Disruption of CXC Motif Chemokine Ligand-14 in Mice Ameliorates Obesity-induced Insulin Resistance
2007; Elsevier BV; Volume: 282; Issue: 42 Linguagem: Inglês
10.1074/jbc.m700412200
ISSN1083-351X
AutoresNoriko Nara, Yuki Nakayama, Shiki Okamoto, Hiroshi Tamura, Mari Kiyono, Masatoshi Muraoka, Kiyoko Tanaka, Choji Taya, Hiroshi Shitara, Rie Ishii, Hiromichi Yonekawa, Yasuhiko Minokoshi, Takahiko Hara,
Tópico(s)Adipose Tissue and Metabolism
ResumoIn obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle. In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle. The number of patients suffering from type 2 diabetes is increasing worldwide, due in large part to the increased incidence of obesity. To date, several key regulators of obesity-induced insulin resistance have been identified. In an obese mouse model, elevation of free fatty acids (FFAs), 4The abbreviations used are: FFA, free fatty acid; WAT, white adipose tissue; CXCL14, CXC motif chemokine ligand-14; HFD, high fat diet; IL-6, interleukin-6; RBP4, retinol-binding protein-4; CCL2, CC motif chemokine ligand-2; BAT, brown adipose tissue; FCS, fetal calf serum; FACS, fluorescence-activated cell sorting; SVCs, stromal and vascular cells; RD, regular diet; IITT, intraperitoneal insulin tolerance test; IGTT, intraperitoneal glucose tolerance test; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcriptase; 2-DG, 2-deoxyglucose; UCP, uncoupling protein; GLUT4, glucose transporter-4.4The abbreviations used are: FFA, free fatty acid; WAT, white adipose tissue; CXCL14, CXC motif chemokine ligand-14; HFD, high fat diet; IL-6, interleukin-6; RBP4, retinol-binding protein-4; CCL2, CC motif chemokine ligand-2; BAT, brown adipose tissue; FCS, fetal calf serum; FACS, fluorescence-activated cell sorting; SVCs, stromal and vascular cells; RD, regular diet; IITT, intraperitoneal insulin tolerance test; IGTT, intraperitoneal glucose tolerance test; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcriptase; 2-DG, 2-deoxyglucose; UCP, uncoupling protein; GLUT4, glucose transporter-4. inflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-6, endoplasmic reticulum stress, and reactive oxygen species inhibit insulin action, in part through the activation of c-Jun N-terminal kinase-1 and NF-κB-mediated signaling pathways, which are critical for obesity-induced insulin resistance (1Uysal K.T. 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Immunity. 2005; 23: 331-342Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Human monocytes acquire CXCL14 responsiveness and lose their chemotactic response to CCL2 after prostaglandin E2 treatment, suggesting that the role of CXCL14 may be distinct from other inflammatory monokines (25Kurth I. Willimann K. Schaerli P. Hunziker T. Clark-Lewis I. Moser B. J. Exp. Med. 2001; 194: 855-861Crossref PubMed Scopus (170) Google Scholar). However, the CXCL14 receptor has not yet been identified, and the physiological roles of CXCL14 have not been fully explored in animal models. In this study, we generated CXCL14-null mice and demonstrated that CXCL14 is involved in the obesity-associated infiltration of macrophages into WAT, alteration of serum adipokine levels, hepatic steatosis, and attenuation of insulin signaling in skeletal muscle, thereby contributing to whole body insulin resistance in HFD-induced obese mice. Generation of CXCL14 Knock-out and Transgenic Mouse Lines—The CXCL14 targeting vector consisted of the genomic DNA sequence of mouse CXCL14 (GenBank™ accession numbers AC165347 and AC124395) containing a deletion of the translation initiation site and exon 2 and a Neo cassette insertion (supplemental Fig. S1A). TT2 embryonic stem cells derived from CBA × C57BL/6 F1 mice were electroporated with linearized targeting vector and cultured in the presence of G418 (350 μg/ml) for 10-12 days. Homologous recombination was assessed by PCR using P1 (5′-CATGCAGATCAACTGATGTCAGACACAGAC-3′) and P2 (5′-CGAATGGGCTGACCGCTTCC-3′) primers (product size 2,193 bp). Embryonic