Nucleoredoxin promotes adipogenic differentiation through regulation of Wnt/β-catenin signaling
2014; Elsevier BV; Volume: 56; Issue: 2 Linguagem: Inglês
10.1194/jlr.m054056
ISSN1539-7262
AutoresYoung Jae Bahn, Kwang‐Pyo Lee, Seung-Min Lee, Jeong Yi Choi, Yeon‐Soo Seo, Ki‐Sun Kwon,
Tópico(s)Heat shock proteins research
ResumoNucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into various tissue types. We found that NRX expression levels were higher in white adipose tissue of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas overexpression increased differentiation. Adipose tissue-specific NRX transgenic mice showed increases in adipocyte size as well as number compared with WT mice. We further confirmed that the Wingless/int-1 class (Wnt)/β-catenin pathway was also involved in NRX-promoted adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes involved in lipid metabolism were downregulated, whereas inflammatory genes, including those encoding macrophage markers, were significantly upregulated, likely contributing to the obesity in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic factor and controls obesity in vivo. Nucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into various tissue types. We found that NRX expression levels were higher in white adipose tissue of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas overexpression increased differentiation. Adipose tissue-specific NRX transgenic mice showed increases in adipocyte size as well as number compared with WT mice. We further confirmed that the Wingless/int-1 class (Wnt)/β-catenin pathway was also involved in NRX-promoted adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes involved in lipid metabolism were downregulated, whereas inflammatory genes, including those encoding macrophage markers, were significantly upregulated, likely contributing to the obesity in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic factor and controls obesity in vivo. Adipose tissues play important roles in the regulation of whole-body energy homeostasis (1Spiegelman B.M. Flier J.S. Obesity and the regulation of energy balance.Cell. 2001; 104: 531-543Abstract Full Text Full Text PDF PubMed Scopus (1939) Google Scholar). Excess energy intake increases the number and size of fat cells (2Kopelman P.G. Obesity as a medical problem.Nature. 2000; 404: 635-643Crossref PubMed Scopus (3652) Google Scholar). These obese adipose tissues facilitate chronic low-grade inflammation and insulin resistance, which result in severe obesity and diabetes (3Wellen K.E. Hotamisligil G.S. Obesity-induced inflammatory changes in adipose tissue.J. Clin. Invest. 2003; 112: 1785-1788Crossref PubMed Scopus (1436) Google Scholar, 4Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. et al.Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5185) Google Scholar). Thus, understanding adipocyte development and adipogenesis could lay the groundwork for the development of efficient therapeutic strategies for preventing and treating metabolic disorders associated with obesity. Adipogenesis is controlled by a balance of internal and external factors that either stimulate or repress adipogenic differentiation. In the early phase of adipogenic differentiation, CCAAT/enhancer binding protein (C/EBP) β and C/EBPδ induce expression of C/EBPα and PPARγ, which are the principal adipogenic transcription factors that control the early differentiation of preadipocytes into lipid-accumulating fat cells (5Farmer S.R. Transcriptional control of adipocyte formation.Cell Metab. 2006; 4: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1393) Google Scholar, 6Rosen E.D. Spiegelman B.M. Molecular regulation of adipogenesis.Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1053) Google Scholar). In addition, PPARγ appears to suppress canonical Wingless/int-1 class (Wnt) signaling by accelerating proteasome-dependent degradation of β-catenin. Conversely, β-catenin, a transcriptional coactivator in the Wnt signaling pathway, blocks adipogenesis by repressing PPARγ and C/EBPα (7Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Inhibition of adipogenesis by Wnt signaling.Science. 2000; 289: 950-953Crossref PubMed Scopus (1527) Google Scholar, 8Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Peroxisome-proliferator-activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis.Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (246) Google Scholar). A number of reports have suggested a relationship between Wnt signaling and diabetes and adipogenesis (9Grant S.F. Thorleifsson G. Reynisdottir I. Benediktsson R. Manolescu A. Sainz J. Helgason A. Stefansson H. Emilsson V. Helgadottir A. et al.Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes.Nat. Genet. 2006; 38: 320-323Crossref PubMed Scopus (1749) Google Scholar, 10Longo K.A. Wright W.S. Kang S. Gerin I. Chiang S.H. Lucas P.C. Opp M.R. MacDougald O.A. Wnt10b inhibits development of white and brown adipose tissues.J. Biol. Chem. 2004; 279: 35503-35509Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Adipose tissue-specific expression of Wnt10b reduces adiposity and improves insulin sensitivity in the ob/ob obesity model (11Wright W.S. Longo K.A. Dolinsky V.W. Gerin I. Kang S. Bennett C.N. Chiang S.H. Prestwich T.C. Gress C. Burant C.F. et al.Wnt10b inhibits obesity in ob/ob and agouti mice.Diabetes. 2007; 56: 295-303Crossref PubMed Scopus (137) Google Scholar). Although the extensive downregulation of β-catenin expression and its antadipogenic effects during adipogenic differentiation have been characterized, less is known about the factors that modulate β-catenin activity during adipogenesis. Wnt signaling is initiated upon binding of Wnt ligands to transmembrane Frizzled receptors. In the canonical Wnt signaling pathway, Frizzled receptors transduce signals through Dishevelled (Dvl) to inhibit glycogen synthase kinase 3 (GSK3β), resulting in hypophosphorylation and subsequent stabilization of β-catenin. Following nuclear translocation, active β-catenin binds to and coactivates members of the T-cell factor/lymphoid-enhancer factor family of transcription factors, leading to activation of target genes (12Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta.Science. 1998; 280: 596-599Crossref PubMed Scopus (1112) Google Scholar, 13Nelson W.J. Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways.Science. 2004; 303: 1483-1487Crossref PubMed Scopus (2241) Google Scholar). Nucleoredoxin (NRX) is member of the thioredoxin (TRX) family of proteins. TRX family proteins commonly possess a pair of redox-active, oxidation-sensitive cysteine residues in the catalytic center that are directly involved in the reduction of disulfide bonds in target proteins (14Lillig C.H. Holmgren A. Thioredoxin and related molecules—from biology to health and disease.Antioxid. Redox Signal. 2007; 9: 25-47Crossref PubMed Scopus (590) Google Scholar). Although NRX activity has been demonstrated in in vitro assays, whether the TRX-related oxidoreductase activity of NRX plays a role in vivo is unknown (15Kurooka H. Kato K. Minoguchi S. Takahashi Y. Ikeda J. Habu S. Osawa N. Buchberg A.M. Moriwaki K. Shisa H. et al.Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin.Genomics. 1997; 39: 331-339Crossref PubMed Scopus (100) Google Scholar). Previous studies have shown that NRX is a multifunction protein that regulates target proteins through its direct binding activity rather than its oxidoreductase activity (16Funato Y. Michiue T. Asashima M. Miki H. The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-beta-catenin signalling through dishevelled.Nat. Cell Biol. 2006; 8: 501-508Crossref PubMed Scopus (306) Google Scholar, 17Hayashi T. Funato Y. Terabayashi T. Morinaka A. Sakamoto R. Ichise H. Fukuda H. Yoshida N. Miki H. Nucleoredoxin negatively regulates Toll-like receptor 4 signaling via recruitment of flightless-I to myeloid differentiation primary response gene (88).J. Biol. Chem. 2010; 285: 18586-18593Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Endogenous NRX protein is predominantly localized in the cytosol of cells, and its transcripts are widely expressed in all adult tissues (18Funato Y. Miki H. Nucleoredoxin, a novel thioredoxin family member involved in cell growth and differentiation.Antioxid. Redox Signal. 2007; 9: 1035-1057Crossref PubMed Scopus (86) Google Scholar). Knockout of the NRX gene in mouse embryos is perinatally lethal; NRX-knockout embryos (day 18.5) are smaller than their WT littermates and exhibit craniofacial defects with short frontal regions (19Funato Y. Terabayashi T. Sakamoto R. Okuzaki D. Ichise H. Nojima H. Yoshida N. Miki H. Nucleoredoxin sustains Wnt/β-catenin signaling by retaining a pool of inactive dishevelled protein.Curr. Biol. 2010; 20: 1945-1952Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Interestingly, a genomic region around the mouse NRX gene is involved in type 1 and type 2 diabetes (20Babaya N. Ikegami H. Fujisawa T. Nojima K. Itoi-Babaya M. Inoue K. Ohno T. Shibata M. Ogihara T. Susceptibility to streptozotocin-induced diabetes is mapped to mouse chromosome 11.Biochem. Biophys. Res. Commun. 2005; 328: 158-164Crossref PubMed Scopus (21) Google Scholar). However, a role for NRX in adipogenesis and obesity has not been reported. Here, we investigated the role of NRX in preadipocyte differentiation and the obesity phenotype using a 3T3-L1 preadipocyte differentiation system and adipose tissue-specific transgenic mice. We show that NRX mediates adipogenesis by modulating β-catenin activity in vitro and in vivo. Our findings suggest that NRX might act as a proadipogenic factor that is involved in adipocyte differentiation and aspects of the obesity phenotype. The loxP-stop-loxP-NRX transgenic mice (LSL-NRX mice) were obtained by microinjection and germ-line transmission of the transgenic construct (supplementary Fig. 1A). The LSL-NRX mouse strain was backcrossed with the C57BL/6 strain for more than eight generations to create a uniform genetic background. Adipose tissue-specific NRX transgenic mice (Adipo-NRX mice) were produced by crossing LSL-NRX mice with adiponectin-Cre transgenic mice, generating mice with adipose tissue-specific overexpression of hNRX. All animal experiments were performed according to protocols approved by the Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Potential founder mice were genotyped by PCR analysis using genomic DNA isolated from mouse tail clips. Primers used for the detection of the transgenic product were 5′-TGC GAG ATT ACA CCA ACC TG-3′ and 5′-CCC ATA TGT CCT TCC GAG TG-3′. Primary adipocytes were isolated from epididymal fat pads using the collagenase method, as described previously (21Soukas A. Socci N.D. Saatkamp B.D. Novelli S. Friedman J.M. Distinct transcriptional profiles of adipogenesis in vivo and in vitro.J. Biol. Chem. 2001; 276: 34167-34174Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Briefly, freshly excised fat pads were minced and digested for 45 min to 1 h at 37°C in Krebs-Ringer bicarbonate (pH 7.4) containing 4%t; BSA and 1.5 mg/ml type I collagenase (Worthington, Lakewood, NJ). The digested tissue was filtered through a 300 μm nylon mesh to remove undigested tissue and centrifuged at 500 g for 5 min. The floating adipocyte fraction was removed, washed with buffer, and recentrifuged to isolate free adipocytes. The stromal-vascular pellet was resuspended in erythrocyte lysis buffer (154 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA), filtered through a 45 μm nylon mesh to remove endothelial cells, and pelleted at 500 g for 5 min. Enriched cells were cultured in a growth medium composed of DMEM, 20% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco-Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO2 atmosphere. The preadipocytes cell line 3T3-L1, derived from mouse embryo fibroblasts, was grown at 37°C in DMEM containing 10% heat-inactivated bovine calf serum (Gibco-Invitrogen), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere. 3T3-L1 cells were induced to differentiate into mature adipocytes according to the procedure of Student et al. (22Student A.K. Hsu R.Y. Lane M.D. Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes.J. Biol. Chem. 1980; 255: 4745-4750Abstract Full Text PDF PubMed Google Scholar), with minor modifications. Briefly, 2 days after reaching confluence (day 0), cells were placed in differentiation medium composed of DMEM, 10% fetal bovine serum, and MDI, a differentiation cocktail consisting of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, and 10 μg/ml insulin (Sigma, St. Louis, MO). The medium was replenished every other day. 3T3-L1 cells stably expressing FLAG-tagged NRX were generated using a lentivirus-mediated infection system. FLAG is a short, hydrophilic fusion tag consisting of eight amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). For expression of NRX, cDNA encoding FLAG-tagged NRX was cloned into the multicloning site of the green fluorescent protein (GFP)-tagged pLentiM1.4 vector. Lentiviruses were subsequently produced by transiently cotransfecting HEK293T cells with pLP1, pLP2, and pVSV-G plasmid (Invitrogen, Carlsbad, CA) using Lipofectamine (Invitrogen). Forty-eight hours after transfection, supernatants containing lentiviral particles were collected and used to infect 3T3-L1 cells in the presence of 4 µg/ml polybrene. Infected cells were selected by incubation with 2 µg/ml puromycin for 2–3 weeks and used in experiments as indicated. NRX cDNA was kindly provided by Dr. Tasuku Honjo (Kyoto University, Japan) (15Kurooka H. Kato K. Minoguchi S. Takahashi Y. Ikeda J. Habu S. Osawa N. Buchberg A.M. Moriwaki K. Shisa H. et al.Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin.Genomics. 1997; 39: 331-339Crossref PubMed Scopus (100) Google Scholar). An NRX mutant in which catalytic Cys205 and Cys208 residues were replaced with Ser (CS-NRX), provided by Dr. Sung-Kyu Ju, was constructed using site-directed mutagenesis. NRX knockdown in 3T3-L1 cells was accomplished using shRNA against mouse NRX in pLKO.1-puro lentiviral vectors obtained from Sigma (clone ID NM_022463.3-1358s1c1 and NM_022463.3-452s1c1), according to the manufacturer's protocol. Briefly, shRNA lentiviral particles were generated in HEK293T cells by transient transfection with pLP1, pLP2, pVSV-G, and shRNA lentiviral vector or pLKO.1-scrambled (control) vector (SHC002V; Sigma) using Lipofectamine. Forty-eight hours after transfection, supernatants containing lentiviral particles were collected and used to infect 3T3-L1 cells in the presence of 4 µg/ml polybrene. Infected cells were selected by incubation with 2 µg/ml puromycin for 2–3 weeks and used in experiments as indicated. An shRNA-resistant NRX incapable of shNRX binding and subsequent NRX degradation was constructed by site-directed mutagenesis of the target region of shNRX. The construct was obtained by standard methods using the primers 5′-CTT TTG TGA ATG ACT TCT TGG CTG AAA AAC TC-3′ and 5′-TCA GGC TTG AGT TTT TCAGCCAAG AAG TCA TT-3′. Underlined regions indicate the sites that were mutated without changing amino acid residues. Total RNA was extracted using the Easy-Blue reagent (iNtRON Biotechnology, Korea), according to the manufacturer's instructions, and treated with RNase-free DNase I (Takara, Shiga, Japan) to remove contaminating genomic DNA. cDNA was then synthesized from total RNA by RT using a DiaStar RT kit (Solgent, Korea). Quantitative RT-PCR analysis was performed using the Step One Plus real-time PCR system (Applied Biosystems, Waltham, MA) with the corresponding primers. Cells were lysed in a lysis buffer containing 20 mM HEPES (pH 7.2), 150 mM NaCl, 0.5% Triton X-100, 0.1 mM Na3VO4, 1 mM NaF, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 5 mg/ml aprotinin (Sigma). Soluble proteins in cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-NRX (R&D Biosystems, Minneapolis, MN, AF5719), anti-PPARγ (Cell Signaling Technology, Danvers, MA, #2430), anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, SC-47778), anti-Dvl-1 (Santa Cruz Biotechnology, SC-8025), anti-lamin A/C (Santa Cruz Biotechnology, SC-6215), anti-cyclin D1 (Cell Signaling Technology, 2922), anti-β-catenin (Cell Signaling Technology, 9562), anti-α-tubulin (Millipore, 05-829), anti-v-akt murine thymoma viral oncogene homology (AKT; Santa Cruz Biotechnology, SC-1618), anti-phospho-AKT (Cell Signaling Technology, 9271L), and anti-fatty acid binding protein 4 (FABP4; Cell Signaling Technology, 2120) antibodies. White adipose tissue (WAT) from ob/ob mice was kindly provided by Dr. Chul-Ho Lee (KRIBB). For immunoprecipitation, lysates were incubated with anti-FLAG agarose (Sigma) or anti-NRX antibody at 4°C for several hours, after which the beads were washed three times with cell lysis buffer. The beads were resuspended in 1× SDS-PAGE sample buffer, and the eluted proteins were resolved by SDS-PAGE. For Amaxa nucleofections, pellets containing 0.5–1.5 × 106 3T3-L1 cells were carefully resuspended in 100 µl of Nucleofector solution (Lonza, Allendale, NJ), mixed with 1–2 µg of plasmids, and subjected to nucleofection using T-30 Amaxa protocols. The cells were then gently transferred into a 6-well plate and cultured until analysis. Luciferase activity was assessed using the Luciferase Assay system (Promega, Madison, WI), according to the manufacturer's instructions. 3T3-L1 cells were transduced with TOPflash and FOPflash exogenous reporter constructs together with pCMV-β-galactosidase. Luciferase activity was normalized to β-galactosidase activity to adjust for transfection efficiency. Cultured cells were washed twice with PBS and fixed by incubating with 3.7% paraformaldehyde for 15 min at room temperature. The cells were then washed with distilled water and stained for 30 min with 0.3% filtered Oil Red O solution in 60% isopropanol. The stained cells were washed twice with distilled water and photomicrographed. Oil Red O staining was then quantified as described previously (23Ramirez-Zacarías J.L. Castro-Munozledo F. Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O.Histochemistry. 1992; 97: 493-497Crossref PubMed Scopus (834) Google Scholar). Incorporated Oil Red O dye was extracted by adding absolute isopropanol to the stained cell-culture dish and shaking the dish for 30 min. Triplicate samples were read at 510 nm using an Ultrospec2000 Spectrophotometer (Pharmacia Biotech, Piscataway, NJ). Adipose tissue, muscle, and liver samples were fixed for 12–16 h at room temperature in 10% formalin (Sigma) and then embedded in paraffin. Five-micron sections cut at 50 μm intervals were mounted on charged glass slides, deparaffinized in xylene, and stained with hematoxylin and eosin (H and E). Adipocyte cross-sectional area was quantified for each adipocyte in each field using National Institutes of Health Image J software (http://rsb.info.nih.gov/ij/). To quantify cell number, adipocytes were isolated from 100 mg of adipose tissue by using 2 mg/ml collagenase type II-S (Sigma) digestion buffer and were counted using a cell counter (Logos Biosystems, Annandale, VA) (24Carnevalli L.S. