• Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads

Views

Total Views
282
On Slideshare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
5
Comments
0
Likes
0

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide

Transcript

  • 1. Molecular and Cellular Endocrinology 287 (2008) 13–19 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Role of Fgf receptor 2c in adipocyte hypertrophy in mesenteric white adipose tissue Morichika Konishi a,1 , Hirotoshi Nakamura a,1 , Hiroyuki Miwa a , Pierre Chambon b , David M. Ornitz c , Nobuyuki Itoh a,∗ a Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Yoshida-Shimoadachi, Sakyo, Kyoto 606-8501, Japan b Institut de G´n´tique et de Biologie Mol´culaire et Cellulaire and Institut Clinique de la Souris, B.P. 10142, 67404 Illkirch, Strasbourg, France e e e c Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO 63110, USA a r t i c l e i n f o a b s t r a c t Article history: Fgf receptor 2c (Fgfr2c) was expressed in mature adipocytes of mouse white adipose tissue (WAT). To Received 16 May 2007 examine the role of Fgfr2c in mature adipocytes, we generated adipocyte-specific Fgfr2 knockout (Fgfr2 Received in revised form 19 December 2007 CKO) mice. The hypertrophy impairment of adipocytes in the mesenteric WAT but not in the subcutaneous Accepted 14 February 2008 WAT and decreased plasma free fatty acid (FFA) levels were observed in Fgfr2 CKO mice. Although the expression of genes involved in adipocyte differentiation and lipid metabolism in the mesenteric WAT was Keywords: essentially unchanged, the expression of uncoupling protein 2 potentially involved in energy dissipation Fgf was significantly increased. Among potential Fgf ligands for Fgfr2c, Fgf9 was preferentially expressed in Fgf receptor White adipose tissue the mesenteric WAT. The present findings indicate that Fgfr2c potentially activated by Fgf9 plays a role in Adipocyte the adipocyte hypertrophy in the mesenteric WAT and FFA metabolism and/or energy dissipation in the Hypertrophy mesenteric WAT might be involved in the hypertrophy impairment. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction proteins, adipokines. Adipokines are involved in numerous phy- siological functions including food intake, energy metabolism and White adipose tissue plays crucial roles in energy homeostasis insulin sensitivity (Ailhaud, 2006). The hypertrophy of mature adi- (Spiegelman and Flier, 1996). Obesity, the excessive development pocytes is associated with dysregulated expression of adipokines, of white adipose tissue, is a risk factor for several diseases inclu- which causes metabolic disorders (Skurk et al., 2007). Therefore, it ding type II diabetes, hypertension, cancer and atherosclerosis is important to elucidate the molecular mechanism underlying the (Kopelman, 2000). Therefore, it is important to understand the regulation of the hypertrophy. molecular and cellular mechanisms by which white adipose tis- Fibroblast growth factors (Fgfs), secreted signaling proteins, play sue develops. The development of white adipose tissue involves important roles in development and metabolism with multiple the proliferation of preadipocytes and their subsequent differentia- biological activities including cell proliferation and differentiation. tion into mature adipocytes, adipogenesis (Rosen and Spiegelman, The Fgf family comprises 22 members (Ornitz and Itoh, 2001; Itoh 2000). Adipogenesis is one of the most intensively studied deve- and Ornitz, 2004). Fgf10−/− mice die shortly after birth (Sekine et lopmental processes. Several transcription factors are involved al., 1999). The embryonic development of white adipose tissue in in adipogenesis. They include members of the CCAAT/enhancer these mice is greatly impaired (Sakaue et al., 2002). Analyses of binding protein (C/EBP) and peroxisome proliferator-activated Fgf10−/− white adipose tissue have indicated that Fgf10 plays cru- receptor (PPAR) families (Rosen, 2005). The hypertrophy of mature cial roles in the proliferation of preadipocytes and their subsequent adipocytes is also an important process for the development of differentiation into mature adipocytes at embryonic stages (Asaki white adipose tissue at postnatal stages. The hypertrophy cau- et al., 2004; Konishi et al., 2006). An Fgf10 receptor is Fgf recep- sed by lipid accumulation results in an increase in the size of tor 2b (Fgfr2b) that is preferentially expressed in preadipocytes mature adipocytes. Mature adipocytes secrete a variety of signaling (Yamasaki et al., 1999). These results indicate that Fgf10 plays roles in the embryonic development of white adipose tissue through the activation of Fgfr2b. There are two alternatively spliced variants of Fgfr2, Fgfr2b and Fgfr2c (Dell and Williams, 1992). Fgfr2c is a major ∗ Corresponding author. Tel.: +81 75 753 4540; fax: +81 75 753 4600. variant. The two differ in their specificity for ligands and pattern E-mail address: itohnobu@pharm.kyoto-u.ac.jp (N. Itoh). 1 These authors contributed equally to this work. of expression (Ornitz et al., 1996; Zhang et al., 2006; Orr-Urtreger 0303-7207/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2008.02.010
  • 2. 14 M. Konishi et al. / Molecular and Cellular Endocrinology 287 (2008) 13–19 et al., 1993). In contrast to Fgfr2b, Fgfr2c is not a receptor for Fgf10 2.4. Detection of Fgfr2 recombination in mice (Zhang et al., 2006). We examined the expression of Fgfr2c in mouse The recombination of Fgfr2 was detected by PCR analysis of genomic DNA puri- white adipose tissue. Fgfr2c was preferentially expressed in mature fied from indicated tissues or cells from control and Fgfr2 CKO mice. Primers P1 adipocytes, indicating that Fgfr2c might play roles in mature adipo- (5 -ATAGGAGCAACAGGCGG-3 ) and P3 (5 -CATAGCACAGGCCAGGTTG-3 ) produced cytes. However, as Fgfr2−/− mice died in the early embryonic stages, a 471 bp fragment indicative of Cre-mediated Fgfr2 recombination (Fig. 2A). E10–E11 (Xu et al., 1998), the role of Fgf2c in mature adipocytes The efficiency of Fgfr2 recombination was assessed by PCR analysis of genomic remains unclear. To examine the role of Fgfr2c in mature adipocytes, DNA of mature adipocytes isolated from control and Fgfr2 CKO mice. Pri- mers P1 (5 -ATAGGAGCAACAGGCGG-3 ) and P2 (5 -TGCAAGAGGCGACCAGTCAG-3 ) we generated adipocyte-specific Fgfr2−/− mice by disrupting Fgfr2 produced a 207 bp fragment of the floxed region of Fgfr2 without recom- selectively in mature adipocytes in postnatal stages. Here, we report bination (Fig. 2A). Primers P4 (5 -GTGGCTCACAACCATCCGTAATG-3 ) and P5 that Fgfr2c plays a role in the hypertrophy of mature adipocytes in (5 -CACTCCTGGCAAGAGCTCAATTT-3 ) produced a 350 bp fragment indicative of the the mesenteric white adipose tissue at postnatal stages. reference (Fig. 2A). The PCR products within the linear range of amplification were separated by electrophoresis on a 1.5% agarose gel, visualized by ethidium bromide 2. Materials and methods staining and quantified with NIH Image software. After correction for the reference levels, the efficiency of Fgfr2 recombination was assessed relative to control litter- 2.1. Expression of genes in adipocytes and stromal-vascular cells examined by mates. reverse transcription-polymerase chain reaction 2.5. Histological analysis Mature adipocytes and stromal-vascular cells were prepared from mouse sub- cutaneous and mesenteric white adipose tissues essentially according to the method Mesenteric and subcutaneous white adipose tissues of 18-week-old control and of Ogawa et al. (1995). Total RNA was extracted from the cells using an RNeasy mini Fgfr2 CKO mice were fixed in Bouin’s fixative, dehydrated, embedded in paraffin and kit (Qiagen). cDNA was synthesized from the RNA as a template in a reaction mix- sectioned. Sections (6 m) were stained with hematoxylin and eosin and examined ture containing moloney murine leukemia virus reverse transcriptase (Gibco BRL) by light microscopy. Images of adipose tissue sections were captured and adipocyte and a random hexadeoxynucleotide primer (Takara, Japan). The cDNA was amplified sizes were measured for at least 150 cells per mice with NIH Image software. by polymerase chain reaction (PCR) with Taq DNA polymerase (Wako, Japan) and primers specific for indicated genes. The nucleotide sequences of primers used are 2.6. Expression of genes in mesenteric white adipose tissue examined by shown in Table 1. semi-quantitative reverse transcription-polymerase chain reaction 2.2. Mutant mice Total RNA was prepared from mesenteric white adipose tissue of 18-week-old control and Fgfr2 CKO mice using an RNeasy mini kit (Qiagen). cDNA was synthe- All mice were maintained in a mixed strain background and housed in a sized as described above. Portions of the reaction mixture were subjected to PCR temperature-controlled environment with 12-h light/dark cycles. The generation (within the linear range of amplification) with Taq DNA polymerase (Wako, Japan) of Fgfr2flox/flox mice and aP2-Cre-ERT2(tg/0) mice has been described previously (Yu et and primers specific for mouse Leptin, peroxisome proliferators-activated receptor al., 2003; Imai et al., 2001). Fgfr2flox/flox mice were mated with aP2-Cre-ERT2(tg/0) mice gamma 2 (PPAR 2), uncoupling protein 2 (UCP2), glucose transporter 1 (GLUT1), glu- to obtain aP2-Cre-ERT2(tg/0) /Fgfr2flox/+ mice. Then the aP2-Cre-ERT2(tg/0) /Fgfr2flox/+ mice cose transporter 4 (GLUT4), lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), were mated with Fgfr2flox/flox mice to obtain aP2-Cre-ERT2(tg/0) /Fgfr2flox/flox mice and fatty acid synthase (FAS), acetyl-CoA carboylase 1 (ACC1) and 18s rRNA. The nucleotide aP2-Cre-ERT2(0/0) /Fgfr2flox/flox littermates. Male aP2-Cre-ERT2(tg/0) /Fgfr2flox/flox mice and sequences of primers used are shown in Table 1. The PCR products were separated male aP2-Cre-ERT2(0/0) /Fgfr2flox/flox littermates were injected intraperitoneally with by electrophoresis on a 1.5–2% agarose gel, visualized by ethidium bromide stai- tamoxifen (Sigma) at a dosage of 0.1 mg/(g day) for 5 consecutive days biweekly ning, and quantified with NIH Image software. After correction for 18S rRNA levels, from 8 to 18 weeks of age. Tamoxifen was dissolved in autoclaved corn oil (Sigma) the abundance of each specific mRNA was expressed as the fold change relative to at a concentration of 25 mg/ml by sonication for 45 min. All mice were individually control littermates. Values are the mean ± S.E.M. obtained from more than four pairs housed and weighed biweekly from 8 to 18 weeks of age. of littermates. 2.3. Genotyping of mice 2.7. Blood parameter analyses and rectal temperature measurement flox/flox For genotyping the Fgfr2 mice, PCR was performed with genomic Blood samples were obtained from 18-week-old control and Fgfr2 CKO mice. DNA using primers, sense (5 -ATAGGAGCAACAGGCGG-3 ) and antisense (5 - Blood glucose was measured by a blood glucose test meter (Glutest R, Sanwa-Kagaku, TGCAAGAGGCGACCAGTCAG-3 ), yielding 207 and 142 bp fragments as described (Yu Japan). Plasma concentrations of triglycerides, cholesterol, and free fatty acids (FFAs) et al., 2003). For genotyping the aP2-cre-ERT2(tg/0) mice, PCR was performed with were measured using triglyceride E-Test, cholesterol E-test and NEFA C-Test kits genomic DNA using primers, sense (5 -ATGTCCAATTTACTGACCG-3 ) and antisense (WAKO, Japan), respectively. The rectal temperature of 17-week-old control and Fgfr2 (5 -CGCCGCATAACCAGTGAAAC-3 ), yielding a 350 bp fragment. CKO mice was measured by a digital thermometer, KN-91 (Natsume, Japan). Table 1 PCR primer sequences used Gene Sense primer Antisense primer Fgfr2 5 -CGCCCACAATGAGGTGGTTA-3 5 -TCACCACCATGCAGGCGATT-3 Fgfr2b 5 -GGGATAAATAGCTCCAATGC-3 5 -TCACAGGCGCTTGCTGTTTG-3 Fgfr2c 5 -GGTGTTAACACCACGGACAA-3 5 -CTCACAGGCGCTGGCAGAACT-3 Leptin 5 -TGCTCCAGCAGCTGCAAGGTGCAAG-3 5 -TCAGCATTCAGGGCTAACATCCAACTGTT-3 UCP2 5 -GCATTGCAGATCTCATCACT-3 5 -CCTTGGTGTAGAACTGTTTG-3 PPAR 2 5 -CCCAGAGCATGGTGCCTT-3 5 -GGCATCTCTGTGTCAACCATGGT-3 Glut1 5 -ATGGATCCCAGCAGCAAGAA-3 5 -GACTTGCCCAGTTTGGAGAA-3 Glut4 5 -AAAACAAGATGCCGTCGGGT-3 5 -CCTGATGTTAGCCCTGAGTA-3 LPL 5 -GGAGTTTGGCTCCAGAGTTT-3 5 -TAGCCAGCTGACACTGGATA-3 HSL 5 -CAGGGCAAAGAAGGATCGAA-3 5 -GTGTGCCACACCCAACAGTT-3 FAS 5 -CCATGGAGGAGGTGGTGATA-3 5 -CGTCTCGGGATCTCTGCTAA-3 ACC1 5 -CACATGAGATCCAGCATGTC-3 5 -TTCTGGGAGTTTCGGGTTCT-3 Fgf4 5 -CCCTATTTGCTCTCGCTACT-3 5 -CTCGTCGGTAAAGAAAGGCA Fgf5 5 -AGAATGAGCCTGTCCTTGCT-3 5 -CTTGGAATCTCTCCCTGAAC-3 Fgf6 5 -GAGGCTGTTCATCACTATGT-3 5 -CCGTTCTACCGTGGAGATCT-3 Fgf8 5 -CCCAACAGGTAACTGTTCAG-3 5 -GGCAATTAGCTTCCCCTTCT-3 Fgf9 5 -GTCCTCTGATGGCTCCCTTA-3 5 -AGACACTGTCTTTGTCAGCTT-3 Fgf17 5 -AAGAAGTCTCTCCAGCGATG-3 5 -TGGCCTCCCTGACTACGTTT-3 Fgf18 5 -TGCCTGTGTGTTTACACTTTCTA-3 5 -TGGTGAAGCCCACATACCAA-3 Fgf20 5 -CCATGGCTCCCTTGACCGAA-3 5 -GGCTCTAGATTCATCAAGTG-3 18S 5 -CTTAGAGGGACAAGTGCA-3 5 -ACGCTGAGCCAGTCAGTGTA-3
  • 3. M. Konishi et al. / Molecular and Cellular Endocrinology 287 (2008) 13–19 15 Fig. 1. Expression of Fgfr2 in white adipose tissue of adult mice. The expression of Fgfr2, Fgfr2b and Fgfr2c in mature adipocytes (mature) and stromal-vascular cells (SV) of subcutaneous (Sub) and mesenteric (Mes) white adipose tissues was deter- mined by RT-PCR using specific primers followed by agarose gel electrophoresis and staining with ethidium bromide. The expression of Leptin and 18S rRNA (18S) was also determined as controls. 2.8. Statistical analysis Data are expressed as mean ± S.E.M. The statistical significance of differences in mean values was assessed with the Student’s t-test. 3. Results Fig. 2. Tamoxifen-induced Fgfr2 gene disruption in adipocytes of adult mice. (A) Schematic representation of the Fgfr2-flox allele (Fgfr2flox ) and the Fgfr2 allele 3.1. Expression of Fgfr2 in white adipose tissue obtained after Cre-mediated excision. Exons encoding the ligand-binding IgIIIb and IgIIIc domains (IIIb and IIIc) and transmembrane domain (TM) were deleted in the Fgfr2 allele. Black boxes, arrowheads and arrows indicate exons, loxP sites and White adipose tissue consists of mature adipocytes and PCR primers, respectively. (B) The generation of tamoxifen-induced Fgfr2 alleles stromal-vascular cells. Stromal-vascular cells mainly consist of was determined by PCR of DNA from the indicated tissues of 18-week-old Fgfr2 preadipocytes (Ailhaud et al., 1992). To address the role of Fgfr2 CKO mice using the primers P1 and P2 followed by agarose gel electrophoresis (left in mouse white adipose tissue at postnatal stages, we examined panel). The generation of tamoxifen-induced Fgfr2 alleles in mature adipocytes the expression of Fgfr2 in mature adipocytes and stromal-vascular (mature) and stromal-vascular cells (SV) of white adipose tissue was also determi- ned (right panel). The gel was stained with ethidium bromide. The PCR products cells of adult mouse inguinal subcutaneous and mesenteric white obtained from genomic DNA using P4 and P5 were also shown as a reference. (C) adipose tissues by reverse transcription-polymerase chain reac- The efficiency of Fgfr2 recombination was assessed by PCR analysis of genomic DNA tion (RT-PCR) (Fig. 1). We also examined the expression of Leptin, of mature adipocytes isolated from subcutaneous (Sub) and mesenteric (Mes) white which was preferentially expressed in mature adipocytes (Ogawa adipose tissues of control and Fgfr2 CKO mice. Fgfr2 alleles were detected by PCR of DNA from the mature adipocytes of 18-week-old control and Fgfr2 CKO mice using et al., 1995), in the white adipose tissues as a control. As expec- the primers P1 and P2. The PCR products obtained using P1 and P3 indicated the ted, Leptin was preferentially expressed in mature adipocytes of Fgfr2-flox alleles, which were not recombined. The products obtained using P4 and the white adipose tissues. Fgfr2 was more abundantly expressed in P5 were also shown as a reference. The products were separated by electropho- mature adipocytes than in stromal-vascular cells of subcutaneous resis on a 1.5% agarose gel and visualized by ethidium bromide staining. The PCR and mesenteric white adipose tissues. Fgfr2 includes two alter- products indicative of the Fgfr2-flox alleles and the reference were quantified with NIH Image software. After correction for the reference levels, the efficiency of Fgfr2 natively spliced variants, Fgfr2b and Fgfr2c, with different ligand recombination was assessed relative to control littermates. specificities (Ornitz et al., 1996; Zhang et al., 2006). We also exa- mined the expression of Fgfr2b and Fgfr2c in mature adipocytes and stromal-vascular cells by RT-PCR. Fgfr2b was preferentially expressed in stromal-vascular cells of both white adipose tissues. In age. We termed them Fgfr2 CKO mice, in which Fgfr2 should be contrast, Fgfr2c was preferentially expressed in mature adipocytes specifically disrupted in mature adipocytes. We also generated aP2- of both white adipose tissues. These results indicate a potential role Cre-ERT2(0/0) /Fgfr2flox/flox mice injected with tamoxifen that were for Fgfr2c in mature adipocytes of white adipose tissue. termed control mice, in which Fgfr2 should not be disrupted. Fgfr2 alleles converted from Fgfr2-flox alleles were detected 3.2. Conditional disruption of Fgfr2 in mature adipocytes at in the white adipose tissue and brown adipose tissue of Fgfr2 CKO postnatal stages mice, whereas no Cre-mediated recombination was observed in the brain, heart, liver, kidney and intestine (Fig. 2B). In the white Mature adipocytes mainly develop and function in the postna- adipose tissue of Fgfr2 CKO mice, Fgfr2 alleles were detected in tal period (Greenwood and Hirsch, 1974). However, Fgfr2−/− mice mature adipocytes but not stromal-vascular cells (Fig. 2B). Most (at died in the early embryonic stages, E10–E11 (Xu et al., 1998). To least more than 50%) of Fgfr2 was ablated from mature adipocytes address the role of Fgfr2c in mature adipocytes, we used the condi- in both the subcutaneous and mesenteric white adipose tissues tional gene silencing approach to disrupt Fgfr2 in mature adipocytes of Fgfr2 CKO mice (Fig. 2C). These results indicate that Fgfr2 was at postnatal stages. We generated aP2-Cre-ERT2(tg/0) /Fgfr2flox/flox specifically ablated in the mature adipocytes in Fgfr2 CKO mice. In mice. The 8-week-old mice were injected intraperitoneally with contrast, no Fgfr2 was ablated from mature adipocytes of control tamoxifen for 5 consecutive days biweekly from 8 to 18 weeks of mice.
  • 4. 16 M. Konishi et al. / Molecular and Cellular Endocrinology 287 (2008) 13–19 indistinguishable (data not shown). We also examined the weights of the heart, liver and interscapular brown adipose tissue, inguinal subcutaneous white adipose tissue and mesenteric white adipose tissue at 18 weeks of age (Fig. 3B). The weights of the heart, liver and brown adipose tissue of Fgfr2 CKO mice were indistinguishable from those of control mice. In addition, the subcutaneous white adipose tissue of Fgfr2 CKO mice was only slightly lighter than that of control mice. In contrast, the mesenteric white adipose tissue of Fgfr2 CKO mice was greatly reduced in weight in comparison with that of control mice (Fig. 3B). 3.4. Analysis of mesenteric white adipose tissue of Fgfr2 CKO mice To examine whether the marked decrease in the weight of the mesenteric white adipose tissue of Fgfr2 CKO mice was caused by impairment of the hypertrophy and/or cell numbers of mature adipocytes, we analyzed the mesenteric white adipose tissue. We also analyzed the subcutaneous white adipose tissue as a control. The average cell area of mature adipocytes in the subcutaneous white adipose tissue of Fgfr2 CKO mice was similar to that of control mice (Fig. 4A). In contrast, the average cell area of mature adipocytes in the mesenteric white adipose tissue of Fgfr2 CKO mice was significantly smaller than that of control mice (Fig. 4A). We also estimated the average cell volume of mature adipocytes from the average cell area. The cell numbers of mature adipo- cytes were also estimated from the white adipose tissue weight and the average cell volume (Fig. 4B). The average cell volume of mature adipocytes of Fgfr2 CKO mice was greatly reduced. In contrast, the cell numbers of mature adipocytes were essentially unchanged. These results indicate that the hypertrophy of mature adipocytes was greatly impaired in the mesenteric white adipose tissue of Fgfr2 CKO mice. Leptin mRNA levels in the white adi- pose tissue were positively associated with the hypertrophy of mature adipocytes (Skurk et al., 2007). We also examined the expression of Leptin in the mesenteric white adipose tissue by semi- quantitative RT-PCR. A great decrease in the Leptin mRNA levels was observed in the mesenteric white adipose tissue of Fgfr2 CKO mice (Fig. 4C). This result also supported that the hypertrophy of mature adipocytes was impaired in the mesenteric white adipose tissue of Fgfr2 CKO mice. 3.5. Expression of genes involved in adipocyte differentiation, energy consumption, glucose transport and lipid metabolism in mesenteric white adipose tissue of Fgfr2 CKO mice To address the role of Fgfr2c in the hypertrophy of mature adi- pocytes, we examined the expression of genes involved in cell differentiation, energy consumption, glucose transport and lipid metabolism in mature adipocytes (Fig. 5). PPAR 2 is a transcrip- tion factor of the peroxisome proliferator-activated receptor family. PPAR 2 plays crucial roles in the hypertrophy of mature adipo- Fig. 3. Body weights and tissue weights of control and Fgfr2 CKO mice. (A) Body cytes as well as the differentiation of adipocytes (Kubota et al., weight gain of control and Fgfr2 CKO mice from 8 to 18 weeks of age. All mice were 1999). UCP2 is expected to enhance energy dissipation in white weighed biweekly from 8 to 18 weeks of age. Results are the mean ± S.E.M. obtained adipose tissue (Surwit et al., 1998). We examined the expression from more than 11 mice per group. (B) The heart, liver, interscapular brown adipose of PPAR 2 and UCP2 in the mesenteric white adipose tissue. The tissue (iBAT), inguinal subcutaneous white adipose tissue (subWAT) and mesente- ric white adipose tissue (mesWAT) weights of 18-week-old control and Fgfr2 CKO expression of PPAR 2 was essentially unchanged. In contrast, the mice. Results are the mean ± S.E.M. obtained from more than eight mice per group, expression of UCP2 was significantly increased in the mesenteric *p < 0.001. white adipose tissue of Fgfr2 CKO mice. Glut1 and Glut4 play roles in glucose accumulation in the white adipose tissue (Moyers et al., 3.3. Analysis of white adipose tissue of Fgfr2 CKO mice 2007; Ishiki and Klip, 2005). LPL is involved in lipid accumulation in the white adipose tissue (Preiss-Landl et al., 2002). The expression We examined the body weights of Fgfr2 CKO and control mice of Glut1, Glut4 and LPL was essentially unchanged in the mesente- from 8 to 18 weeks of age. The body weights of Fgfr2 CKO mice were ric white adipose tissue. HSL is involved in lipolysis in the white essentially indistinguishable from those of control mice at all stages adipose tissue (Langin et al., 1996). The expression of HSL was also examined (Fig. 3A). Their food consumption was also essentially essentially unchanged. FAS and ACC1 are directly involved in fatty
  • 5. M. Konishi et al. / Molecular and Cellular Endocrinology 287 (2008) 13–19 17 Fig. 5. Expression of genes involved in adipocyte differentiation, energy consump- tion, glucose transport and lipid metabolism in mesenteric white adipose tissue of Fgfr2 CKO mice. The expression of the indicated genes was analyzed by RT-PCR using total RNA extracted from the mesenteric white adipose tissue of 18-week-old control and Fgfr2 CKO mice. The PCR products were separated by electrophoresis on a 1.5–2% agarose gel, visualized by ethidium bromide staining, and quantified with NIH Image software. The expression of the indicated genes was determined after correction for the expression of 18S rRNA as a control. Results are the mean ± S.E.M. obtained from more than four pairs of littermates. Table 2 Blood parameters and rectal temperature in control and Fgfr2 CKO mice Control Fgfr2 CKO Glucose (mg/dl) 127 ± 9 106 ± 5 Total cholesterol (mg/dl) 55.0 ± 4.0 55.5 ± 6.8 Triglycerides (mg/dl) 84.0 ± 14.3 91.6 ± 12.7 Free fatty acids (mequiv./l) 0.71 ± 0.12 0.49 ± 0.05 Rectal temperature (◦ C) 37.6 ± 0.5 37.3 ± 0.5 Results are the mean ± S.E.M. obtained from five pairs of littermates. acid synthesis (Wakil et al., 1983). We also examined the expres- sion of ACC1 and FAS in the mesenteric white adipose tissue. The expression of ACC1 was slightly decreased in the mesenteric white adipose tissue of Fgfr2 CKO mice. In contrast, the expression of FAS was rather increased in the mesenteric white adipose tissue of Fgfr2 CKO mice. 3.6. Blood parameters and rectal temperature in Fgfr2 CKO mice As the hypertrophy of mature adipocytes in the Fgfr2 CKO mesenteric white adipose tissue was impaired, we also examined blood glucose and plasma triglyceride, cholesterol, and FFA levels of Fgfr2 CKO mice. We found blood glucose and plasma triglyceride and cholesterol levels essentially unchanged (Table 2). In contrast, decreased plasma FFA levels of Fgfr2 CKO mice were observed. We also examined the rectal temperature of Fgfr2 CKO mice, which was essentially unchanged (Table 2). Fig. 4. Histological analysis of mesenteric white adipose tissue of Fgfr2 CKO mice. (A) Paraffin sections of the mesenteric white adipose tissue (mesWAT) and mice. Mature adipocyte cell numbers were also estimated from the white adipose subcutaneous white adipose tissue (subWAT) of 18-week-old control and Fgfr2 CKO tissue weight and the mature adipose cell volume. Results are the mean ± S.E.M. mice were stained with hematoxylin and eosin. Scale bar=100 m. The average obtained from more than three mice per group, *p = 0.06. (C) The expression of Lep- adipocyte cell areas in the mesenteric and subcutaneous white adipose tissue of tin in the mesenteric white adipose tissue was determined by RT-PCR followed by control and Fgfr2 CKO mice were measured using at least 150 cells per mouse with agarose gel electrophoresis, and staining with ethidium bromide, and quantified NIH Image software. Results are the mean ± S.E.M. obtained from more than three with NIH Image software. The expression of Leptin was determined after correction mice per group, *p < 0.05. (B) Mature adipose cell volumes were estimated from for the expression of 18S rRNA as a control. Results are the mean ± S.E.M. obtained each adipocyte cell area in mesenteric white adipose tissue of contorol and Fgfr2 CKO from four pairs of littermates, **p < 0.001.
  • 6. 18 M. Konishi et al. / Molecular and Cellular Endocrinology 287 (2008) 13–19 using adipocyte-specific Fgfr2 knockout mice, Fgfr2 CKO mice, at postnatal stages. The mesenteric white adipose tissue of Fgfr2 CKO mice was greatly decreased in weight. The decrease was mainly caused by impairment of the hypertrophy of mature adipocytes and not by a reduction in the cell numbers. In contrast, the hypertrophy of mature adipocytes was not significantly impaired in the subcu- taneous white adipose tissue, indicating that Fgfr2c is selectively essential in the mesenteric white adipose tissue. To understand the role of Fgfr2c in the hypertrophy of adipo- cytes of the mesenteric white adipose tissue, the expression of genes related to adipocyte differentiation, energy consumption, glucose transport and lipid metabolism were determined. These factors potentially play roles in the hypertrophy of mature adipo- cytes. The expression of most of genes was essentially unchanged in mesenteric white adipose tissue of Fgfr2 CKO mice. Howe- ver, the expression of UCP2 was significantly increased in the mesenteric white adipose tissue of Fgfr2 CKO mice. As UCP2 is expected to enhance energy dissipation in the white adipose tis- sue (Surwit et al., 1998), the impairment of the hypertrophy might be caused at least in part by an enhancement of energy dis- sipation mediated by UCP2 in mature adipocytes. In addition, decreased plasma FFA levels of Fgfr2 CKO mice were observed, indicating that FFA release from the mesenteric white adipose tissue of Fgfr2 CKO mice might be decreased. As the expres- sion of the enzymes involved in lipolysis and FFA synthesis was Fig. 6. Expression of Fgfs in white adipose tissue of adult mice. The expression of Fgf4, essentially unchanged in the mesenteric white adipose tissue, the Fgf5, Fgf6, Fgf8, Fgf9, Fgf17, Fgf18 and Fgf20 in the subcutaneous (Sub) and mesen- mechanism of decrease in plasma FFA levels remains to be elucida- teric (Mes) white adipose tissues of adult mice was determined by RT-PCR using ted. specific primers followed by agarose gel electrophoresis and staining with ethidium The expression of Fgfr2c in mature adipocytes of the mesenteric bromide. The expression of 18S rRNA (18S) was also determined as a control. white adipose tissue was only slightly stronger than that of mature adipocytes of the subcutaneous white adipose tissue. In addition, 3.7. Expression of Fgfs in white adipose tissue the efficiency with which Fgfr2 was deleted in both mature adi- pocytes was essentially similar. Therefore, the selective effect of Fgfr2c could be activated by Fgf4, Fgf5, Fgf6, Fgf8, Fgf9, Fgf17, deletion of Fgfr2 on the mesenteric white adipose tissue was not Fgf18 and Fgf20 (Ornitz et al., 1996; Zhang et al., 2006). As Fgfs caused by the selective expression of Fgfr2c or the efficiency of the usually activate Fgfrs in a paracrine manner (Ornitz and Itoh, 2001), deletion in the mesenteric white adipose tissue. Fgfs usually acti- we examined the expression of these Fgfs in the mesenteric and vate their receptors, Fgfrs, in a paracrine manner (Ornitz and Itoh, subcutaneous white adipose tissues by RT-PCR. Their expression 2001). Several Fgfs have been reported to activate Fgfr2c (Ornitz et was also examined in the embryos and brains as a positive control, al., 1996; Zhang et al., 2006). We examined the expression of these and was significantly detected (data not shown). In contrast, the Fgfs in mesenteric and subcutaneous white adipose tissues. Among expression of Fgf4, Fgf5, Fgf6, Fgf8, Fgf17, Fgf18 and Fgf20 was barely them, Fgf9 was preferentially expressed in the stromal-vascular detected in the white adipose tissues (Fig. 6); however, the expres- cells of the mesenteric white adipose tissue but not in those of the sion of Fgf9 was preferentially detected in stromal-vascular cells subcutaneous white adipose tissue. In contrast, other Fgfs examined of the mesenteric white adipose tissue but not the subcutaneous were expressed at much lower levels. These results are essentially white adipose tissue. We also examined the expression of Fgf9 in consistent with previous results that Fgf9 was expressed at higher the mesenteric white adipose tissue of Fgfr2 CKO mice by RT-PCR, levels in the visceral adipose tissue than in the subcutaneous adi- and found it essentially unchanged (data not shown). pose tissue in obese humans (Gabrielsson et al., 2002) and that Fgf9 protein was detected in mesothelial cells of the visceral adi- 4. Discussion pose tissue by immunohistochemistry (Gabrielsson et al., 2002). These findings indicate that Fgf9 might promote the hypertrophy Fgf signaling plays crucial roles in developmental and metabo- of mature adipocytes through Fgfr2c activation in the mesenteric lic processes at both embryonic and postnatal stages (Ornitz and white adipose tissue; however, as Fgf9−/− mice die shortly after Itoh, 2001; Itoh and Ornitz, 2004). In the development of white birth (Colvin et al., 2001), the role of Fgf9 in the hypertrophy adipose tissue at embryonic stages, Fgf10 signaling through Fgfr2b remains unclear. plays crucial roles in the proliferation of preadipocytes and their In conclusion, Fgf signaling through Fgfr2c plays a role in the differentiation into mature adipocytes (Sakaue et al., 2002; Asaki et hypertrophy of mature adipocytes in mesenteric white adipose tis- al., 2004; Konishi et al., 2006). White adipose tissue mainly deve- sue at postnatal stages. This is the first report that Fgf signaling lops at postnatal stages (Greenwood and Hirsch, 1974). Fgfr2c, a potentially plays a role in the hypertrophy of mature adipocytes major variant of Fgfr2, is preferentially expressed in mature adi- in vivo at postnatal stages. The present findings together with our pocytes at postnatal stages, indicating that Fgfr2c might play a previous findings (Asaki et al., 2004; Konishi et al., 2006) indicate role in the white adipose tissue at postnatal stages. However, the that Fgf signaling through Fgfr2b and Fgfr2c plays distinct roles in role of Fgfr2c in white adipose tissue at postnatal stages remains the development of white adipose tissue. Fgf10 signaling through unclear. Fgfr2b plays roles in the proliferation and differentiation of prea- Fgfr2−/− mice died in the early embryonic stages (Xu et al., 1998). dipocytes. In contrast, Fgf signaling through Fgfr2c play a role in Therefore, we examined the role of Fgfr2c in mature adipocytes the hypertrophy of mature adipocytes. Fgf9 is a potential ligand
  • 7. M. Konishi et al. / Molecular and Cellular Endocrinology 287 (2008) 13–19 19 for Fgfr2c in mature adipocytes of mesenteric white adipose tissue. Langin, D., Holm, C., Lafontan, M., 1996. Adipocyte hormone-sensitive lipase: a major Our findings will provide new insight into the molecular mecha- regulator of lipid metabolism. Proc. Nutr. Soc. 55, 93–109. Moyers, J.S., Shinyanova, T.L., Mehrbod, F., Dunbbar, J.D., Noblitt, T.W., Otto, K.A., nism underlying the development of the white adipose tissue at Reifel-Miller, A., Kharitonenkov, A., 2007. Molecular determinants of FGF-21 postnatal stages. activity-synergy and cross-talk with PPAR signaling. J. Cell. Physiol. 210, 1–6. Ogawa, Y., Masuzaki, H., Isse, N., Okazaki, K., Mori, K., Shigemoto, M., Satoh, N., Acknowledgements Tamura, N., Hosoda, K., Yoshimasa, Y., Jingami, H., Kawada, T., Nakao, K., 1995. Molecular cloning of rat Obese cDNA and augmented gene expression in geneti- This work was supported by a grant-in-aid for scientific research cally Obese Zucker Fath (fa/fa) rats. J. Clin. Invest. 96, 1647–1652. Ornitz, D.M., Itoh, N., 2001. Fibroblast growth factors. Genome Biol. 2, from the Ministry of Education, Science, Culture, Sports, and Tech- 3005.1–3005.12. nology, Japan (M.K., N.I.) and by grants from the Takeda Science Ornitz, D.M., Xu, J., Colvin, J.S., McEwen, D.G., MacArthur, C.A., Coulier, F., Gao, G., Foundation (N.I.) and the Mitsubishi Foundation (N.I.). Goldfarb, M., 1996. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271, 15292–15297. Orr-Urtreger, A., Bedford, M.T., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, References D., Lonai, P., 1993. Developmental localization of the splicing alternatives of Fibroblast growth factor receptor-2 (FGFR2). Dev. Biol. 158, 475–486. Ailhaud, G., 2006. Adipose tissue as a secretory organ: from adipogenesis to the Preiss-Landl, K., Zimmermann, R., Hammerle, G., Zechner, R., 2002. Lipoproteinli- metabolic syndrome. C.R. Biol. 329, 570–577. pase: the regulation of tissue specific expression and its role in lipid and energy Ailhaud, G., Grimaldi, P., Negrel, R., 1992. Cellular and molecular aspects of adipose metabolism. Curr. Opin. Lipidol. 13, 471–481. tissue development. Annu. Rev. Nutr. 12, 207–233. Rosen, E.D., 2005. The transcriptional basis of adipocyte development. Prostaglan- Asaki, T., Konishi, M., Miyake, A., Kato, S., Tomizawa, M., Itoh, N., 2004. Roles of dins Leukot. Essent. Fatty Acids 73, 31–34. fibroblast growth factor 10 (Fgf10) in adipogenesis in vivo. Mol. Cell. Endocrinol. Rosen, E.D., Spiegelman, B.M., 2000. Molecular regulation of adipogenesis. Annu. 218, 119–128. Rev. Cell Dev. Biol. 16, 145–171. Colvin, J.S., Green, R.P., Schmahl, J., Capel, B., Ornitz, D.M., 2001. Male-to-female sex Sakaue, H., Konishi, M., Ogawa, W., Asaki, T., Mori, T., Yamasaki, M., Takata, M., Ueno, reversal in mice lacking fibroblast growh factor 9. Cell 104, 875–889. H., Kato, S., Kasuga, M., Itoh, N., 2002. Requirement of fibroblast growth factor Dell, K.R., Williams, L.T., 1992. A novel form of fibroblast growth factor receptor 10 in development of white adipose tissue. Genes Dev. 16, 908–912. 2. Alternative splicing of the third immunoglobulin-like domain confers ligand Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, binding specificity. J. Biol. Chem. 267, 21225–21229. N., Matsui, D., Koga, Y., Itoh, N., Kato, S., 1999. Fgf10 is essential for limb and lung Gabrielsson, B.G., Johansson, J.M., Jennische, E., Jernas, M., Itoh, Y., Peltonen, M., formation. Nat. Genet. 21, 138–141. Olbers, T., Lonn, L., Lonroth, H., Sjostorm, L., Carlsson, B., Carlsson, L.M., Lonn, M., Skurk, T., Alberti-Huber, C., Herder, C., Hauner, H., 2007. Relationship between adi- 2002. Depot-specific expression of fibroblast growth factors in human adipose pocyte size and adipokine expression and secretion. J. Clin. Endocrin. Metab. 92, tissue. Obes. Res. 10, 608–616. 1023–1033. Greenwood, M.R., Hirsch, J., 1974. Postnatal development of adipocyte cellularity in Spiegelman, B.M., Flier, J.S., 1996. Adipogenesis and obesity; rounding out the big the normal rat. J. Lipid Res. 15, 474–483. picture. Cell 87, 377–389. Imai, T., Jiang, M., Chambon, P., Metzger, D., 2001. Impaired adipogenesis and lipolysis Surwit, R.S., Wang, S., Petro, A.E., Sanchis, D., Raimbault, S., Ricquier, D., Collins, in the mouse upon selective ablation of retinoid X receptor alpha mediated by S., 1998. Diet-induced changes in uncoupling proteins in obesity-prone and tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes. Proc. obesity-resistant starins of mice. Proc. Natl. Acad. Sci. U.S.A. 95, 4061–4065. Natl. Acad. Sci. U.S.A. 98, 224–228. Wakil, S.J., Stoops, J.K., Joshi, V.C., 1983. Fatty acid synthesis and its regulation. Annu. Ishiki, M., Klip, A., 2005. Minireview: recent developments in the regulation of glu- Rev. Biochem. 52, 537–579. cose transporter-4 traffic: new signals, locations, and partners. Endocrinology Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R.I., Ornitz, D.M., Leder, P., Deng, C., 146, 5071–5078. 1998. Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regula- Itoh, N., Ornitz, D.M., 2004. Evolution of the Fgf and Fgfr gene families. Trends Genet. tion loop between FGF8 and FGF10 is essential for limb induction. Development 20, 563–569. 125, 753–765. Konishi, M., Asaki, T., Koike, N., Miwa, H., Miyake, A., Itoh, N., 2006. Role of Fgf10 Yamasaki, M., Emoto, H., Konishi, M., Mikami, T., ohuchi, H., Nakao, K., Itoh, N., 1999. in cell proliferation in white adipose tissue. Mol. Cell. Endocrinol. 249, 71– FGF-10 is a growth factor for preadipocytes in white adipose tissue. Biochem. 77. Biophys. Res. Commun. 258, 109–112. Kopelman, P.G., 2000. Obesity as a medical problem. Nature 404, 635–643. Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E.N., Towler, D.A., Ornitz, D.M., 2003. Kubota, N., Terauchi, Y., Miki, H., Takemoto, H., Yamauchi, T., Komeda, K., Satoh, S., Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signa- Nakano, R., Ishii, C., Sugiyama, Y., Eto, K., Tsubamoto, Y., Okuno, A., Murakami, K., ling in the regulation of osteoblast function and bone growth. Development 130, Sekihara, H., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K., Kita- 3063–3074. mura, T., Fujita, T., Ezaki, O., Aizawa, S., Nagai, R., Tobe, K., Kimura, S., Kadwaki, T., Zhang, X., Ibrahimi, O.A., Olsen, S.K., Umemori, H., Mohammadi, M., Ornitz, D.M., 1999. PPAR mediates high-fat diet induced adipocyte hypertrophy and insulin 2006. Receptor specificity of the fibroblast growth factor family. The complete resistance. Mol. Cell. 4, 597–609. mammalian FGF family. J. Biol. Chem. 281, 15694–15700.