stem cell clones were microinjected into 8-cell-stage eggs of ICR mice to generate chimeras, which were bred to C57BL/6 mice to obtain F1 heterozygous mutant mice (C57BL/6-CBA mixed background). F1 and F2 mice were intercrossed to obtain CXCL14+/+, CXCL14+/-, and CXCL14-/- littermates. For genotyping, PCR was carried out on tail fragment-derived DNA, using wild-type primers (5′-AGACCTGTACGGCGGCGACTC-3′ and 5′-GTCCGATCTAACCCTAGGTTG-3′, 210-bp product) and mutant primers (5′-CTGCATCTGCGTGTTCGAAT-3′ and 5′-GTCCGATCTAACCCTAGGTTG-3′, 251-bp product). As CXCL14+/+ and CXCL14+/- mice were phenotypically indistinguishable, most experiments were performed using CXCL14+/- and CXCL14-/- littermates of F2 and F3 generations. To generate the CXCL14 transgenic mouse line, the open reading frame of the mouse CXCL14 cDNA was amplified by PCR and inserted downstream of the 6.5-kb version of the mouse MCK promoter/enhancer region (26Cox G.A. Cole N.M. Matsumura K. Phelps S.F. Hauschka S.D. Campbell K.P. Faulkner J.A. Chamberlain J.S. Nature. 1993; 364: 725-729Crossref PubMed Scopus (260) Google Scholar). The SV40-derived polyadenylation signal from pcDNA3.1 (Invitrogen) was then added to the 3′ end. The DNA fragment was gel-purified, and microinjected into the pronucleus of fertilized eggs of C57BL/6 mice. The following set of PCR primers was used to detect the mck-CXCL14 transgene in tail fragment-derived DNA: 5′-CAGGATGTGGCCACATCAGGCAACTTGGGC-3′ and 5′-CCAGGCATTGTACCACTTGA-3′ (430-bp product). All mice were maintained under a 12-h light, 12-h dark cycle in a pathogen-free animal facility. All experimental procedures with mice were pre-approved by the ethical committee of the institute. Southern Blot Analysis—High molecular weight DNA (10 μg) extracted from the spleen was digested with EcoRI. Southern blot hybridization was carried out according to standard procedures. The 390-bp [32P]dCTP-labeled probe was generated by PCR using the following primers: 5′-GCTTCCAGATGTGAGATCCAG-3′ and 5′-AGTAGACTGAGTTCCTCTA-3′. Northern Blot Analysis—Total RNA (20 μg) was isolated from the indicated tissues and organs using TRIzol (Invitrogen). Northern blot analysis was carried out using a PCR-amplified cDNA fragment as a probe. Sequences of the gene-specific primer sets used for PCR are listed in supplemental Table S1. Cell Fractionation—Fragmented periovarian fat pads were digested in a solution of collagenase/dispase (1 mg/ml PBS; Roche Applied Science) for 45 min at 37 °C with gentle agitation. After centrifugation at 1200 rpm for 5 min, floating cells (adipocytes) were recovered, and cells in the pellet were incubated in a solution of biotinylated anti-Mac1 antibody (M1/70, 1:50 dilution; BD Biosciences) in PBS containing 5% fetal calf serum (FCS) on ice for 30 min, followed by incubation with fluorescein isothiocyanate-conjugated streptavidin (BD Biosciences) (10 μg/ml). Fluorescence intensity and number of viable cells were measured by fluorescence activated cell sorting (FACS), using a FACSAria system (BD Biosciences). Mac1-positive (Mac1+) cells and Mac1-negative (Mac1-) cells were collected and designated as macrophages and stromal and vascular cells (SVCs), respectively. Immunohistochemistry—For histological analysis, fat pads were fixed in 4% paraformaldehyde in PBS, and paraffin-embedded sections were prepared (5-μm sections). Sections were incubated with biotinylated F4/80 antibody (1:20 dilution; BM8, Caltag) at room temperature for 30 min, followed by incubation with horseradish peroxidase-conjugated streptavidin (1:50 dilution; Molecular Probes) and diaminobenzidine (Sigma), as described previously (27Nakayama Y. Nara N. Kawakita Y. Takeshima Y. Arakawa M. Katoh M. Morita S. Iwatsuki K. Tanaka K. Okamoto S. Kitamura T. Seki N. Matsuda R. Matsuo M. Saito K. Hara T. Am. J. Pathol. 