Tài liệu Báo cáo khoa học: Pronounced adipogenesis and increased insulin sensitivity caused by overproduction of prostaglandin D2 in vivo pptx

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Pronounced adipogenesis and increased insulin sensitivitycaused by overproduction of prostaglandin D2in vivoYasushi Fujitani1,*, Kosuke Aritake1, Yoshihide Kanaoka1,2, Tsuyoshi Goto3, Nobuyuki Takahashi3,Ko Fujimori1,4and Teruo Kawada31 Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Japan2 Department of Medicine, Harvard Medical School, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital,Boston, MA, USA3 Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,Japan4 Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, JapanIntroductionThe 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 isaltered in various physiological or pathological condi-tions [1]. The increased adipose tissue mass associatedwith obesity results from an increase in the numberKeywordsadipocytes; H-PGDS; obesity; PGD2;transgenic mouseCorrespondenceK. Fujimori, Laboratory of Biodefense andRegulation, Osaka University ofPharmaceutical Sciences, 4-20-1 Nasahara,Takatsuki, Osaka 569-1094, JapanFax: +81 726 690 1055Tel: +81 726 690 1055E-mail: fujimori@gly.oups.ac.jp*Present addressPharmaceutical Research Division, TakedaPharmaceutical Co. Ltd., Osaka, Japan(Received 28 October 2009, revised 22December 2009, accepted 4 January2010)doi:10.1111/j.1742-4658.2010.07565.xLipocalin-type prostaglandin (PG) D synthase is expressed in adiposetissues and involved in the regulation of glucose tolerance and atherosclero-sis in type 2 diabetes. However, the physiological roles of PGD2in adipo-genesis in vivo are not clear, as lipocalin-type prostaglandin D synthase canalso 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 PGD2in adipogenesis.PGD2production in white adipose tissue of H-PGDS TG mice wasincreased approximately seven-fold as compared with that in wild-type(WT) mice. With a high-fat diet, H-PGDS TG mice gained more bodyweight than WT mice. Serum leptin and insulin levels were increased inH-PGDS TG mice, and the triglyceride level was decreased by about 50%as compared with WT mice. Furthermore, in the white adipose tissue ofH-PGDS TG mice, transcription levels of peroxisome proliferator-activatedreceptor c, fatty acid binding protein 4 and lipoprotein lipase wereincreased approximately two-fold to five-fold as compared with those ofWT mice. Finally, H-PGDS TG mice showed clear hypoglycemia afterinsulin clamp. These results indicate that TG mice overexpressing H-PGDSabundantly produced PGD2in adipose tissues, resulting in pronounced adi-pogenesis and increased insulin sensitivity. The present study provides thefirst evidence that PGD2participates in the differentiation of adipocytesand in insulin sensitivity in vivo, and the H-PGDS TG mice could consti-tute a novel model mouse for diabetes studies.Abbreviations15d-PGJ2, 15-deoxy-D12,14prostaglandin J2; ACC, acetyl-CoA carboxylase; aP2, fatty acid-binding protein 4, adipocyte; BAT, brown adiposetissue; 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-CoAdesaturase; SEM, standard error of the mean; TG, transgenic; WAT, white adipose tissue.1410 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBSand size of adipocytes. A major role of adipocytes isto store large amounts of triglycerides during periodsof energy excess and to mobilize these depots duringperiods of nutritional deprivation. The number ofadipocytes is thought to increase as a result of differ-entiation of adipocytes. Moreover, adipocytes arehighly specialized cells that secrete various adipocyto-kines, whose release largely reflects the amounts ofstored triglyceride. Insights in the molecular mecha-nisms underlying adipogenesis may lead to the devel-opment of strategies for reducing the prevalence ofobesity.Adipogenesis is a complex process accompanied byvarious changes in hormone