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

Pronounced adipogenesis and increased insulin sensitivity caused by overproduction of prostaglandin D 2 in vivo Yasushi Fujitani 1, *, Kosuke Aritake 1 , Yoshihide Kanaoka 1,2 , Tsuyoshi Goto 3 , Nobuyuki Takahashi 3 , Ko Fujimori 1,4 and Teruo Kawada 3 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; PGD 2 ; 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 PGD 2 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 PGD 2 in adipogenesis. PGD 2 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 PGD 2 in adipose tissues, resulting in pronounced adi- pogenesis and increased insulin sensitivity. The present study provides the first evidence that PGD 2 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-D 12,14 prostaglandin 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. 1410 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS and 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 PGD 2 enhances adipocyte differentiation [2], but that PGE 2 and PGF 2a suppress 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 PGD 2 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 PGD 2 production and transportation of lipophilic molecules, such as retinoids [25], biliverdin, bilirubin [26], gangliosides [27], and amyloid b-peptides [28], with high affinities (K d = 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 PGD 2 regulates the differentiation of adipocytes in vivo remains to be elucidated. In the present study, we have generated transgenic (TG) mice, which produce abundant PGD 2 by overex- pression of human H-PGDS, and used them to investi- gate the physiological significance of PGD 2 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 PGD 2 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 Y. Fujitani et al. Roles of prostaglandin D 2 in adipogenesis in vivo FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1411 significant 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 (C T) 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 PGD 2 in WAT after 6 weeks of the HF diet. WAT of TG (S55) mice con- tained significantly more PGD 2 (approximately seven- fold) than that of WT mice (Fig. 3A). To examine the effects of the increased PGD 2 level 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 SalI NotI WT S41 S55 S66 WT S41 S55 S66 WT S41 S55 S66 5 10 WT WT Liver WAT 0 5 PGDS activity (nmol·min –1 ·mg –1 protein) S55 S55 WT S41 S55 S66 promoter IntronCMV promoter A B CD 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. Roles of prostaglandin D 2 in adipogenesis in vivo Y. Fujitani et al. 1412 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS adipocyte (aP2), lipoprotein lipase (LPL), stearoyl- CoA desaturase (SCD), CD36 and acetyl-CoA carbox- ylase (ACC) in WAT of TG mice were significantly upregulated by approximately 2.5-fold, three-fold, five- fold, 8.6-fold, 1.2-fold and 22-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 WT TG WT TG Increased body weight (g) ** ** * * ** * 1 1·5 ** WT (n = 18) S41 (n = 8) S55 (n = 8) * * Normal diet HF diet Normal diet HF diet 10 15 10 15 6420 6420 Duration (week) * Duration (week) 0 0·5 Tissue weight (g) Liver BAT Epididymal fat Perirenal fat * * * 0 5 0 5 Increased weight (g) Visceral fat 0123456 ** ** ** ** ** ** 0 1 2 3 4 5 Duration ( week ) ** 0123456 ** ** ** ** ** Subcutaneous fat 0 1 2 3 4 5 0123456 Muscle (with organs) 0 1 2 3 4 5 A B C 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. Y. Fujitani et al. Roles of prostaglandin D 2 in adipogenesis in vivo FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1413 Relative mRNA level (/β-actin mRNA level) Relative mRNA level (/β-actin mRNA level) 0 0.2 0.4 0.4 0.8 PPARγ 0 10 20 30 0 ** ** * aP2 LPL WT TG WT TG WT TG SCD CD36 ACC 0 1 2 3 * PGD 2 (ng·g –1 tissue) WT TG 4 40 8 0.5 ** ** 0 0 WT TG WT TG WT TG 10 20 30 2 4 6 0 0.2 0.3 0.4 0.1 ** AB Fig. 3. PGD 2 production 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. Glucose level (% of change) Time after in j ection ( min ) 0 50 100 150 0 30 60 90 120 0 50 100 150 0306090120 Insulin (0.75 U kg –1 ) Insulin (3.0 U kg –1 ) * * * InsulinTriglyceride Glucose Leptin 10 20 30 40 50 0 40 80 120 Concentration (mg·dL –1 ) 0 40 80 120 0 0.5 1.0 1.5 0 * ** * * ** ** WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TG Normal HFNormal HFNormal HFNormal HF A B Fig. 4. Serum markers and insulin 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 