2004; 164: 1773-1782Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). For fatty liver analysis, fixed cryostat sections (7 μm thick) were with stained with Oil Red O (Sigma; 1.5 mg/ml in 60% isopropyl alcohol) for 10 min at room temperature. Chemotaxis Assay—Mac1+ mononuclear cells in the peripheral blood of 5-month-old C57BL/6 female mice were sorted by FACSAria and applied to the D-6 chamber (width, 260 μm; depth, 5 μm) of EZ-TAXIScan™ MIC-1000 (Effector Cell Institute, Tokyo, Japan) (28Kanegasaki S. Nomura Y. Nitta N. Akiyama S. Tamatani T. Goshoh Y. Yoshida T. Sato T. Kikuchi Y. J. Immunol. Methods. 2003; 282: 1-11Crossref PubMed Scopus (109) Google Scholar) in RPMI 1640 medium (Sigma) containing 0.1% BSA in 20 mm HEPES, pH 8.0. Human CXCL14 (PeproTech, 100 nm) was added to a lower well of the chamber, and cell migration was recorded for 3 h at 37 °C. Diet Study and Metabolic Measurements—Mice were fed a regular diet (RD) (12% fat, CE-2; CLEA, Tokyo) or an HFD (32% fat, HFD-32; CLEA). Initial studies were carried out using female mice fed an RD alone (11 months) or an HFD (6 months with RD and 3 months with HFD). We also used younger mice fed an HFD (2 months with RD and 3 months with HFD) and obtained similar results. For the intraperitoneal insulin tolerance test (IITT) and intraperitoneal glucose tolerance test (IGTT), mice were fasted for 16 h and then injected intraperitoneally with human insulin (0.75 milliunits/g; Sigma) or glucose (0.75 mg/g; Wako Pure Chemical Industries Ltd., Osaka, Japan), respectively. To measure insulin concentrations for the IGTT, glucose (2 mg/g) was administered. Blood glucose was measured using a Glutest Ace R glucometer (Sanwa Chemical, Nagoya, Japan). Levels of serum cholesterol, serum, and hepatic triglyceride and serum FFAs were analyzed using the corresponding enzyme-based measurement kits (Wako). To determine hepatic triglyceride concentrations, liver fragments (100 mg) were homogenized in 1 ml of 0.1 m KCl, and lipids were extracted from the homogenate with chloroform:methanol (2:1, v/v) (29Akiyama T.E. Lambert G. Nicol C.J. Matsusue K. Peters J.M. Brewer Jr., H.B. Gonzalez F.J. J. Biol. Chem. 2004; 279: 20874-20881Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). An aliquot of the organic phase was collected, dried, and resuspended in isopropyl alcohol containing 1% Triton X-100. Serum concentrations of insulin, leptin, and adiponectin were determined using enzyme-linked immunosorbent assay kits from Shibayagi (Shibukawa, Gumma, Japan), Morinaga Chemical (Yokohama, Japan), and Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. Statistical Analysis—Statistical analysis was carried out using the unpaired Student's t test and Statview J5.0 software (Abacus Concepts). A p value of <0.05 was considered to be significant. CT Scan Analysis—The amount of visceral fat and subcutaneous fat of HFD-fed mice was examined by radiography using a CT apparatus (LaTheta LCT-100M, Aloka, Tokyo), according to the manufacturer's protocol. CT scanning was done at 2-mm intervals from the diaphragm to the bottom of the abdominal cavity. Western Blot Analysis—For the in vivo analysis, mice were fasted for 16 h, injected with human insulin (10 milliunits/g) intraperitoneally, and then sacrificed 4 min after injection. Liver and skeletal muscle (tibialis anterior and gastrocnemius muscles) were dissected and immediately frozen in liquid nitrogen. For in vitro analysis, confluent C2C12 myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% horse serum for 4 days to induce myotube differentiation. After 16 h of serum starvation, cells were incubated with or without CXCL14 (100 nm) for 1 h followed by stimulation with insulin (10 nm) at 37 °C for 10 min in the presence or absence of CXCL14. Total protein (20 μg) was subjected to Western blot analysis using a polyclonal antibody to phosphorylated Akt (Akt-pSer473) or nonphosphorylated Akt (Cell Signaling) according to the manufacturer's instructions. CXCL14 in the brain and RBP4 in serum were detected using sheep anti-mouse CXCL14 antibody (R & D Systems) and rabbit anti-human RBP4 antibody (DAKO), respectively. Reverse Transcriptase (RT)-PCR—First-strand cDNAs were prepared from total RNA (5 μg) using SuperScript II reverse transcriptase and a random primer (Invitrogen). One percent of the cDNA mixture was subjected to 30-40 cycles of PCR using the primer sets listed in supplemental Table S1, an annealing temperature of 57 °C, and ExTaq DNA polymerase (Takara, Otsu, Japan). Measurement of 2-Deoxyglucose (2-DG) Uptake—Differentiated C2C12 myocytes were generated as described for Western blot analysis. 