sensitivity and gene expres-sion caused by many stimuli, including lipid mediators.Prostaglandins (PGs) are involved in the regulation ofadipocyte differentiation. In vitro studies have shownthat PGD2enhances adipocyte differentiation [2], butthat PGE2and PGF2asuppress adipogenesis [3–5].PGD synthase (PGDS) consists of two types of pro-tein [6]. One is lipocalin-type PGDS (L-PGDS), and theother is hematopoietic PGDS (H-PGDS). H-PGDS wasoriginally purified from rat spleen as a cytosolic, gluta-thione-requiring enzyme [7,8], responsible for the bio-synthesis of PGD2in antigen-presenting cells [9], mastcells [10,11], megakaryocytes [12,13], and type 2 helperT-lymphocytes [14]. There have been extensive biochem-ical and genetic analyses of H-PGDS [15], and H-PGDSwas crystallized with its specific inhibitor at 1.7 A˚reso-lution by X-ray diffraction analysis [16]. H-PGDS wasshown to be a member of the sigma-class glutathioneS-transferase (GST) family, and is also called GSTS1[17]. On the other hand, L-PGDS has been purifiedfrom rat brain [18], and is expressed in brain, heart, andmale genital organs, as well as in adipocytes and omen-tal adipose tissues [19–22]. The different types of PGDShave 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 abifunctional protein, having enzymatic activity withregard to both PGD2production and transportation oflipophilic 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 inhibitedadipocyte 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 theaorta after receiving a high-fat (HF) diet [29]. Adipo-cytes of the L-PGDS knockout mice were significantlylarger than those of wild-type (WT) mice [29]. Anotherrecent study demonstrated that L-PGDS knockout micedid 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-PGDSin adipogenesis and diabetes-related phenotypes is notclear. Moreover, because of the dual functions ofL-PGDS, whether PGD2regulates the differentiation ofadipocytes in vivo remains to be elucidated.In the present study, we have generated transgenic(TG) mice, which produce abundant PGD2by overex-pression of human H-PGDS, and used them to investi-gate the physiological significance of PGD2inadipogenesis in vivo. The H-PGDS TG mice showedobesity, pronounced adipogenesis, and increased insu-lin sensitivity when on the HF diet.ResultsGeneration of H-PGDS TG miceHuman H-PGDS cDNA under the regulatory controlof the chicken b-actin promoter and cytomegalovirus(CMV) enhancer (Fig. 1A) was microinjected into thenuclei of fertilized eggs from FVB mice. We establishedthree lines of H-PGDS TG mice, termed S41, S55, andS66. Northern blot analysis for estimation of mRNAexpression of the transgene revealed higher expressionin S41 and S55 mice and lower expression in S66 mice inthe liver, white adipose tissue (WAT), and brown adi-pose tissue (BAT), although H-PGDS was not expressedin each tissue of WT mice (Fig. 1B). The expression ofhuman H-PGDS in hepatocytes and adipocytes of theH-PGDS TG mice (S55) was confirmed by immunohis-tochemistry, using a specific antibody against humanH-PGDS (Fig. 1C). Liver homogenates from WT andTG mice were used for PGDS activity assays. As shownin Fig. 1D, the tissue homogenates of TG mice showedhigher levels of PGD2production than those of WTmice (approximately 18-fold, 25-fold and five-fold inS41, S55 and S66 mice, respectively). These results indi-cate that the H-PGDS TG mice overexpress humanH-PGDS transcripts, proteins and activities in varioustissues. In further experiments, we decided to use S41and S55 mice as TG mice, because these mice showedmore abundant mRNA expression and enzymaticactivity of human H-PGDS.HF diet studyIn order to examine the effects of PGDS overexpres-sion on adipogenesis, WT and TG mice were fed anormal or HF diet for 6 weeks after delactation(Fig. 2A). TG mice showed normal growth and noY. Fujitani et al. Roles of prostaglandin D2in adipogenesis in vivoFEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1411significant differences in spontaneous locomotor activ-ity, rectal temperature and amount of food intakeunder either normal or HF diet conditions in compari-son with WT mice (data not shown). The body weightsof WT and TG mice were almost the same at the startof this experiment (21.2 ± 0.3 g, 20.5 ± 0.7 g and20.2 ± 0.5 g for WT, S41 and S55 mice, respectively).The body weights of both WT and TG mice increasedin a similar manner under normal diet conditions(Fig. 2B). In contrast, under HF diet conditions, thebody weights of both S41 and S55 mice increasedmore, with statistically significant differences from WTmice (Fig. 2B). Next, we measured tissue weights ofthe liver, WATs (epididymal and perirenal fat) andBAT under HF diet condition. WAT weights of TGmice were significantly increased, by 20–30%, as com-pared with those of WT mice. The BAT mass of TGmice was larger than that of WT mice. On the otherhand, liver weights showed no difference between WTand 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 adiposetissue mass under HF diet conditions.Body distribution of adipose tissues asdetermined by computed tomography (C T) analysisTo further assess the effect of H-PGDS overexpressionon the increase in adipose tissues, the weights ofsubcutaneous and visceral adipose tissues, as well as ofmuscle, of WT and TG (S55) mice were analyzed with amicro-CT scanner under HF diet conditions. Visceraland subcutaneous adipose tissue weights of TG micewere significantly increased after 1 week of the HF dietin comparison with those of WT mice (Fig. 2D). Theweights of visceral and subcutaneous adipose tissues ofTG mice were approximately 1.5-fold and 1.4-fold,respectively, of those of WT mice after 6 weeks of theHF diet (Fig. 2D). In contrast, the weight of musclewith organ, but without fats, showed no significant dif-ference between WT and TG mice (Fig. 2D). Theseresults 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 ofTG miceWe measured the amounts of PGD2in WAT after6 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 theeffects of the increased PGD2level on peroxisome pro-liferator-activated receptor (PPAR) c activation, weperformed quantitative RT-PCR to measure themRNA expression levels of adipogenic genes, includingseveral PPARc-target genes, the transcription ofwhich is enhanced in adipogenesis [31,32]. The expres-sion levels of PPARc, fatty acid-binding protein 4,Human H-PGDS cDNAChicken β-actin enhancerLiver WAT BATβ-globinPolyASalINotIWTS41S55S66WTS41S55S66WTS41S55S66510WTWTLiver WAT05PGDS activity(nmol·min–1·mg–1 protein) S55S55WTS41S55S66promoterIntronCMV promoterABCDFig. 1. Generation of human H-PGDS TGmice. (A) Schematic representation ofhuman H-PGDS transfer vector. TheSalI–NotI fragment was microinjected intofertilized eggs of FVB mice. (B) Northernblot analysis of transgene expression in theliver, WAT, and BAT. Ten micrograms oftotal RNA was subjected to agarose gelelectrophoresis, blotted onto a nylon mem-brane, and hybridized with the32P-labeledfull-length cDNA for human H-PGDS. (C)Immunohistochemical analysis of transgeneexpression in the liver and WAT. Paraffinsections of liver and WAT from WT and TGmice (S55) were stained with antibodyagainst human H-PGDS. Bars: 100 lm.(D) PGDS activity in liver of WT and S41,S55 and S66 TG mice.Roles of prostaglandin D2in adipogenesis in vivo Y. Fujitani et al.1412 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBSadipocyte (aP2), lipoprotein lipase (LPL), stearoyl-CoA desaturase (SCD), CD36 and acetyl-CoA carbox-ylase (ACC) in WAT of TG mice were significantlyupregulated by approximately 2.5-fold, three-fold, five-fold, 8.6-fold, 1.2-fold and 22-fold, respectively, incomparison with those in WT mice (Fig. 3B). Theseresults indicate that mRNA expression of PPARc tar-get genes is increased in WAT of TG mice, suggestingthat PPARc might be activated more in WAT of TGmice than in WAT of WT