of normal or HF diet, serum levels of
triglyceride, glucose, leptin and insulin were
deter-mined (Fig 4A) Under both dietary conditions, triglyceride levels in TG (S55) mice were lower than those in WT mice by about 50%, whereas glucose levels were unchanged Interestingly, serum leptin lev-els were markedly increased in TG mice by approxi-mately 1.7-fold and 3.3-fold after the normal and HF diet, respectively, in comparison with WT mice Fur-thermore, insulin levels in TG mice were also increased as compared with those in WT mice by approximately 2.6-fold and two-fold after the normal and HF diet, respectively We next examined poten-tial alterations of insulin sensitivity in TG mice TG mice fed the HF diet for 12 weeks showed clear hypoglycemia after insulin loading as compared with
WT mice (Fig 4B) The same results were obtained
in TG mice fed a normal diet These results clearly
****
*
*
**
*
1 1·5
**
WT (n = 18) S41 (n = 8) S55 (n = 8)
*
10
15
10 15
6 4 2
Duration (week)
*
Duration (week)
0
0·5
rirenal fa
*
*
0
5
0 5
Visceral fat
0 1 2 3 4 5 6
**
**
**
**
**
**
0 1 2 3 4 5
Duration (week)
**
0 1 2 3 4 5 6
**
**
**
**
**
Subcutaneous fat
0 1 2 3 4 5
0 1 2 3 4 5 6
Muscle (with organs)
0 1 2 3 4 5
A
D
Fig 2 Body weight increase in mice when
on the normal and HF diets (A) After
delac-tation, WT and H-PGDS TG (S55) mice were
fed either the normal or the HF diet for
6 weeks A representative male mouse
from each group is shown (B) Body weight
was monitored every week for 6 weeks.
Closed circles (n = 61), squares (n = 22) and
triangles (n = 36) indicate WT, S41 and S55
mice, respectively Values are expressed as
means ± SEMs *P < 0.05, **P < 0.01 as
compared with WT mice (C) Tissue weights
of epididymal and perirenal fat, BAT, and liver.
Values are expressed as means ± SEMs.
*P < 0.05, **P < 0.01 as compared with
WT mice (D) Changes in the weights of
visceral and subcutaneous fat and muscle
with organ, but without fat, of WT and
H-PGDS TG mice (n = 6) Continuous
dissections of mouse fat and bone in the
whole body were quantified by use of a
micro-CT scanner and LATHETA software
(Aloka) Open and closed circles correspond
to WT and H-PGDS TG mice, respectively.
**P < 0.01 as compared with WT mice.
Trang 50 0.2
0.4
0.4 0.8 PPARγ
0 10 20 30
0
**
**
*
0
1
2
–1 tissue)
4
**
**
0 0
10 20 30
2 4 6
0
0.2 0.3 0.4
0.1
**
Fig 3 PGD2production and expression of adipogenic genes (A) Predominant produc-tion of PGD 2 in TG mice PGD 2 levels in WAT of WT and TG mice after the HF diet were measured by enzyme immunoassay (B) Transcription levels of adipogenic genes (encoding PPARc, aP2, LPL, SCD, CD36, and ACC) in WAT After being fed the HF diet for 6 weeks, mice were killed, and total RNA was isolated from WAT Expression levels of the target genes were normalized
to those of the b-actin mRNA level as an internal control, and calculated as fold inten-sity Values are expressed as means ± SEMs (n = 4–6) *P < 0.05, **P < 0.01 as compared with WT mice.
Time after injection (min) 0
50
100
150
0 50 100 150
Insulin (0.75 U kg –1 ) Insulin (3.0 U kg –1 )
*
*
*
Insulin
10 20 30 40 50
0 40 80 120
0
40
80
120
0 0.5 1.0 1.5
**
*
*
**
**
Normal HF Normal HF
Normal HF Normal HF
A
sensitiv-ity test (A) After being fed a normal or HF diet for 6 weeks, mice were killed and blood was collected Values are expressed as the means ± SEMs (n = 4–6) *P < 0.05,
**P < 0.01 as compared with WT mice (B) WT (open circles) and TG (closed circles) mice were injected with 0.75 UÆkg)1and 3.0 UÆkg)1of insulin after being fed a nor-mal or HF diet, respectively The y-axis indi-cates the percentage change in blood glucose level as compared with the value before injection (100% at t = 0) Values are expressed as the means ± SEMs (n = 6–7).
*P < 0.05 as compared with WT mice.
