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Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes
FEBS Letters 582 (2008) 573–578
Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes Michiko Takahashia, Yutaka Takahashia,*, Kenichi Takahashib, Fyodor N. Zolotaryovb, Kyoung Su Hongb, Riko Kitazawac, Keiji Iidaa, Yasuhiko Okimurad, Hidesuke Kajie, Sohei Kitazawac, Masato Kasugaa, Kazuo Chiharaa a
Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-2, Kusunoki-cho, Chu-o-ku, Kobe 650-0017, Japan b Research Affairs Center, Research Division, JCR Pharmaceuticals Co. Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan c Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Japan d Department of Basic Allied Medicine, Kobe University School of Medicine, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0142, Japan e Division of Physiology/Metabolism, University of Hyogo, 13-71, Kita-Oji-cho, Akashi 673-8588, Japan Received 15 December 2007; revised 5 January 2008; accepted 14 January 2008 Available online 31 January 2008 Edited by Laszlo Nagy
Abstract To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Adipokine; Chemerin; Insulin signaling; Adipocyte differentiation; 3T3-L1 adipocyte; Insulin sensitivity
1. Introduction Obesity is an emerging health hazard and contributes to the increased morbidity and mortality particularly in Western societies [1]. Obesity is frequently associated with the metabolic syndrome that includes glucose intolerance, insulin resistance, hypertension, hypertriglyceridemia, low HDL cholesterol, and type2 diabetes (T2DM). Adipokines secreted from adipose tissue play an essential role in these conditions [2,3]. Adipocytes also secrete inflammatory cytokines which are typically produced by macrophages, such as tumor necrosis factor-a (TNF-a), interleukin-6 and the chemokine family members, monocyte chemotactic protein-1 (MCP-1) that is related to insulin resistance [4,5]. These adipokines exert their effect via endocrine, autocrine and paracrine fashion and regulate metabolic status.
* Corresponding author. Fax: +81 78 382 5899. E-mail address: [email protected] (Y. Takahashi).
The 3T3-L1 and 3T3-F422A culture lines, derived from disaggregated Swiss 3T3 mouse embryos [6] are the most widely used culture in vitro adipocyte models. Especially, the 3T3-L1 cell line is one of the most well-characterized and reliable models for studying the conversion of preadipocytes into adipocytes. The expression of many adipokines is observed in the process of differentiation. To elucidate molecular mechanisms of obesity related disease such as diabetes and metabolic syndrome, it is important to understand the function of these adipokines. The 3T3-L1 adipocytes are very useful tool for these purposes. Chemerin, also known as tazarotene-induced gene 2 (TIG2) [7], has been recently characterized as a chemotactic agent that was identified as the ligand for an orphan G protein-coupled receptor, ChemR23 expressed by immature dendritic cells (DCs) and macrophages [8]. There have been several reports that chemerin plays a potential role in controlling immune responses at sites of inflammation or tissue injury as a chemotactic factor for specific subsets of immunoregulatory antigenpresenting cells [9,10]. However, it has not been elucidated the role of chemerin in metabolic regulation. More recently, it has been reported that chemerin is an adipokine and regulates adipocyte differentiation [11], the serum chemerin level was associated with body mass index, circulating triglycerides, and blood pressure [12], and chemerin stimulates ERK1/2 and lipolysis in the 3T3L1 adipocytes [13]. Here we also identified TIG2/chemerin as a novel adipokine. We further demonstrate that chemerin is an autocirne/ paracrine factor and stimulates insulin-dependent glucose uptake concomitant with the enhanced insulin signaling in adipocytes.
2. Materials and methods 2.1. Materials Mouse recombinant Chemerin and anti-human ChemR23 monoclonal antibody (MAB362) were purchased from R&D systems (Minneapolis, MN). Human insulin was obtained from Eli Lilly Japan (Kobe, Japan). Dexamethasone and IBMX were purchased from Sigma (St. Louis, MO). Anti IRS-1 and anti phosphotyrosine (RC20H) antibody were purchased from Cell Signaling Technology (Boston, MA) and BD Bioscience (San Jose, CA), respectively.
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.01.023
574 2.2. Cell culture The 3T3-L1 cells and Cos7 cells were purchased from American Type Culture Collection (Manassas, VA). The 3T3-L1 and 3T3F442A [14] preadipocytes were maintained and differentiated as previously described [14,15]. Differentiated adipocytes were used for the experiments on the day 10 after the initiation of differentiation. 2.3. Animals Mouse experiments were performed according to the guidelines of the animal ethics committee of Kobe University Graduate School of Medicine. Male C57BL/6J mice and db/db mice (10–12 weeks old) were purchased from CLEA Japan (Osaka, Japan). The mice were kept on 12-h day/night cycle, were housed in cages and had free access to water and normal chow. 2.4. Differential display To identify adipocyte differentiation-related gene, differential display was conducted as described previously [16,17]. In brief, heat-denatured total RNA (0.5 lg) from the 3T3-L1 adipocytes at the day 0 and 7 were applied for reverse transcription, using the oligo (dT) primer T12MA, T12MC, T12MG, or T12MT (M, a mixture of A, C, and G) as anchor primer. PCR was performed using 1 lM anchor primer (T12MN), and 0.5 lM arbitrary. Radio-labeled PCR amplification products were analyzed by electrophoresis using denaturing 6% polyacrylamide gels. The PCR fragments with different densities were extracted and reamplified using the same primer set used for the differential display reactions. The fragments were subcloned into a pGEM-T Easy vector (Promega, MI) and sequencing analysis was performed. 2.5. Northern blot analysis and semiquantitative RT-PCR analysis Total RNA was extracted from indicated tissues of 10-week-old male C57BL/6J mice using TRIzol (Invitrogen, Carlsbad, CA). For Northern blotting, total RNA (15 lg) was subjected and analyzed with chemerin-specific probe (full length mouse TIG2 cDNA) as previously described [18]. For semiquantitative PCR analysis, total RNA was extracted from indicated tissues of 10-months-old male db/db mice. First strand cDNA was synthesized from 1 lg of total RNA using ThermoScript RT-PCR System (Invitrogen). The primers for mouse 36B4 were: 5 0 -GAGGAATCAGATGAGGATATGGGA-3 0 and 5 0 -AAGCAGGCTGACTTGGTTGC-3 0 and for mouse chemerin were 5 0 -GTGCACAATCAAACCAAACG-3 0 and 5 0 -GGCAAACTGTCCAGGTAGGA-3 0 , respectively. 2.6. Cloning and in vitro expression of chemerin in Cos7 cells We cloned mouse chemerin using cDNA from 3T3-L1 adipocytes as a template. PCR was performed with following primers; m-chemerin-S; 0 5 -CACCATGAAGTGCTTGCTGATCTCC-3 0 and m-chemerin-AS; 5 0 -TTTGGTTCTCAGGGGCCCTGG-3 0 . The control pcDNA3.1D/ V5-His-TOPO vector and the vector containing full-length chemerin cDNA were transfected into Cos7 cell using Fugene (Boehringer Mannheim) and the culture media was collected 48 h after transfection. The media was immunoprecipitated using anti V5 antibody (Invitrogen) and immunoblot was performed using the same antibody. 2.7. Generation of recombinant human chemerin using baculovirus Human chemerin cDNA was cloned using PCR from human adipose tissue cDNA (Clontech, Mountain View, CA) into TOPO TA cloning vector (Invitrogen). pFastBac1-Msp was constructed to express a fusion protein with the N-terminal honey melittin signal peptide as described [19]. The recombinant bacmid was transfected into Sf9 cells using Cellfectin reagent (Life Technologies). Then, High Fiveä Cells (Invitrogen) were infected with the recombinant viruses. After incubation for 3 days at 27 °C, the supernatant was harvested. After dialysis, the recombinant human chemerin was purified by HiTrap SP XL and Tricorn Superdex 75 100/300 (Amersham Biosciences, Piscataway, NJ). Purity of the protein was verified using SDS–PAGE (Fig. 3A). 2.8. Preparation of antiserum Human chemerin cDNA was subcloned into the pTE-14b vector containing His tag. The expression vector was transformed into a Competent AD494(DE3) Escherichia coli. After IPTG induction, the
M. Takahashi et al. / FEBS Letters 582 (2008) 573–578 pelleted bacteria were washed, suspended in 20 mM Sodium phosphate (pH 7.5), containing 500 mM NaCl, 0.1% TritonX-100 and 8 M urea, and sonicated. The clear lysate was purified and eluted by imidazol buffer using a HiTrap Chelating HP (Amersham Biosciences, Uppsala, Sweden). The rabbit was immunized intracutaneously with 150 lg histagged chemerin emulsified in FreundÕs complete adjuvant. Booster doses of 300 lg his-tagged chemerin emulsified in FCA were administered at 2 week intervals until the antibody titer was observed. The rabbit was bled 10 days after immunization. The specificity of the antibody was verified by immunoblotting of recombinant human chemerin (Fig. 3A) and absorption experiments (data not shown). 2.9. Glucose uptake in the 3T3-L1 adipocytes The 3T3-L1 adipocytes were incubated with or without 100 n/ml chemerin in DulbeccoÕs modified EagleÕs medium (DMEM) containing 10% FCS for 12 h before the glucose uptake assay. The assay was essentially performed as described previously [17]. Briefly, the cells were washed several times with HanksÕ solution, and treated with different concentrations of insulin for 15 min at 37 °C. Then 0.5 lCi of [3H]-2-deoxy-D -glucose (NEN Life Science Products, Boston, MA) was added to the medium containing 0.1 mM unlabeled 2-deoxy-D -glucose, and the cells were incubated further for 10 min. The cells were then washed with phosphate-buffered saline, solubilized with 0.2 M NaOH, and the radioactivity was counted using a liquid scintillation counter. Data were representative of three experiments, and each value was corrected for protein content. 2.10. Immunoblot analysis and immunohitochemistry Fifty ng of recombinant human chemerin was analyzed using antihuman chemerin rabbit polyclonal antibody as primary antibody in 1:10000 dilution. As a second antibody, anti-rabbit IgG antibody conjugated with HRP (Amersham) was used as previously described [20]. To detect secreted endogenous chemerin in the 3T3-L1 culture media, heat inactivated 10 ll of supernatant was applied for SDS– PAGE and analyzed as described above. To examine IRb and IRS-1 tyrosine phosphorylation and detection of IRS-1-associated IRb, immunoblot analysis was performed as previously described [21]. For immunohitochemistry, the 3T3-L1 adipocytes on coverslips were permeabilized and blocked with the blocking solution (the MaxiTags immunoperoxidase system; Themo Shandon, Pittsburgh, PA), and incubated with the anti-human chemerin rabbit polyclonal antibody or anti-human ChemR23 mouse monoclonal antibody was used as the primary antibody in 1:100 dilutions, respectively. Biotinylated anti-rabbit or mouse immunoglobulins (ENVISION kit; Dako, Carpinteria, CA) were used as secondary antibodies. The avidin– biotin-peroxidase complex method was used, and the samples were developed with DAB as chromogen (Dako), counterstained with hematoxylin as previously described [22,23]. 2.11. Statistical analysis The results were expressed as the means ± SEM of at least 3 experiments performed independently unless stated otherwise. Statistical analysis was performed by Student-t test. A p-value less than 0.05 denoted the presence of a statistically significant difference.
3. Results 3.1. The expression of chemerin was strongly induced during adipocyte differentiation and secreted into media Among the 318 genes whose expression levels were changed during 3T3-L1 adipocyte differentiation, as detected by differential display analysis, we found 12 putative secreted molecules, including adiponectin and three unknown signal sequence containing molecules. One of these three unknown molecules showed a protein of 164 amino acids with 16 putative signal peptides (NCBI gene accession No. AK002298), which was previously reported as TIG2 [7] and Chemerin [24] (Fig. 1A). Then we performed Northern blot analysis to validate the results obtained by the differential display. As
M. Takahashi et al. / FEBS Letters 582 (2008) 573–578
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showed a substantial expression in the adipose tissue including visceral, epididymal, and brown adipose tissue (Fig. 2A). The abundant expression in the adipose tissue in vivo supported the characteristics of chemerin as an adipokine. The expression of many adipokines is regulated in the changes of metabolic status, such as obesity and diabetes. Thus, we analyzed the changes in the expression of chemerin in the liver and adipose tissue (Fig. 2B) in db/db mice that show marked obesity and diabetes because of the lack of leptin receptor. Interestingly, in the liver, the expression of chemerin was not changed, however, in epididymal fat, the expression of chemerin was markedly reduced. These results indicated that the expression of chemerin was differentially regulated depending on each tissue. To investigate the physiological function, we generated recombinant human chemerin that shares 65% amino acid identity to the mouse orthologue. Also chemerin-specific antibody was raised in rabbit using bacteria-derived human recombinant chemerin as an antigen. As shown in Fig. 3A, we successfully
Br a He in art Lu ng Li ve Sp r lee Ki n dn ey Sk ele T e tal m V i stis u s cle sc era Ep l fa t idi Br dym ow al f su n bc ad at uta ip ne ose o u ti s s a su dip e os et
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Fig. 1. Identification, expression of TIG2/chemerin in the 3T3-L1 adipocytes: (A) Differential display for identifying adipocyte differentiation-related gene in the 3T3-L1 adipocytes. Arrow indicates PCR fragment equivalent to chemerin. (B) Northern blotting analysis of chemerin mRNA in the 3T3-L1 and 3T3-F442A adipocytes. Chemerin mRNA was markedly induced during adipocyte differentiation both in the 3T3-L1 and 3T3-F442A adipocytes. (C) Immunoblotting of chemerin protein in cell culture medium. Cos7 cells were transfected with chemerin expression vector and the protein in culture media was detected using ant c-terminal V5 antibody.
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shown in Fig. 1B, Northern blot analysis successfully demonstrated that this gene is indeed strongly induced during adipocyte differentiation not only in the 3T3-L1 adipocytes but also in the 3T3-F442A adipocytes, indicating its common expression pattern in adipocyte differentiation. Next, we cloned full-length cDNA using total RNA derived from the 3T3-L1 adipocytes, inserted into an expression vector, expressed in Cos7 cells, and performed immunoblotting analysis using cell culture media. Immunobloting analysis using c-terminal V5 tag clearly showed that this molecule was secreted into the culture media in a predicted size. This molecule was initially reported as Tazarotene-induced gene 2 (TIG2) [7] and later identified as a ligand for the orphan G protein coupled receptor ChemR23 [8] and was named chemerin because of its chemoattractant activity to immature dendrite cells and macrophages [8]. 3.2. The expression of chemerin was differently regulated in liver and adipose tissue We next evaluated chemerin gene expression in the mouse. It has been reported that abundant chemerin transcripts were found in the liver, lung, pituitary, and ovary [8], however, in addition to these tissues, Northern blotting analysis clearly
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16kD Fig. 2. (A) Tissue distribution of TIG2/chemerin expression in mice. Fifteen micrograms of total RNA from mice tissue was applied and analyzed by Northern blotting using mouse chemerin specific probe. There was abundant expression in the liver, adipose tissue including visceral, epididymal, and brown adipose tissue. (B) Semiquantitative PCR analysis of the expression of chemerin in the liver and epididymal fat in db/db mice. In the liver, the expression of chemerin was slightly increased compared to that in control mice. In contrast, the expression of chemerin was markedly reduced in the epididymal fat. 36B4 expression was used as a control. (C) In db/db mice, the serum chemerin levels were decreased compared to the control.
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generated and purified recombinant human chemerin (left panel) and the antibody specifically recognized the protein in immunoblotting analysis (right panel) in the predicted size. Using this chemerin-specific antibody, we analyzed the serum levels of chemerin in the control and db/db mice. Intriguingly, The serum levels of chemerin were substantially decreased in diabetic db/db mice (Fig. 2C), suggesting that serum chemerin levels are associated with obesity and diabetic status. 3.3. Endogenous chemerin was secreted from the 3T3-L1 adipocytes Using this chemerin-specific antibody, we conducted immunoblotting analysis to clarify if the endogenous chemerin is secreted into the supernatant of 3T3-L1 adipocytes culture media. It has been reported that chemerin was secereted into media as 18 kDa prochemerin and processed enzymatically to be activated as chemerin that was truncated in its c-terminals. As shown in Fig. 3B, after adipocyte differentiation, chemerin was secreted into the culture media of the 3T3-L1 adipocytes in a predicted size of cleaved active form.
M. Takahashi et al. / FEBS Letters 582 (2008) 573–578
Generally, adipokines exert its effect not only in endocrine manner as a hormone but also in autocrine/paracrine manner in adipose tissue. We analyzed the protein expression pattern of chemerin and its receptor/ChemR23 using immunohistochemistry. The chemerin-specific antibody recognized the protein expression in the differentiated 3T3-L1 adipocytes (Fig. 3C). The immunohitochemical analysis demonstrated that ChemR23 was expressed in the differentiated 3T3-L1 adipocytes as well as the ligand. These results imply that chemerin exerts its biological action in autocrine/paracrine manner in the adipocytes. 3.4. Chemerin potentiated insulin-stimulated glucose uptake in 3T3-L1 adipocytes The fact that 3T3-L1 cells secrete chemerin and its receptor is expressed in the 3T3-L1 adipocytes suggested that this factor might modulate adipocyte function such as insulin-stimulated glucose transport through autocrine/paracrine mechanism. We incubated the 3T3-L1 adipocytes with recombinant human chemerin and measured the uptake of 2-deoxyglucose in the
Fig. 3. (A) Generation of recombinant human chemerin and specific antibody. Recombinant human chemerin was generated in baculovirus system in a soluble form. Purified 3 or 5 lg of recombinant human chemerin in each lane was applied to SDS–PAGE and visualized by Coomassie-staining (left panel). Immunoblotting analysis using the antibody raised by recombinant human chemerin specifically recognized the protein (right panel). (B) Endogenous chemerin was present in the culture media in the differentiated 3T3-L1 adipocytes. Recombinant human chemerin was applied as a positive control. A presence of the bands corresponding to the high molecular weight suggests that chemerin forms multimer. (C) Chemerin was expressed during adipocyte differentiation. ChemR23 was also expressed in the mature adipocytes.
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basal state and after stimulation of insulin. Intriguingly, incubation with 100 ng/ml recombinant human chemerin significantly potentiated insulin-stimulated glucose uptake by 41% (Fig. 4A). Similar results were obtained using commercially available recombinant mouse chemerin (data not shown). In addition, inubation with chemerin potentiated insulin-stimulated tyrosine phosphorylation of IRS-1 (Fig. 4B). These results clearly demonstrated that chemerin regulates insulin sensitivity in the 3T3L1 adipocytes in autocrine/paracrine manner.
Bozaoglu et al. demonstrated that the expression of chemerin in adipose tissue of obese and type 2 diabetic Psammomys obesus was significantly higher than that in lean, normoglycemic P. obesus [12]. In contrast, we found that the expression of chemerin in epididymal fat was markedly reduced in db/db mice. Although the precise reason of this discrepancy is unclear, this could be due to the species difference. We further demonstrated that the serum chemerin concentration was substantially reduced concomitant with the reduction in the expression in the adipose tissue, suggesting that the adipose tissue is a main source of serum chemerin. ChemR23 also known as Dez in mice was an orphan G protein-coupled receptor related to chemokine receptor expressed in macrophages and immature DCs [25]. Chemerin was originally reported as a tazarotene-induced gene in skin raft with unknown function [7] and has recently been reported as a ligand for ChemR23 and was abundantly present in human inflammatory fluids [8]. Chemerin was shown to be activated by proteolytic cleavage and promote calcium mobilization and chemotaxis of immature DCs and macrophages [8,26]. These data suggested that chemerin plays a potential role in regulating inflammation by recruiting antigen-presenting cells. However, it has not been elucidated the essential role of chemerin especially in metabolic regulation. Recently, it has been reported that macrophage accumulation and associated chronic inflammation in adipose tissue are related to the pathogenesis of obesity and insulin resistance [27,28] Emerging evidences indicate that obesity is an inflammatory disease as well as atherosclerosis [29]. In this aspect, it is intriguing that this novel adipokine, chemerin regulates the inflammation and macrophage recruitment as well as insulin sensitivity in adipose tissue. More recently, chemerin has been reported as a adipokine and regulates adipocyte differentiation [11], serum chemerin level was associated with body mass index, circulating triglycerides and blood pressure [12], and chemerin stimulates ERK1/2 and lipolysis in the 3T3L1 adipocytes [13]. Together with our data, it is suggested that chemerin is a novel adipokine that regulates adipocyte differentiation and function and is related to the pathogenesis of metabolic syndrome.
4. Discussion To explore a novel adipokine, we screened adipocyte differentiation-related gene using differential display and found that chemerin is strongly induced during the adipocyte differentiation. We also demonstrated that chemerin was secreted during adipocyte differentiation in vitro and was expressed abundantly in adipose tissue in vivo. Chemerin and its receptor/ ChemR23 were expressed during the adipocyte differentiation, suggesting that chemerin exerted in autocrine/paracrine fashion. These data were compatible with the recent report [11–13]; however we further show the functional and physiological relevance of this novel adipokine chemerin. Intriguingly, the expression of chemerin in vivo was differently regulated in the liver and adipose tissue in db/db mice. We further demonstrated that the serum chemerin concentration was decreased in db/db mice. In addition, chemerin potentiated insulin-stimulated glucose uptake in the 3T3-L1 adipocytes concomitant with enhancing insulin signaling. These data suggest pathophysiological significance of chemerin in obesity and diabetes. It is also demonstrated that chemerin is a novel adipokine that regulates insulin sensitivity in adipose tissue.
Acknowledgement: We thank C. Ogata, K. Imura, S. Matsuda, and N. Sakamoto for excellent technical assistance, lab members for discussion, and H. Iwakabe for support. This work was supported in part by a Grant-in-Aid for Scientific Research from Japanese Ministry of Education Science, Sports and Culture and grants from the Foundation for Growth Science, Ono Foundation and a grant for 21st Century COE Program, ‘‘Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model’’ from Ministry of Education, Culture, Sports, Science and Technology of Japan.
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M. Takahashi et al. / FEBS Letters 582 (2008) 573–578 [18] Takahashi, Y., Carpino, N., Cross, J.C., Torres, M., Parganas, E. and Ihle, J.N. (2003) SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation. Embo J. 22, 372–384. [19] Tachibana, K. et al. (2000) Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins. J. Cell Biol. 150, 1161–1176. [20] Takahashi, Y., Kaji, H., Okimura, Y., Goji, K., Abe, H. and Chihara, K. (1996) Brief report: short stature caused by a mutant growth hormone. New Engl. J. Med. 334, 432–436. [21] Takahashi, Y., Okimura, Y., Mizuno, I., Iida, K., Takahashi, T., Kaji, H., Abe, H. and Chihara, K. (1997) Leptin induces mitogenactivated protein kinase-dependent proliferation of C3H10T1/2 cells. J. Biol. Chem. 272, 12897–12900. [22] Kishimoto, K., Kitazawa, R., Kurosaka, M., Maeda, S. and Kitazawa, S. (2006) Expression profile of genes related to osteoclastogenesis in mouse growth plate and articular cartilage. Histochem. Cell Biol. 125, 593–602. [23] Takahashi, Y. et al. (2007) Growth hormone reverses nonalcoholic steatohepatitis in a patient with adult growth hormone deficiency. Gastroenterology 132, 938–943. [24] Parmentier, M. (2004) Characterization of new chemoattractant agents in leukocytes. Bull. Mem. Acad. R Med. Belg. 159, 515– 520, discussion 521. [25] Samson, M. et al. (1998) ChemR23, a putative chemoattractant receptor, is expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV1 strains. Eur. J. Immunol. 28, 1689–1700. [26] Wittamer, V., Gregoire, F., Robberecht, P., Vassart, G., Communi, D. and Parmentier, M. (2004) The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency. J. Biol. Chem. 279, 9956–9962. [27] Xu, H. et al. (2003) Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J. Clin. Invest. 112, 1821–1830. [28] Weisberg, S.P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R.L. and Ferrante Jr., A.W. (2003) Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 112, 1796–1808. [29] Wellen, K.E. and Hotamisligil, G.S. (2003) Obesity-induced inflammatory changes in adipose tissue. J. Clin. Invest. 112, 1785–1788.
Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes Michiko Takahashia, Yutaka Takahashia,*, Kenichi Takahashib, Fyodor N. Zolotaryovb, Kyoung Su Hongb, Riko Kitazawac, Keiji Iidaa, Yasuhiko Okimurad, Hidesuke Kajie, Sohei Kitazawac, Masato Kasugaa, Kazuo Chiharaa a
Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-2, Kusunoki-cho, Chu-o-ku, Kobe 650-0017, Japan b Research Affairs Center, Research Division, JCR Pharmaceuticals Co. Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan c Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Japan d Department of Basic Allied Medicine, Kobe University School of Medicine, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0142, Japan e Division of Physiology/Metabolism, University of Hyogo, 13-71, Kita-Oji-cho, Akashi 673-8588, Japan Received 15 December 2007; revised 5 January 2008; accepted 14 January 2008 Available online 31 January 2008 Edited by Laszlo Nagy
Abstract To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Adipokine; Chemerin; Insulin signaling; Adipocyte differentiation; 3T3-L1 adipocyte; Insulin sensitivity
1. Introduction Obesity is an emerging health hazard and contributes to the increased morbidity and mortality particularly in Western societies [1]. Obesity is frequently associated with the metabolic syndrome that includes glucose intolerance, insulin resistance, hypertension, hypertriglyceridemia, low HDL cholesterol, and type2 diabetes (T2DM). Adipokines secreted from adipose tissue play an essential role in these conditions [2,3]. Adipocytes also secrete inflammatory cytokines which are typically produced by macrophages, such as tumor necrosis factor-a (TNF-a), interleukin-6 and the chemokine family members, monocyte chemotactic protein-1 (MCP-1) that is related to insulin resistance [4,5]. These adipokines exert their effect via endocrine, autocrine and paracrine fashion and regulate metabolic status.
* Corresponding author. Fax: +81 78 382 5899. E-mail address: [email protected] (Y. Takahashi).
The 3T3-L1 and 3T3-F422A culture lines, derived from disaggregated Swiss 3T3 mouse embryos [6] are the most widely used culture in vitro adipocyte models. Especially, the 3T3-L1 cell line is one of the most well-characterized and reliable models for studying the conversion of preadipocytes into adipocytes. The expression of many adipokines is observed in the process of differentiation. To elucidate molecular mechanisms of obesity related disease such as diabetes and metabolic syndrome, it is important to understand the function of these adipokines. The 3T3-L1 adipocytes are very useful tool for these purposes. Chemerin, also known as tazarotene-induced gene 2 (TIG2) [7], has been recently characterized as a chemotactic agent that was identified as the ligand for an orphan G protein-coupled receptor, ChemR23 expressed by immature dendritic cells (DCs) and macrophages [8]. There have been several reports that chemerin plays a potential role in controlling immune responses at sites of inflammation or tissue injury as a chemotactic factor for specific subsets of immunoregulatory antigenpresenting cells [9,10]. However, it has not been elucidated the role of chemerin in metabolic regulation. More recently, it has been reported that chemerin is an adipokine and regulates adipocyte differentiation [11], the serum chemerin level was associated with body mass index, circulating triglycerides, and blood pressure [12], and chemerin stimulates ERK1/2 and lipolysis in the 3T3L1 adipocytes [13]. Here we also identified TIG2/chemerin as a novel adipokine. We further demonstrate that chemerin is an autocirne/ paracrine factor and stimulates insulin-dependent glucose uptake concomitant with the enhanced insulin signaling in adipocytes.
2. Materials and methods 2.1. Materials Mouse recombinant Chemerin and anti-human ChemR23 monoclonal antibody (MAB362) were purchased from R&D systems (Minneapolis, MN). Human insulin was obtained from Eli Lilly Japan (Kobe, Japan). Dexamethasone and IBMX were purchased from Sigma (St. Louis, MO). Anti IRS-1 and anti phosphotyrosine (RC20H) antibody were purchased from Cell Signaling Technology (Boston, MA) and BD Bioscience (San Jose, CA), respectively.
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.01.023
574 2.2. Cell culture The 3T3-L1 cells and Cos7 cells were purchased from American Type Culture Collection (Manassas, VA). The 3T3-L1 and 3T3F442A [14] preadipocytes were maintained and differentiated as previously described [14,15]. Differentiated adipocytes were used for the experiments on the day 10 after the initiation of differentiation. 2.3. Animals Mouse experiments were performed according to the guidelines of the animal ethics committee of Kobe University Graduate School of Medicine. Male C57BL/6J mice and db/db mice (10–12 weeks old) were purchased from CLEA Japan (Osaka, Japan). The mice were kept on 12-h day/night cycle, were housed in cages and had free access to water and normal chow. 2.4. Differential display To identify adipocyte differentiation-related gene, differential display was conducted as described previously [16,17]. In brief, heat-denatured total RNA (0.5 lg) from the 3T3-L1 adipocytes at the day 0 and 7 were applied for reverse transcription, using the oligo (dT) primer T12MA, T12MC, T12MG, or T12MT (M, a mixture of A, C, and G) as anchor primer. PCR was performed using 1 lM anchor primer (T12MN), and 0.5 lM arbitrary. Radio-labeled PCR amplification products were analyzed by electrophoresis using denaturing 6% polyacrylamide gels. The PCR fragments with different densities were extracted and reamplified using the same primer set used for the differential display reactions. The fragments were subcloned into a pGEM-T Easy vector (Promega, MI) and sequencing analysis was performed. 2.5. Northern blot analysis and semiquantitative RT-PCR analysis Total RNA was extracted from indicated tissues of 10-week-old male C57BL/6J mice using TRIzol (Invitrogen, Carlsbad, CA). For Northern blotting, total RNA (15 lg) was subjected and analyzed with chemerin-specific probe (full length mouse TIG2 cDNA) as previously described [18]. For semiquantitative PCR analysis, total RNA was extracted from indicated tissues of 10-months-old male db/db mice. First strand cDNA was synthesized from 1 lg of total RNA using ThermoScript RT-PCR System (Invitrogen). The primers for mouse 36B4 were: 5 0 -GAGGAATCAGATGAGGATATGGGA-3 0 and 5 0 -AAGCAGGCTGACTTGGTTGC-3 0 and for mouse chemerin were 5 0 -GTGCACAATCAAACCAAACG-3 0 and 5 0 -GGCAAACTGTCCAGGTAGGA-3 0 , respectively. 2.6. Cloning and in vitro expression of chemerin in Cos7 cells We cloned mouse chemerin using cDNA from 3T3-L1 adipocytes as a template. PCR was performed with following primers; m-chemerin-S; 0 5 -CACCATGAAGTGCTTGCTGATCTCC-3 0 and m-chemerin-AS; 5 0 -TTTGGTTCTCAGGGGCCCTGG-3 0 . The control pcDNA3.1D/ V5-His-TOPO vector and the vector containing full-length chemerin cDNA were transfected into Cos7 cell using Fugene (Boehringer Mannheim) and the culture media was collected 48 h after transfection. The media was immunoprecipitated using anti V5 antibody (Invitrogen) and immunoblot was performed using the same antibody. 2.7. Generation of recombinant human chemerin using baculovirus Human chemerin cDNA was cloned using PCR from human adipose tissue cDNA (Clontech, Mountain View, CA) into TOPO TA cloning vector (Invitrogen). pFastBac1-Msp was constructed to express a fusion protein with the N-terminal honey melittin signal peptide as described [19]. The recombinant bacmid was transfected into Sf9 cells using Cellfectin reagent (Life Technologies). Then, High Fiveä Cells (Invitrogen) were infected with the recombinant viruses. After incubation for 3 days at 27 °C, the supernatant was harvested. After dialysis, the recombinant human chemerin was purified by HiTrap SP XL and Tricorn Superdex 75 100/300 (Amersham Biosciences, Piscataway, NJ). Purity of the protein was verified using SDS–PAGE (Fig. 3A). 2.8. Preparation of antiserum Human chemerin cDNA was subcloned into the pTE-14b vector containing His tag. The expression vector was transformed into a Competent AD494(DE3) Escherichia coli. After IPTG induction, the
M. Takahashi et al. / FEBS Letters 582 (2008) 573–578 pelleted bacteria were washed, suspended in 20 mM Sodium phosphate (pH 7.5), containing 500 mM NaCl, 0.1% TritonX-100 and 8 M urea, and sonicated. The clear lysate was purified and eluted by imidazol buffer using a HiTrap Chelating HP (Amersham Biosciences, Uppsala, Sweden). The rabbit was immunized intracutaneously with 150 lg histagged chemerin emulsified in FreundÕs complete adjuvant. Booster doses of 300 lg his-tagged chemerin emulsified in FCA were administered at 2 week intervals until the antibody titer was observed. The rabbit was bled 10 days after immunization. The specificity of the antibody was verified by immunoblotting of recombinant human chemerin (Fig. 3A) and absorption experiments (data not shown). 2.9. Glucose uptake in the 3T3-L1 adipocytes The 3T3-L1 adipocytes were incubated with or without 100 n/ml chemerin in DulbeccoÕs modified EagleÕs medium (DMEM) containing 10% FCS for 12 h before the glucose uptake assay. The assay was essentially performed as described previously [17]. Briefly, the cells were washed several times with HanksÕ solution, and treated with different concentrations of insulin for 15 min at 37 °C. Then 0.5 lCi of [3H]-2-deoxy-D -glucose (NEN Life Science Products, Boston, MA) was added to the medium containing 0.1 mM unlabeled 2-deoxy-D -glucose, and the cells were incubated further for 10 min. The cells were then washed with phosphate-buffered saline, solubilized with 0.2 M NaOH, and the radioactivity was counted using a liquid scintillation counter. Data were representative of three experiments, and each value was corrected for protein content. 2.10. Immunoblot analysis and immunohitochemistry Fifty ng of recombinant human chemerin was analyzed using antihuman chemerin rabbit polyclonal antibody as primary antibody in 1:10000 dilution. As a second antibody, anti-rabbit IgG antibody conjugated with HRP (Amersham) was used as previously described [20]. To detect secreted endogenous chemerin in the 3T3-L1 culture media, heat inactivated 10 ll of supernatant was applied for SDS– PAGE and analyzed as described above. To examine IRb and IRS-1 tyrosine phosphorylation and detection of IRS-1-associated IRb, immunoblot analysis was performed as previously described [21]. For immunohitochemistry, the 3T3-L1 adipocytes on coverslips were permeabilized and blocked with the blocking solution (the MaxiTags immunoperoxidase system; Themo Shandon, Pittsburgh, PA), and incubated with the anti-human chemerin rabbit polyclonal antibody or anti-human ChemR23 mouse monoclonal antibody was used as the primary antibody in 1:100 dilutions, respectively. Biotinylated anti-rabbit or mouse immunoglobulins (ENVISION kit; Dako, Carpinteria, CA) were used as secondary antibodies. The avidin– biotin-peroxidase complex method was used, and the samples were developed with DAB as chromogen (Dako), counterstained with hematoxylin as previously described [22,23]. 2.11. Statistical analysis The results were expressed as the means ± SEM of at least 3 experiments performed independently unless stated otherwise. Statistical analysis was performed by Student-t test. A p-value less than 0.05 denoted the presence of a statistically significant difference.
3. Results 3.1. The expression of chemerin was strongly induced during adipocyte differentiation and secreted into media Among the 318 genes whose expression levels were changed during 3T3-L1 adipocyte differentiation, as detected by differential display analysis, we found 12 putative secreted molecules, including adiponectin and three unknown signal sequence containing molecules. One of these three unknown molecules showed a protein of 164 amino acids with 16 putative signal peptides (NCBI gene accession No. AK002298), which was previously reported as TIG2 [7] and Chemerin [24] (Fig. 1A). Then we performed Northern blot analysis to validate the results obtained by the differential display. As
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showed a substantial expression in the adipose tissue including visceral, epididymal, and brown adipose tissue (Fig. 2A). The abundant expression in the adipose tissue in vivo supported the characteristics of chemerin as an adipokine. The expression of many adipokines is regulated in the changes of metabolic status, such as obesity and diabetes. Thus, we analyzed the changes in the expression of chemerin in the liver and adipose tissue (Fig. 2B) in db/db mice that show marked obesity and diabetes because of the lack of leptin receptor. Interestingly, in the liver, the expression of chemerin was not changed, however, in epididymal fat, the expression of chemerin was markedly reduced. These results indicated that the expression of chemerin was differentially regulated depending on each tissue. To investigate the physiological function, we generated recombinant human chemerin that shares 65% amino acid identity to the mouse orthologue. Also chemerin-specific antibody was raised in rabbit using bacteria-derived human recombinant chemerin as an antigen. As shown in Fig. 3A, we successfully
Br a He in art Lu ng Li ve Sp r lee Ki n dn ey Sk ele T e tal m V i stis u s cle sc era Ep l fa t idi Br dym ow al f su n bc ad at uta ip ne ose o u ti s s a su dip e os et
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Fig. 1. Identification, expression of TIG2/chemerin in the 3T3-L1 adipocytes: (A) Differential display for identifying adipocyte differentiation-related gene in the 3T3-L1 adipocytes. Arrow indicates PCR fragment equivalent to chemerin. (B) Northern blotting analysis of chemerin mRNA in the 3T3-L1 and 3T3-F442A adipocytes. Chemerin mRNA was markedly induced during adipocyte differentiation both in the 3T3-L1 and 3T3-F442A adipocytes. (C) Immunoblotting of chemerin protein in cell culture medium. Cos7 cells were transfected with chemerin expression vector and the protein in culture media was detected using ant c-terminal V5 antibody.
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shown in Fig. 1B, Northern blot analysis successfully demonstrated that this gene is indeed strongly induced during adipocyte differentiation not only in the 3T3-L1 adipocytes but also in the 3T3-F442A adipocytes, indicating its common expression pattern in adipocyte differentiation. Next, we cloned full-length cDNA using total RNA derived from the 3T3-L1 adipocytes, inserted into an expression vector, expressed in Cos7 cells, and performed immunoblotting analysis using cell culture media. Immunobloting analysis using c-terminal V5 tag clearly showed that this molecule was secreted into the culture media in a predicted size. This molecule was initially reported as Tazarotene-induced gene 2 (TIG2) [7] and later identified as a ligand for the orphan G protein coupled receptor ChemR23 [8] and was named chemerin because of its chemoattractant activity to immature dendrite cells and macrophages [8]. 3.2. The expression of chemerin was differently regulated in liver and adipose tissue We next evaluated chemerin gene expression in the mouse. It has been reported that abundant chemerin transcripts were found in the liver, lung, pituitary, and ovary [8], however, in addition to these tissues, Northern blotting analysis clearly
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16kD Fig. 2. (A) Tissue distribution of TIG2/chemerin expression in mice. Fifteen micrograms of total RNA from mice tissue was applied and analyzed by Northern blotting using mouse chemerin specific probe. There was abundant expression in the liver, adipose tissue including visceral, epididymal, and brown adipose tissue. (B) Semiquantitative PCR analysis of the expression of chemerin in the liver and epididymal fat in db/db mice. In the liver, the expression of chemerin was slightly increased compared to that in control mice. In contrast, the expression of chemerin was markedly reduced in the epididymal fat. 36B4 expression was used as a control. (C) In db/db mice, the serum chemerin levels were decreased compared to the control.
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generated and purified recombinant human chemerin (left panel) and the antibody specifically recognized the protein in immunoblotting analysis (right panel) in the predicted size. Using this chemerin-specific antibody, we analyzed the serum levels of chemerin in the control and db/db mice. Intriguingly, The serum levels of chemerin were substantially decreased in diabetic db/db mice (Fig. 2C), suggesting that serum chemerin levels are associated with obesity and diabetic status. 3.3. Endogenous chemerin was secreted from the 3T3-L1 adipocytes Using this chemerin-specific antibody, we conducted immunoblotting analysis to clarify if the endogenous chemerin is secreted into the supernatant of 3T3-L1 adipocytes culture media. It has been reported that chemerin was secereted into media as 18 kDa prochemerin and processed enzymatically to be activated as chemerin that was truncated in its c-terminals. As shown in Fig. 3B, after adipocyte differentiation, chemerin was secreted into the culture media of the 3T3-L1 adipocytes in a predicted size of cleaved active form.
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Generally, adipokines exert its effect not only in endocrine manner as a hormone but also in autocrine/paracrine manner in adipose tissue. We analyzed the protein expression pattern of chemerin and its receptor/ChemR23 using immunohistochemistry. The chemerin-specific antibody recognized the protein expression in the differentiated 3T3-L1 adipocytes (Fig. 3C). The immunohitochemical analysis demonstrated that ChemR23 was expressed in the differentiated 3T3-L1 adipocytes as well as the ligand. These results imply that chemerin exerts its biological action in autocrine/paracrine manner in the adipocytes. 3.4. Chemerin potentiated insulin-stimulated glucose uptake in 3T3-L1 adipocytes The fact that 3T3-L1 cells secrete chemerin and its receptor is expressed in the 3T3-L1 adipocytes suggested that this factor might modulate adipocyte function such as insulin-stimulated glucose transport through autocrine/paracrine mechanism. We incubated the 3T3-L1 adipocytes with recombinant human chemerin and measured the uptake of 2-deoxyglucose in the
Fig. 3. (A) Generation of recombinant human chemerin and specific antibody. Recombinant human chemerin was generated in baculovirus system in a soluble form. Purified 3 or 5 lg of recombinant human chemerin in each lane was applied to SDS–PAGE and visualized by Coomassie-staining (left panel). Immunoblotting analysis using the antibody raised by recombinant human chemerin specifically recognized the protein (right panel). (B) Endogenous chemerin was present in the culture media in the differentiated 3T3-L1 adipocytes. Recombinant human chemerin was applied as a positive control. A presence of the bands corresponding to the high molecular weight suggests that chemerin forms multimer. (C) Chemerin was expressed during adipocyte differentiation. ChemR23 was also expressed in the mature adipocytes.
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basal state and after stimulation of insulin. Intriguingly, incubation with 100 ng/ml recombinant human chemerin significantly potentiated insulin-stimulated glucose uptake by 41% (Fig. 4A). Similar results were obtained using commercially available recombinant mouse chemerin (data not shown). In addition, inubation with chemerin potentiated insulin-stimulated tyrosine phosphorylation of IRS-1 (Fig. 4B). These results clearly demonstrated that chemerin regulates insulin sensitivity in the 3T3L1 adipocytes in autocrine/paracrine manner.
Bozaoglu et al. demonstrated that the expression of chemerin in adipose tissue of obese and type 2 diabetic Psammomys obesus was significantly higher than that in lean, normoglycemic P. obesus [12]. In contrast, we found that the expression of chemerin in epididymal fat was markedly reduced in db/db mice. Although the precise reason of this discrepancy is unclear, this could be due to the species difference. We further demonstrated that the serum chemerin concentration was substantially reduced concomitant with the reduction in the expression in the adipose tissue, suggesting that the adipose tissue is a main source of serum chemerin. ChemR23 also known as Dez in mice was an orphan G protein-coupled receptor related to chemokine receptor expressed in macrophages and immature DCs [25]. Chemerin was originally reported as a tazarotene-induced gene in skin raft with unknown function [7] and has recently been reported as a ligand for ChemR23 and was abundantly present in human inflammatory fluids [8]. Chemerin was shown to be activated by proteolytic cleavage and promote calcium mobilization and chemotaxis of immature DCs and macrophages [8,26]. These data suggested that chemerin plays a potential role in regulating inflammation by recruiting antigen-presenting cells. However, it has not been elucidated the essential role of chemerin especially in metabolic regulation. Recently, it has been reported that macrophage accumulation and associated chronic inflammation in adipose tissue are related to the pathogenesis of obesity and insulin resistance [27,28] Emerging evidences indicate that obesity is an inflammatory disease as well as atherosclerosis [29]. In this aspect, it is intriguing that this novel adipokine, chemerin regulates the inflammation and macrophage recruitment as well as insulin sensitivity in adipose tissue. More recently, chemerin has been reported as a adipokine and regulates adipocyte differentiation [11], serum chemerin level was associated with body mass index, circulating triglycerides and blood pressure [12], and chemerin stimulates ERK1/2 and lipolysis in the 3T3L1 adipocytes [13]. Together with our data, it is suggested that chemerin is a novel adipokine that regulates adipocyte differentiation and function and is related to the pathogenesis of metabolic syndrome.
4. Discussion To explore a novel adipokine, we screened adipocyte differentiation-related gene using differential display and found that chemerin is strongly induced during the adipocyte differentiation. We also demonstrated that chemerin was secreted during adipocyte differentiation in vitro and was expressed abundantly in adipose tissue in vivo. Chemerin and its receptor/ ChemR23 were expressed during the adipocyte differentiation, suggesting that chemerin exerted in autocrine/paracrine fashion. These data were compatible with the recent report [11–13]; however we further show the functional and physiological relevance of this novel adipokine chemerin. Intriguingly, the expression of chemerin in vivo was differently regulated in the liver and adipose tissue in db/db mice. We further demonstrated that the serum chemerin concentration was decreased in db/db mice. In addition, chemerin potentiated insulin-stimulated glucose uptake in the 3T3-L1 adipocytes concomitant with enhancing insulin signaling. These data suggest pathophysiological significance of chemerin in obesity and diabetes. It is also demonstrated that chemerin is a novel adipokine that regulates insulin sensitivity in adipose tissue.
Acknowledgement: We thank C. Ogata, K. Imura, S. Matsuda, and N. Sakamoto for excellent technical assistance, lab members for discussion, and H. Iwakabe for support. This work was supported in part by a Grant-in-Aid for Scientific Research from Japanese Ministry of Education Science, Sports and Culture and grants from the Foundation for Growth Science, Ono Foundation and a grant for 21st Century COE Program, ‘‘Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model’’ from Ministry of Education, Culture, Sports, Science and Technology of Japan.
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