mice.Serum levels of triglyceride, glucose, leptin andinsulin, and insulin sensitivity, in TG miceAfter 6 weeks of normal or HF diet, serum levels oftriglyceride, glucose, leptin and insulin were deter-mined (Fig. 4A). Under both dietary conditions,triglyceride levels in TG (S55) mice were lower thanthose in WT mice by about 50%, whereas glucoselevels 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 HFdiet, respectively, in comparison with WT mice. Fur-thermore, insulin levels in TG mice were alsoincreased as compared with those in WT mice byapproximately 2.6-fold and two-fold after the normaland HF diet, respectively. We next examined poten-tial alterations of insulin sensitivity in TG mice. TGmice fed the HF diet for 12 weeks showed clearhypoglycemia after insulin loading as compared withWT mice (Fig. 4B). The same results were obtainedin TG mice fed a normal diet. These results clearlyWT TG WT TGIncreased body weight (g)*********11·5**WT (n = 18)S41 (n = 8)S55 (n = 8)**Normal diet HF dietNormal diet HF diet101510156420 6420Duration (week)*Duration (week)00·5Tissue weight (g)LiverBATEpididymalfatPerirenalfat***0505Increased weight (g)Visceral fat0123456************012345Duration (week)**0123456**********Subcutaneous fat0123450123456Muscle (with organs)012345ABCDFig. 2. Body weight increase in mice whenon the normal and HF diets. (A) After delac-tation, WT and H-PGDS TG (S55) mice werefed either the normal or the HF diet for6 weeks. A representative male mousefrom each group is shown. (B) Body weightwas monitored every week for 6 weeks.Closed circles (n = 61), squares (n = 22) andtriangles (n = 36) indicate WT, S41 and S55mice, respectively. Values are expressed asmeans ± SEMs. *P < 0.05, **P < 0.01 ascompared with WT mice. (C) Tissue weightsof epididymal and perirenal fat, BAT, and liver.Values are expressed as means ± SEMs.*P < 0.05, **P < 0.01 as compared withWT mice. (D) Changes in the weights ofvisceral and subcutaneous fat and musclewith organ, but without fat, of WT andH-PGDS TG mice (n = 6). Continuousdissections of mouse fat and bone in thewhole body were quantified by use of amicro-CT scanner andLATHETA software(Aloka). Open and closed circles correspondto WT and H-PGDS TG mice, respectively.**P < 0.01 as compared with WT mice.Y. Fujitani et al. Roles of prostaglandin D2in adipogenesis in vivoFEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1413Relative mRNA level(/β-actin mRNA level)Relative mRNA level(/β-actin mRNA level)γ01020300*****aP2 LPLWT TG WT TG WT TGSCDCD36 ACC0123*PGD2 (ng·g–1 tissue)WT TG44080.5****00WT TG WT TG WT TG10203024600.**ABFig. 3. PGD2production and expression ofadipogenic genes. (A) Predominant produc-tion of PGD2in TG mice. PGD2levels inWAT of WT and TG mice after the HF dietwere measured by enzyme immunoassay.(B) Transcription levels of adipogenic genes(encoding PPARc, aP2, LPL, SCD, CD36,and ACC) in WAT. After being fed the HFdiet for 6 weeks, mice were killed, and totalRNA was isolated from WAT. Expressionlevels of the target genes were normalizedto those of the b-actin mRNA level as aninternal control, and calculated as fold inten-sity. Values are expressed as means ±SEMs (n = 4–6). *P < 0.05, **P < 0.01 ascompared with WT mice.Glucose level (% of change)Time after injection (min)0501001500 30 60 90 1200501001500306090120Insulin (0.75 U kg–1) Insulin (3.0 U kg–1)***InsulinTriglyceride Glucose Leptin102030405004080120Concentration (mg·dL–1)0408012000.51.01.50*********WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TGNormal HFNormal HFNormal HFNormal HFABFig. 4. Serum markers and insulin sensitiv-ity test. (A) After being fed a normal or HFdiet for 6 weeks, mice were killed and bloodwas collected. Values are expressed as themeans ± 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 UÆ kg)1and3.0 UÆkg)1of insulin after being fed a nor-mal or HF diet, respectively. The y-axis indi-cates the percentage change in bloodglucose level as compared with the valuebefore injection (100% at t = 0). Values areexpressed as the means ± SEMs (n = 6–7).*P < 0.05 as compared with WT mice.Roles of prostaglandin D2in adipogenesis in vivo Y. Fujitani et al.1414 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBSindicate that overexpression of H-PGDS increasesinsulin sensitivity in vivo.Adipocyte differentiation ex vivoFinally, we examined whether the overexpression of H-PGDS also promotes ex vivo differentiation of adipo-cytes. Preadipocytes prepared from WATs of WT orTG (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 afterinduction of differentiation, the differentiated adipo-cytes prepared from WAT of TG mice accumulatedapparently greater amounts of lipid droplets than thoseof 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 TGmouse-derived adipocytes was increased by approxi-mately two-fold as compared with WT mouse-derivedcells (Fig. 5C). Therefore, these results suggest that theoverproduction of PGD2promotes adipocyte differen-tiation, thereby regulating adipogenesis.DiscussionIn this study, we generated H-PGDS TG mice over-producing PGD2, and showed that PGD2acts as anactivator in adipogenesis in vivo. We used H-PGDSTG mice to elucidate the functions of PGD2in adipo-genesis in vivo, because L-PGDS is a bifunctional pro-tein, both producing PGD2and acting as a carrierprotein for small lipophilic molecules [23], even thoughL-PGDS, but not H-PGDS, was detected in adipocytes[2,19]. Investigations using L-PGDS knockout micehave demonstrated that L-PGDS is involved in theregulation of glucose tolerance and atherosclerosis intype 2 diabetes [29,33], and showed induction of obes-ity [30]. However, it is not known which functions ofL-PGDS are associated with these phenotypes.15-Deoxy-D12,14PGJ2(15d-PGJ2), which is one ofthe metabolites of PGD2, has been identified as aligand for PPARc that can activate the differentiationof adipose cells [34,35]. However, the concentrations of15d-PGJ2used 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-Parikhet al. [36] demonstrated that 15d-PGJ2was present at alow level that is insufficient for activation of adipocytedifferentiation. Thus, the contribution of 15d-PGJ2toin vivo adipogenesis remains to be clarified.H-PGDS TG mice gained more body weight thanWT mice when on the HF diet (Fig. 2A,B,D), and theWAT weight of TG mice was larger than that of WTmice (Fig. 2C); this was accompanied by upregulationof the expression of adipogenic genes in WAT(Fig. 3B), suggesting pronounced differentiation ofadipocytes and subsequent obesity in H-PGDS TGmice. Furthermore, we observed a drastic increase inPGD2levels in WAT of H-PGDS TG mice (Fig. 3A),whereas PGE2and PGF2alevels were not significantlyaltered in WAT in TG mice as compared with those inWT mice (data not shown); these results are consistentwith the previous result showing that, even if PGD2production was decreased, the biosynthesis of otherPGs 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 andincrease body weight, but act as antidiabetic drugs toimprove insulin sensitivity [37]. Indeed, the overexpres-sion of H-PGDS improved insulin resistance (TG miceshowed clear hypoglycemia in response to insulinclamp, as shown in Fig. 4B). Thus, PGD2and ⁄ orPGD2metabolites might be involved in the regulationof adipogenesis through PPARc in vivo. Further stud-ies to investigate the precise mechanism, including theTriglyceride (mg·well–1)LPL mRNA level(/β-actin mRNA level)WT TG300.3**WT TG0102000.10.2WT TGABCFig. 5. Adipocyte differentiation ex vivo. (A) Primary cultured adipo-cytes from WAT of WT and H-PGDS TG mice were cultured in thepresence of DEX, IBMX and insulin for 7 days, and stained for lipiddroplet 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-actinas a control, and calculated as fold intensity. Values are expressedas the means ± SEMs (n = 4–6). *P < 0.05 as compared with WTmice.Y. Fujitani et al. Roles of prostaglandin D2in adipogenesis in vivoFEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1415changes oin uptake of fatty acids and the number ofadipocytes, 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, TGmice showed higher insulin concentrations in blood,whereas the basal glucose level was not different fromthat 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 ispossible that PGD2stimulates pancreatic islets toincrease insulin secretion. Indeed, serum insulin levelswere increased after treatment with thiazolidinedionesin diabetic mice through regulation of insulin produc-tion in pancreatic islet cells [39–41]. Thus, theincreased insulin level seen in H-PGDS TG mice whenon the HF diet might be due to effects of PGD2onpancreatic islet cells. The precise mechanism needs tobe elucidated in further investigations that includeanalysis of pancreatic islet cells.The H-PGDS TG mouse is a novel obesity modelwith which to investigate the mechanism of adipogene-sis. As is the case for obese people with overnutritionand energy imbalance, as is common in advancedcountries, H-PGDS TG mice become obese after theHF diet but not after the normal diet. This phenotypeis distinct from that seen in the well-known obesitymodel mice, such as db ⁄ db and ob ⁄ ob mice, which aredeficient in the leptin receptor and leptin genes, respec-tively [42].In summary, H-PGDS TG mice produced substan-tial amounts of PGD2as compared with WT mice,and showed obesity, pronounced adipogenesis, andincreased insulin sensitivity when on the HF diet.Thus, we show, for the first time, that PGD2isinvolved in the activation of adipogenesis and regula-tion of insulin sensitivity in vivo. Further characteriza-tion of the role of PGD2in adipocyte differentiationand function is an important goal, with possible thera-peutic implications for the treatment of metabolic dis-orders, such as diabetes and obesity. Moreover, theTG mouse expressing PGDS is a useful model for thestudy of obesity.Experimental proceduresGeneration of H-PGDS TG miceThe coding region of human H-PGDS was cloned into thedownstream sites of the chicken b-actin promoter and theCMV enhancer of the pCAGGS expression vector [43]. A3.6 kb SalI–NotI fragment from pCAGGS containing theH-PGDS expression cassette was microinjected intopronuclei of fertilized eggs of FVB mice (Taconic, Hudson,NY, USA). Transgene-positive founder mice were identifiedby Southern blot analysis of genomic DNA isolated fromthe tail. Each founder was further bred with FVB mice,and transgene-positive male and female mice were used andcompared with WT littermates. Mice were maintainedunder specific pathogen-free conditions in isolated cageswith a 12 h light ⁄ 12 h dark photoperiod in a humidity-con-trolled and temperature-controlled room (55% at 24 °C).Water and food were available ad libitum. The protocolsused for all animal experiments in this study were approvedby the Animal Research Committee of Osaka BioscienceInstitute.HF dietImmediately after delactation, mice were fed a normal chowdiet (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 wasmonitored every week.CT analysisAfter mice were anesthetized with intravenous sodiumpentobarbital (Nembutal; 50 mg Ækg)1; Abbott Laboratories,North Chicago, IL, USA), CT analysis was performed witha 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 animage at the level of the umbilicus. Subcutaneous fat wasdefined 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 visceraland subcutaneous fat weights were determined by auto-matic planimetry. All experiments were performed at leastthree times.Immunohistochemical analysisParaffin-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 antibodyagainst human H-PGDS overnight at 4 °C. 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 theavidin–biotin–peroxidase complex system (Vectastain ABCKit; Vector Laboratories). Immunohistochemical signalswere visualized with peroxidase, using 3¢,3¢-diamino-benzidine hydrochloride cromogen (Sigma, St Louis, MO,USA).Roles of prostaglandin D2in adipogenesis in vivo Y. Fujitani et al.1416 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBSMeasurement of serum levels of leptin, insulin,triglyceride, and glucoseBlood was collected from the abdominal aorta. Triglycerideand glucose levels were determined by using TriglycerideTest Wako (Wako Pure Chemical, Osaka, Japan) andAntsense II (Bayer Medical, Tokyo, Japan), respectively.Plasma leptin and insulin levels were measured by usingELISA kits (Morinaga Institute of Biological Science,Yokohama, Japan), according to the manufacturer’sinstructions.RNA analysisPreparation of total RNA and synthesis of first-strandcDNAs were performed as described previously [44]. North-ern blot analysis was performed as described previously[45].Expression levels of PPAR c , aP2 and LPL genes werequantified by using the LightCycler system (Roche Diag-nostics, Mannheim, Germany) with LightCycler FastStartDNA Master SYBR Green I (Roche Diagnostics) and thefollowing PCR primer sets: 5¢-GGAGATCTCCAGTGATATCGACCA-3¢ and 5¢-ACGGCTTCTACGGATCGAAACT-3¢ for PPARc ,5¢-AAGACAGCTCCTCCTCGAAGGTT-3¢ and 5¢-TGACCAAATCCCCATTTACGC-3¢ for aP2,5¢-ATCCATGGATGGACGGTAACG-3¢ and 5¢-CTGGATCCCAATACTTCGACCA-3¢ for LPL, 5¢-TGGGTTGGCTGCTTGTG-3¢ and 5¢-GCGTGGGCAGGATGAAG-3¢for SCD, 5¢-GATGTGGAACCCATAACTGGATTCAC-3¢and 5¢-GGTCCCAGTCTCATTTAGCCACAGTA-3¢ forCD36, 5¢-GCGTCGGGTAGATCCAGTT-3¢ and 5¢-CTCAGTGGGGCTTAGCTCTG-3¢ for ACC, and 5¢-AACACCCCAGCCATGTACGTAG-3¢ and 5¢-TGTCAAAGAAAGGGTGTAAAACGC-3¢ for b-actin. Expression levels ofthe target genes were normalized to that of b-actin.Insulin sensitivity testMice were fed a normal or HF diet for 12 weeks afterdelactation. Basal blood was collected from the tail vein(t = 0 min) and immediately measured for glucose, usingan Antsense II. Porcine insulin was injected subcutaneously,and blood was collected at 30, 60, 90 and 120 min afterinjection.Measurement of PGDS activity and PGD2contentPGDS 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 sampleswere then separated by HPLC (Gilson, Middleton, WI,USA), as described previously [47]. The amounts of PGD2in tissues were determined by using the PGD2-MOX EIAKit (Cayman Chemical, Ann Arbor, MI, USA), asdescribed previously [16,46].Preparation of primary cultured adipose cells andinduction of adipogenic differentiationPrimary culture of adipose cells was performed as describedpreviously [48], from epididymal adipose tissues collectedfrom six WT and six TG mice (8–10 weeks of age). Cellswere seeded on six-well tissue culture plates (type I colla-gen-precoated; AGC Techno Glass, Chiba, Japan) at a den-sity of 2 · 105cells per well, and incubated in the growthmedium at 37 °C under a humidified atmosphere of 95%air and 5% CO2. After confluence had been reached, thegrowth medium was replaced with the differentiation med-ium containing insulin (10 lgÆ mL)1; Sigma), 1 lm DEX(Sigma) and 0.5 mm IBMX (Sigma) for 2 days as describedpreviously [2]. The cells were then cultured in the growthmedium containing insulin (5 lgÆmL)1) and 200 lm ascor-bic acid for 7 days. Lipid accumulation was observed bymicroscopy with Oil-Red O staining [2]. Triglyceride con-tents in the cells were measured by the Wako triglyceridetest, according to the manufacturer’s instruction.StatisticsThe data are presented as means ± standard errors of themean (SEMs), and were statistically analyzed by means ofthe unpaired t-test or the Welch t-test when variances wereheterogeneous. 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Pronounced adipogenesis and increased insulin sensitivity caused by overproduction of prostaglandin D2 in vivo Yasushi Fujitani1,*,. leptin and insulin, and insulin sensitivity, in TG miceAfter 6 weeks of normal or HF diet, serum levels of triglyceride, glucose, leptin and insulin were
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