3T3-L1 preadipocytes were cultured in DMEM containing 10% FCS at 37 °C. Two days after reaching confluence, cells were treated with 0.25 μm dexamethasone and 0.5 mm 3-isobutyl-1-methylxanthine for 48 h, followed by 10 μm insulin alone for an additional 48 h. The cells were then maintained for 6 days in DMEM containing 10% FCS. For the 2-DG uptake assay, C2C12-derived myocytes or 3T3-L1-derived adipocytes were serum-starved for 16 h, and then incubated with 0, 0.1, 1, or 10 nm CXCL14 at 37 °C for 1 h. Cells were stimulated with insulin (10 nm) for 30 min and incubated with 100 nm [1-3H]2-DG (0.8 μCi/ml, Amersham Biosciences) at 37 °C for 10 min in the presence or absence of CXCL14. Specific uptake of 2-DG was measured as described previously (30Wang L. Huang J. Saha P. Kulkarni R.N. Hu M. Kim Y. Park K. Chan L. Rajan A.S. Lee I. Moore D.D. Mol. Endocrinol. 2006; 20: 2671-2681Crossref PubMed Scopus (38) Google Scholar). Obesity-associated Up-regulation of CXCL14 mRNA—In adult mice, CXCL14 was expressed in the brain, lung, kidney, ovary, skeletal muscle, uterus, and WAT (Fig. 1A). Expression of CXCL14 was strikingly elevated in WAT, BAT, and skeletal muscle (tibialis anterior muscle) in obese mice fed an HFD (Fig. 1B), and in ob/ob mice (Fig. 1C) compared with mice fed an RD. This was the first demonstration that CXCL14 is an obesity-induced gene. In WAT, the expression level of CXCL14 mRNA in SVCs and macrophages was equivalent in RD-fed and HFD-fed mice, whereas in adipocytes, CXCL14 transcripts were detectable only in HFD-fed mice (Fig. 1D). The WAT of HFD-fed mice was infiltrated by a larger number of SVCs and macrophages than the WAT of RD-fed mice (see Fig. 3A); thus, all three types of cells in WAT may contribute to the increased level of expression of CXCL14 in WAT of obese mice. Currently available antibodies were not sensitive enough to quantitate CXCL14 protein levels in WAT or other tissues.FIGURE 3Reduced infiltration of macrophages into WAT of HFD-fed CXCL14-/- mice. A, cells were enzymatically dissociated from periovarian fat pads of RD-fed or HFD-fed CXCL14+/- or CXCL14-/- female mice, and the total numbers of Mac1+ and Mac1- cells in WAT were calculated based on FACS analysis. RD, n = 6 for each group; HFD, n = 4 for CXCL14+/-; n = 5 for CXCL14-/-. B, paraffin sections of periovarian fat pads from representative RD-fed or HFD-fed CXCL14+/- and CXCL14-/- female mice were immunostained with anti-F4/80 antibody. Brown signals surrounding adipocytes represent F4/80+ macrophages. Magnification, ×200. C, chemotactic response of peripheral blood Mac1+ cells to CXCL14 was examined using an EZ-TAXIScan™ apparatus. The percentage of cells that migrated further than 43 μm from their origin is presented. Data represent the means ± S.E. (n = 4). *, p < 0.01; n.s., not significant compared with the cells in the absence of CXCL14.View Large Image Figure ViewerDownload Hi-res image Download (PPT) General Properties of CXCL14-deficient Mice—To understand the physiological role of CXCL14, we generated heterozygous (CXCL14+/-) and homozygous (CXCL14-/-) CXCL14 mutant mice, according to standard gene targeting strategies (supplemental Fig. S1A). Southern, Northern, and Western blot analyses confirmed that region-specific recombination occurred and that CXCL14 transcripts and protein were absent in null mutant mice (supplemental Fig. S1, B-D). Although the birth ratio of CXCL14-/- mice was lower than the expected Mendelian frequency, CXCL14-/- adult mice were fertile and asymptomatic for