UÆ kg )1 and 3.0 UÆkg )1 of 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. Roles of prostaglandin D 2 in adipogenesis in vivo Y. Fujitani et al. 1414 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS indicate 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 PGD 2 promotes adipocyte differen- tiation, thereby regulating adipogenesis. Discussion In this study, we generated H-PGDS TG mice over- producing PGD 2 , and showed that PGD 2 acts as an activator in adipogenesis in vivo. We used H-PGDS TG mice to elucidate the functions of PGD 2 in adipo- genesis in vivo, because L-PGDS is a bifunctional pro- tein, both producing PGD 2 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-D 12,14 PGJ 2 (15d-PGJ 2 ), which is one of the metabolites of PGD 2 , has been identified as a ligand for PPARc that can activate the differentiation of adipose cells [34,35]. However, the concentrations of 15d-PGJ 2 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-PGJ 2 was present at a low level that is insufficient for activation of adipocyte differentiation. Thus, the contribution of 15d-PGJ 2 to in vivo adipogenesis 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 PGD 2 levels in WAT of H-PGDS TG mice (Fig. 3A), whereas PGE 2 and PGF 2a 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 PGD 2 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, PGD 2 and ⁄ or PGD 2 metabolites might be involved in the regulation of adipogenesis through PPARc in vivo. Further stud- ies to investigate the precise mechanism, including the Triglyceride (mg·well –1 ) LPL mRNA level (/β-actin mRNA level) WT TG 30 0.3 * * WT TG 0 10 20 0 0.1 0.2 WT TG A BC 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. Y. Fujitani et al. Roles of prostaglandin D 2 in adipogenesis in vivo FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1415 changes 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 PGD 2 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 PGD 2 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 PGD 2 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 PGD 2 is involved in the activation of adipogenesis and regula- tion of insulin sensitivity in vivo. Further characteriza- tion of the role of PGD 2 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-controlled room (55% at 24 °C). 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 mg Ækg )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 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 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). Roles of prostaglandin D 2 in adipogenesis in vivo Y. Fujitani et al. 1416 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS Measurement 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 PPAR c , 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 PGD 2 content 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 PGD 2 in tissues were determined by using the PGD 2 -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 · 10 5 cells per well, and incubated in the growth medium at 37 °C under a humidified atmosphere of 95% air and 5% CO 2 . After confluence had been reached, the growth 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 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). References 1 Spiegelman BM & Flier JS (2001) Obesity and the regu- lation of energy balance. Cell 104, 531–543. 2 Fujimori K, Aritake K & Urade Y (2007) A novel path- way to enhance adipocyte differentiation of 3T3-L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol Y. Fujitani et al. Roles of prostaglandin D 2 in adipogenesis in vivo FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1417 regulatory element-binding protein-1c. J Biol Chem 282, 18458–18466. 3 Casimir DA, Miller CW & Ntambi JM (1996) Prea- dipocyte differentiation blocked by prostaglandin stimu- lation of prostanoid FP2 receptor in murine 3T3-L1 cells. Differentiation 60, 203–210. 4 Miller CW, Casimir DA & Ntambi JM (1996) The mecha- nism of inhibition of 3T3-L1 preadipocyte differentiation by prostaglandin F2alpha. Endocrinology 137, 5641– 5650. 5 Tsuboi H, Sugimoto Y, Kainoh T & Ichikawa A (2004) Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation. Biochem Biophys Res Commun 322, 1066–1072. 6 Urade Y & Hayaishi O (2000) Prostaglandin D syn- thase: structure and function. Vitam Horm 58, 89–120. 7 Christ-Hazelhof E & Nugteren DH (1979) Purification and characterisation of prostaglandin endoperoxide D- isomerase, a cytoplasmic, glutathione-requiring enzyme. Biochim Biophys Acta 572, 43–51. 8 Urade Y, Fujimoto N, Ujihara M & Hayaishi O (1987) Biochemical and immunological characterization of rat spleen prostaglandin D synthetase. J Biol Chem 262, 3820–3825. 9 Murakami M, Matsumoto R, Urade Y, Austen KF & Arm JP (1995) c-kit ligand mediates increased expres- sion of cytosolic phospholipase A2, prostaglandin endo- peroxide synthase-1, and hematopoietic prostaglandin D2 synthase and increased IgE-dependent prostaglandin D2 generation in immature mouse mast cells. J Biol Chem 270, 3239–3246. 10 Urade Y, Ujihara M, Horiguchi Y, Igarashi M, Nagata A, Ikai K & Hayaishi O (1990) Mast cells contain spleen-type prostaglandin D synthetase. J Biol Chem 265, 371–375. 11 Urade Y, Ujihara M, Horiguchi Y, Ikai K & Hayaishi O (1989) The major source of endogenous prostaglan- din D2 production is likely antigen-presenting cells. Localization of glutathione-requiring prostaglandin D synthetase in histiocytes, dendritic, and Kupffer cells in various rat tissues. J Immunol 143, 2982–2989. 12 Fujimori K, Kanaoka Y, Sakaguchi Y & Urade Y (2000) Transcriptional activation of the human hemato- poietic prostaglandin D synthase gene in megakaryo- blastic cells. Roles of the Oct-1 element in the 5¢- flanking region and the AP-2 element in the untrans- lated exon 1. J Biol Chem 275, 40511–40516. 13 Mahmud I, Ueda N, Yamaguchi H, Yamashita R, Yamamoto S, Kanaoka Y, Urade Y & Hayaishi O (1997) Prostaglandin D synthase in human mega- karyoblastic cells. J Biol Chem 272, 28263–28266. 14 Tanaka K, Ogawa K, Sugamura K, Nakamura M, Takano S & Nagata K (2000) Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets. J Immunol 164, 2277–2280. 15 Kanaoka Y & Urade Y (2003) Hematopoietic prosta- glandin D synthase. Prostaglandins Leukot Essent Fatty Acids 69, 163–167. 16 Aritake K, Kado Y, Inoue T, Miyano M & Urade Y (2006) Structural and functional characterization of HQL-79, an orally selective inhibitor of human hemato- poietic prostaglandin D synthase. J Biol Chem 281, 15277–15286. 17 Hayes JD, Flanagan JU & Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45, 51–88. 18 Urade Y, Fujimoto N & Hayaishi O (1985) Purification and characterization of rat brain prostaglandin D synthetase. J Biol Chem 260, 12410–12415. 19 Jowsey IR, Murdock PR, Moore GB, Murphy GJ, Smith SA & Hayes JD (2003) Prostaglandin D2 syn- thase enzymes and PPARgamma are co-expressed in mouse 3T3-L1 adipocytes and human tissues. Prosta- glandins Other Lipid Mediat 70, 267–284. 20 Quinkler M, Bujalska IJ, Tomlinson JW, Smith DM & Stewart PM (2006) Depot-specific prostaglandin synthe- sis in human adipose tissue: a novel possible mechanism of adipogenesis. Gene 380, 137–143. 21 Urade Y & Hayaishi O (2000) Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase. Biochim Biophys Acta 1482, 259–271. 22 Xie Y, Kang X, Ackerman WE, Belury MA, Koster C, Rovin BH, Landon MB & Kniss DA (2006) Differenti- ation-dependent regulation of the cyclooxygenase cascade during adipogenesis suggests a complex role for prostaglandins. Diabetes Obes Metab 8, 83–93. 23 Urade Y & Aritake K (2007) Multifunctional Properties of Lipocalin-type Prostaglandin D Synthase. Research Signpost, Kerala. 24 Urade Y & Eguchi N (2002) Lipocalin-type and hema- topoietic prostaglandin D synthases as a novel example of functional convergence. Prostaglandins Other Lipid Mediat 68–69, 375–382. 25 Tanaka T, Urade Y, Kimura H, Eguchi N, Nishikawa A & Hayaishi O (1997) Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of reti- noid transporter. J Biol Chem 272, 15789–15795. 26 Beuckmann CT, Aoyagi M, Okazaki I, Hiroike T, Toh H, Hayaishi O & Urade Y (1999) Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type pros- taglandin D synthase. Biochemistry 38 , 8006–8013. 27 Mohri I, Taniike M, Okazaki I, Kagitani-Shimono K, Aritake K, Kanekiyo T, Yagi T, Takikita S, Kim HS, Urade Y et al. (2006) Lipocalin-type prostaglandin D synthase is up-regulated in oligodendrocytes in lyso- somal storage diseases and binds gangliosides. J Neuro- chem 97, 641–651. 28 Kanekiyo T, Ban T, Aritake K, Huang ZL, Qu WM, Okazaki I, Mohri I, Murayama S, Ozono K, Taniike M et al. (2007) Lipocalin-type prostaglandin D Roles of prostaglandin D 2 in adipogenesis in vivo Y. Fujitani et al. 1418 FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS synthase ⁄ beta-trace is a major amyloid beta-chaperone in human cerebrospinal fluid. Proc Natl Acad Sci USA 104, 6412–6417. 29 Ragolia L, Palaia T, Hall CE, Maesaka JK, Eguchi N & Urade Y (2005) Accelerated glucose intolerance, nephropathy, and atherosclerosis in prostaglandin D2 synthase knock-out mice. J Biol Chem 280, 29946– 29955. 30 Tanaka R, Miwa Y, Mou K, Tomikawa M, Eguchi N, Urade Y, Takahashi-Yanaga F, Morimoto S, Wake N & Sasaguri T (2009) Knockout of the l-pgds gene aggra- vates obesity and atherosclerosis in mice. Biochem Biophys Res Commun 378, 851–856. 31 Kang S, Bajnok L, Longo KA, Petersen RK, Hansen JB, Kristiansen K & MacDougald OA (2005) Effects of Wnt signaling on brown adipocyte differentiation and metabolism mediated by PGC-1alpha. Mol Cell Biol 25, 1272–1282. 32 Rosen ED (2005) The transcriptional basis of adipocyte development. Prostaglandins Leukot Essent Fatty Acids 73, 31–34. 33 Ragolia L, Hall CE & Palaia T (2008) Lipocalin-type prostaglandin D(2) synthase stimulates glucose trans- port via enhanced GLUT4 translocation. Prostaglandins Other Lipid Mediat 87, 34–41. 34 Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM & Evans RM (1995) 15-Deoxy- delta12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 83, 803–812. 35 Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC & Lehmann JM (1995) A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 83, 813–819. 36 Bell-Parikh LC, Ide T, Lawson JA, McNamara P, Reilly M & FitzGerald GA (2003) Biosynthesis of 15- deoxy-delta12,14-PGJ2 and the ligation of PPAR- gamma. J Clin Invest 112, 945–955. 37 Yamauchi T, Kamon J, Waki H, Murakami K, Motojima K, Komeda K, Ide T, Kubota N, Terauchi Y, Tobe K et al. (2001) The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance. J Biol Chem 276, 41245–41254. 38 Jowsey IR, Thomson AM, Flanagan JU, Murdock PR, Moore GB, Meyer DJ, Murphy GJ, Smith SA & Hayes JD (2001) Mammalian class Sigma glutathione S-trans- ferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases. Biochem J 359, 507–516. 39 Evans-Molina C, Robbins RD, Kono T, Tersey SA, Vestermark GL, Nunemaker CS, Garmey JC, Deering TG, Keller SR, Maier B et al. (2009) Peroxisome prolif- erator-activated receptor gamma activation restores islet function in diabetic mice through reduction of endo- plasmic reticulum stress and maintenance of euchroma- tin structure. Mol Cell Biol 29, 2053–2067. 40 Wajchenberg BL (2007) Beta-cell failure in diabetes and preservation by clinical treatment. Endocr Rev 28, 187– 218. 41 Yildirim S, Bolkent S & Sundler F (2008) The role of rosiglitazone treatment in the modulation of islet hor- mones and hormone-like peptides: a combined in situ hybridization and immunohistochemical study. J Mol Histol 39, 635–642. 42 Speakman J, Hambly C, Mitchell S & Krol E (2008) The contribution of animal models to the study of obesity. Lab Anim 42, 413–432. 43 Niwa H, Yamamura K & Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199. 44 Fujimori K, Kadoyama K & Urade Y (2005) Protein kinase C activates human lipocalin-type prostaglandin D synthase gene expression through de-repression of Notch-Hes signaling and enhancement of AP-2 beta function in brain-derived TE671 cells. J Biol Chem 280, 18452–18461. 45 Kanaoka Y, Fujimori K, Kikuno R, Sakaguchi Y, Urade Y & Hayaishi O (2000) Structure and chromo- somal localization of human and mouse genes for hematopoietic prostaglandin D synthase. Conservation of the ancestral genomic structure of sigma-class gluta- thione S-transferase. Eur J Biochem 267, 3315–3322. 46 Fujitani Y, Kanaoka Y, Aritake K, Uodome N, Okazaki-Hatake K & Urade Y (2002) Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase trans- genic mice. J Immunol 168, 443–449. 47 Pinzar E, Kanaoka Y, Inui T, Eguchi N, Urade Y & Hayaishi O (2000) Prostaglandin D synthase gene is involved in the regulation of non-rapid eye movement sleep. Proc Natl Acad Sci USA 97, 4903–4907. 48 Ohyama M, Matsuda K, Torii S, Matsui T, Yano H, Kawada T & Ishihara T (1998) The interaction between vitamin A and thiazolidinedione on bovine adipocyte differentiation in primary culture. J Anim Sci 76, 61–65. Y. Fujitani et al. Roles of prostaglandin D 2 in adipogenesis in vivo FEBS Journal 277 (2010) 1410–1419 ª 2010 The Authors Journal compilation ª 2010 FEBS 1419 . Pronounced adipogenesis and increased insulin sensitivity caused by overproduction of prostaglandin D 2 in vivo Yasushi Fujitani 1, *,. 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