Trang 6indicate that overexpression of H-PGDS increases
insulin sensitivity in vivo
Adipocyte differentiation ex vivo
Finally, we examined whether the overexpression of
H-PGDS also promotes ex vivo differentiation of
adipo-cytes Preadipocytes prepared from WATs of WT or
TG (S55) mice were differentiated with 1 lm
dexa-methasone (DEX), 0.5 mm 3-isobutyl-1-methylxanthine
(IBMX), and insulin (10 lgÆmL)1) Ten days after
induction of differentiation, the differentiated
adipo-cytes prepared from WAT of TG mice accumulated
apparently greater amounts of lipid droplets than those
of WT mice (Fig 5A) Intracellular triglyceride
con-tents in TG mouse-derived adipocytes were
signifi-cantly larger than in WT mouse-derived cells (Fig 5B)
Moreover, the mRNA expression level of LPL in TG
mouse-derived adipocytes was increased by
approxi-mately two-fold as compared with WT mouse-derived
cells (Fig 5C) Therefore, these results suggest that the
overproduction of PGD2 promotes adipocyte
differen-tiation, thereby regulating adipogenesis
Discussion
In this study, we generated H-PGDS TG mice over-producing PGD2, and showed that PGD2 acts as an activator in adipogenesis in vivo We used H-PGDS
TG mice to elucidate the functions of PGD2 in adipo-genesis in vivo, because L-PGDS is a bifunctional pro-tein, both producing PGD2 and acting as a carrier protein for small lipophilic molecules [23], even though L-PGDS, but not H-PGDS, was detected in adipocytes [2,19] Investigations using L-PGDS knockout mice have demonstrated that L-PGDS is involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes [29,33], and showed induction of obes-ity [30] However, it is not known which functions of L-PGDS are associated with these phenotypes
15-Deoxy-D12,14PGJ2 (15d-PGJ2), which is one of the metabolites of PGD2, has been identified as a ligand for PPARc that can activate the differentiation
of adipose cells [34,35] However, the concentrations of 15d-PGJ2 used for activation of PPARc in most stud-ies are much higher (2.5–100 lm) than those of conven-tional PGs (picomolar range) Moreover, Bell-Parikh
et al.[36] demonstrated that 15d-PGJ2was present at a low level that is insufficient for activation of adipocyte differentiation Thus, the contribution of 15d-PGJ2 to
in vivoadipogenesis remains to be clarified
H-PGDS TG mice gained more body weight than
WT mice when on the HF diet (Fig 2A,B,D), and the WAT weight of TG mice was larger than that of WT mice (Fig 2C); this was accompanied by upregulation
of the expression of adipogenic genes in WAT (Fig 3B), suggesting pronounced differentiation of adipocytes and subsequent obesity in H-PGDS TG mice Furthermore, we observed a drastic increase in PGD2 levels in WAT of H-PGDS TG mice (Fig 3A), whereas PGE2 and PGF2a levels were not significantly altered in WAT in TG mice as compared with those in
WT mice (data not shown); these results are consistent with the previous result showing that, even if PGD2 production was decreased, the biosynthesis of other PGs was not significantly affected [16]
The phenotypes seen in H-PGDS TG mice are con-sistent with the findings that thiazolidinediones, PPARc agonists, enhance adipocyte differentiation and increase body weight, but act as antidiabetic drugs to improve insulin sensitivity [37] Indeed, the overexpres-sion of H-PGDS improved insulin resistance (TG mice showed clear hypoglycemia in response to insulin clamp, as shown in Fig 4B) Thus, PGD2 and⁄ or PGD2 metabolites might be involved in the regulation
of adipogenesis through PPARc in vivo Further stud-ies to investigate the precise mechanism, including the
*
*
0
10
20
0 0.1 0.2
A
Fig 5 Adipocyte differentiation ex vivo (A) Primary cultured
adipo-cytes from WAT of WT and H-PGDS TG mice were cultured in the
presence of DEX, IBMX and insulin for 7 days, and stained for lipid
droplet accumulation with Oil Red O (B) Triglyceride levels in
pri-mary cultured adipocytes Values are expressed as means ± SEMs
(n = 4) **P < 0.01 as compared with WT mice (C) The
transcrip-tion level of the LPL gene in WAT was normalized to that of b-actin
as a control, and calculated as fold intensity Values are expressed
as the means ± SEMs (n = 4–6) *P < 0.05 as compared with WT
mice.
Trang 7changes oin uptake of fatty acids and the number of
adipocytes, are needed In addition, we need to
eluci-date the effects of GST activity in H-PGDS TG mice,
because H-PGDS also has GST activity [38]
In contrast to their increased insulin sensitivity, TG
mice showed higher insulin concentrations in blood,
whereas the basal glucose level was not different from
that of WT mice (Fig 4A) In the H-PGDS TG mice,
apart from the improvement in peripheral insulin
resis-tance through the activation of PPARc in WAT, it is
possible that PGD2 stimulates pancreatic islets to
increase insulin secretion Indeed, serum insulin levels
were increased after treatment with thiazolidinediones
in diabetic mice through regulation of insulin
produc-tion in pancreatic islet cells [39–41] Thus, the
increased insulin level seen in H-PGDS TG mice when
on the HF diet might be due to effects of PGD2 on
pancreatic islet cells The precise mechanism needs to
be elucidated in further investigations that include
analysis of pancreatic islet cells
The H-PGDS TG mouse is a novel obesity model
with which to investigate the mechanism of
adipogene-sis As is the case for obese people with overnutrition
and energy imbalance, as is common in advanced
countries, H-PGDS TG mice become obese after the
HF diet but not after the normal diet This phenotype
is distinct from that seen in the well-known obesity
model mice, such as db⁄ db and ob ⁄ ob mice, which are
deficient in the leptin receptor and leptin genes,
respec-tively [42]
In summary, H-PGDS TG mice produced
substan-tial amounts of PGD2 as compared with WT mice,
and showed obesity, pronounced adipogenesis, and
increased insulin sensitivity when on the HF diet
Thus, we show, for the first time, that PGD2 is
involved in the activation of adipogenesis and
regula-tion of insulin sensitivity in vivo Further
characteriza-tion of the role of PGD2 in adipocyte differentiation
and function is an important goal, with possible
thera-peutic implications for the treatment of metabolic
dis-orders, such as diabetes and obesity Moreover, the
TG mouse expressing PGDS is a useful model for the
study of obesity
Experimental procedures
Generation of H-PGDS TG mice
The coding region of human H-PGDS was cloned into the
downstream sites of the chicken b-actin promoter and the
CMV enhancer of the pCAGGS expression vector [43] A
3.6 kb SalI–NotI fragment from pCAGGS containing the
H-PGDS expression cassette was microinjected into
pronuclei of fertilized eggs of FVB mice (Taconic, Hudson,
NY, USA) Transgene-positive founder mice were identified
by Southern blot analysis of genomic DNA isolated from the tail Each founder was further bred with FVB mice, and transgene-positive male and female mice were used and compared with WT littermates Mice were maintained under specific pathogen-free conditions in isolated cages with a 12 h light⁄ 12 h dark photoperiod in a humidity-con-trolled and temperature-conhumidity-con-trolled room (55% at 24C) Water and food were available ad libitum The protocols used for all animal experiments in this study were approved
by the Animal Research Committee of Osaka Bioscience Institute
HF diet
Immediately after delactation, mice were fed a normal chow diet (Oriental Yeast, Tokyo, Japan) or an HF diet contain-ing casein (20%; w⁄ w), a-cornstarch (30.2%), sucrose (10%), lard (25%), corn oil (5%), minerals (3.5%), vita-mins (1%), cellulose powder (5%), and d⁄ l-methionine (0.3%) For 6 weeks after delactation, body weight was monitored every week
CT analysis
After mice were anesthetized with intravenous sodium pentobarbital (Nembutal; 50 mgÆkg)1; Abbott Laboratories, North Chicago, IL, USA), CT analysis was performed with
a micro-CT scanner (LaTheta LCT-100; Aloka, Tokyo, Japan) Data were analyzed using latheta software (Alo-ka) The fat and muscle weights were determined from an image at the level of the umbilicus Subcutaneous fat was defined as the extraperitoneal fat between skin and muscle The intraperitoneal part with the same density as the sub-cutaneous fat layer was defined as visceral fat The visceral and subcutaneous fat weights were determined by auto-matic planimetry All experiments were performed at least three times
Immunohistochemical analysis
Paraffin-embedded sections were treated with 0.3% (v⁄ v) hydrogen peroxide in methanol for 30 min to block endo-genous peroxidase, and then 0.02 m glycine for 10 min Sections were incubated with rabbit polyclonal antibody against human H-PGDS overnight at 4C After washing, the sections were incubated with the biotinylated goat anti-(rabbit IgG) for 30 min (Vector Laboratories, Burlingame,
CA, USA), and this was followed by staining with the avidin–biotin–peroxidase complex system (Vectastain ABC Kit; Vector Laboratories) Immunohistochemical signals were visualized with peroxidase, using 3¢,3¢-diamino-benzidine hydrochloride cromogen (Sigma, St Louis, MO, USA)
Trang 8Measurement of serum levels of leptin, insulin,
triglyceride, and glucose
Blood was collected from the abdominal aorta Triglyceride
and glucose levels were determined by using Triglyceride
Test Wako (Wako Pure Chemical, Osaka, Japan) and
Antsense II (Bayer Medical, Tokyo, Japan), respectively
Plasma leptin and insulin levels were measured by using
ELISA kits (Morinaga Institute of Biological Science,
Yokohama, Japan), according to the manufacturer’s
instructions
RNA analysis
Preparation of total RNA and synthesis of first-strand
cDNAs were performed as described previously [44]
North-ern blot analysis was performed as described previously
[45]
Expression levels of PPARc, aP2 and LPL genes were
quantified by using the LightCycler system (Roche
Diag-nostics, Mannheim, Germany) with LightCycler FastStart
DNA Master SYBR Green I (Roche Diagnostics) and the
following PCR primer sets: 5¢-GGAGATCTCCAGTGA
TATCGACCA-3¢ and 5¢-ACGGCTTCTACGGATCGAA
ACT-3¢ for PPARc, 5¢-AAGACAGCTCCTCCTCGAAGG
TT-3¢ and 5¢-TGACCAAATCCCCATTTACGC-3¢ for aP2,
5¢-ATCCATGGATGGACGGTAACG-3¢ and 5¢-CTGGA
TCCCAATACTTCGACCA-3¢ for LPL, 5¢-TGGGTTGG
CTGCTTGTG-3¢ and 5¢-GCGTGGGCAGGATGAAG-3¢
for SCD, 5¢-GATGTGGAACCCATAACTGGATTCAC-3¢
and 5¢-GGTCCCAGTCTCATTTAGCCACAGTA-3¢ for
CD36, 5¢-GCGTCGGGTAGATCCAGTT-3¢ and 5¢-CTC
AGTGGGGCTTAGCTCTG-3¢ for ACC, and 5¢-AACAC
CCCAGCCATGTACGTAG-3¢ and 5¢-TGTCAAAGAAA
GGGTGTAAAACGC-3¢ for b-actin Expression levels of
the target genes were normalized to that of b-actin
Insulin sensitivity test
Mice were fed a normal or HF diet for 12 weeks after
delactation Basal blood was collected from the tail vein
(t = 0 min) and immediately measured for glucose, using
an Antsense II Porcine insulin was injected subcutaneously,
and blood was collected at 30, 60, 90 and 120 min after
injection
Measurement of PGDS activity and PGD2content
PGDS activity was measured as described previously
[16,46] The PGs in tissues were extracted with ethyl
ace-tate, which was evaporated under nitrogen, and the samples
were then separated by HPLC (Gilson, Middleton, WI,
USA), as described previously [47] The amounts of PGD2
in tissues were determined by using the PGD2-MOX EIA
Kit (Cayman Chemical, Ann Arbor, MI, USA), as described previously [16,46]
Preparation of primary cultured adipose cells and induction of adipogenic differentiation
Primary culture of adipose cells was performed as described previously [48], from epididymal adipose tissues collected from six WT and six TG mice (8–10 weeks of age) Cells were seeded on six-well tissue culture plates (type I colla-gen-precoated; AGC Techno Glass, Chiba, Japan) at a den-sity of 2· 105
cells per well, and incubated in the growth medium at 37C under a humidified atmosphere of 95% air and 5% CO2 After confluence had been reached, the growth medium was replaced with the differentiation med-ium containing insulin (10 lgÆmL)1; Sigma), 1 lm DEX (Sigma) and 0.5 mm IBMX (Sigma) for 2 days as described previously [2] The cells were then cultured in the growth medium containing insulin (5 lgÆmL)1) and 200 lm ascor-bic acid for 7 days Lipid accumulation was observed by microscopy with Oil-Red O staining [2] Triglyceride con-tents in the cells were measured by the Wako triglyceride test, according to the manufacturer’s instruction
Statistics
The data are presented as means ± standard errors of the mean (SEMs), and were statistically analyzed by means of the unpaired t-test or the Welch t-test when variances were heterogeneous P-values < 0.05 considered to be significant
Acknowledgements
We acknowledge Y Urade (Osaka Bioscience Institute, Osaka, Japan) for valuable discussions This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K Fuji-mori and K Aritake), and Research for Promoting Technological Seeds from Japan Science and Technol-ogy Agency, the Suzuken Memorial Foundation, the Sumitomo Foundation, the Gushinkai Foundation (to K Fujimori), and the Takeda Science Foundation (to K Fujimori and Y Fujitani)
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