severe diseases. However, CXCL14-/- and CXCL14+/- mice exhibited several differences in body weight and metabolic parameters. Female CXCL14-/- mice showed a more pronounced phenotype than male CXCL14-/- mice, particularly under HFD feeding conditions. Thus, the following experiments were conducted using female homozygous CXCL14-/- and heterozygous CXCL14+/- littermates. Under RD feeding conditions, the mean body weight of CXCL14-/- mice (26.3 ± 1.3 g) was 24.6% less than that of CXCL14+/- mice (34.9 ± 2.3 g) (Fig. 2A). Periovarian WAT mass in RD-fed CXCL14-/- mice was significantly reduced, to 25.4% of CXCL14+/- mice (Fig. 2, B and C). The weights of the liver and kidney were comparable between the two strains of RD-fed mice (Fig. 2C). Under RD feeding conditions, CXCL14-/- mice ate 19.3% less than CXCL14+/- mice (Fig. 2F), which indicated that the lean phenotype and WAT hypoplasia of CXCL14-/- mice is because of decreased appetite. The total macrophage number in WAT was not significantly different between RD-fed CXCL14-/- and CXCL14+/- mice (see Fig. 3A); thus, the decreased appetite of CXCL14-/- mice appeared to be independent of the macrophage chemoattractant activity of CXCL14 in adipose tissues. Under HFD feeding conditions, whereas both CXCL14-/- and CXCL14+/- mice gained a significant amount of weight (Fig. 2, A and B), CXCL14-/- mice were 20.8% lighter than CXCL14+/- mice (Fig. 2A). In contrast to RD-feeding conditions, the periovarian WAT mass of CXCL14-/- mice increased under HFD-feeding conditions and was not significantly different from that of CXCL14+/- mice (3.4 ± 0.4 and 4.7 ± 0.6 g, respectively; p = 0.107) (Fig. 2, B and D). To accurately determine the adiposity of HFD-fed CXCL14-/- mice, we analyzed a second group of HFD-fed female mice by CT scan. The estimated weights of total visceral fat and subcutaneous fat in the body were not statistically different (p = 0.0671 and 0.958, respectively) (Fig. 2E). Next, we compared fat cell size in HFD-fed CXCL14-/- mice and HFD-fed CXCL14+/- mice. As shown in supplemental Fig. S2, A-D, there was an HFD feeding-dependent hypertrophy of adipocytes in WAT of CXCL14-/- mice. These data collectively indicated that CXCL14-/- mice are not defective in adipogenesis and WAT development under HFD-feeding conditions. Although the WAT was enlarged in HFD-fed CXCL14-/- mice compared with HFD-fed CXCL14+/- mice, the dimensions (supplemental Fig. S3A) and mass (1.6 ± 0.1 and 3.8 ± 0.3 g, respectively) (Fig. 2D) of the livers were smaller in HFD-fed CXCL14-/- mice. The livers of HFD-fed CXCL14-/- mouse had less Oil Red O-positive lipid deposits compared with HFD-fed CXCL14+/+ or CXCL14+/- mice (supplemental Fig. S3B). The amount of triglyceride in the livers of HFD-fed CXCL14-/- mice was 20.0% less than HFD-fed CXCL14+/- mice (supplemental Fig. S3C). These results indicated that CXCL14-deficient mice are partially protected from HFD-induced hepatic steatosis. In contrast to adipose tissue and skeletal muscle, HFD-induced up-regulation of CXCL14 mRNA was not observed in the livers of CXCL14+/- mice (supplemental Fig. S3D). Thus, the smaller size and decreased fat content in the livers of HFD-fed CXCL14-/- mice were likely an indirect effect of the CXCL14 deficiency. The kidney mass of HFD-fed CXCL14-/- mice was also smaller than HFD-fed CXCL14+/- mice (0.36 ± 0.02 and 0.45 ± 0.02 g, respectively) (Fig. 2D). The level of food intake of HFD-fed CXCL14-/- mice was not significantly different from that of HFD-fed CXCL14+/- mice (p = 0.097) (Fig. 2F). As mentioned above, although HFD-fed CXCL14-/- mice were able to convert excess energy into WAT, their body weight and organ masses were smaller than HFD-fed CXCL14+/- mice. This unusual phenotype implied that CXCL14-/- mice have central nervous system defects as well, because CXCL14 mRNA was also observed in the brain (Fig. 1A). The mean nasal-anal length of HFD-fed CXCL14-/- mice (9.22 ± 0.086 cm) was signi
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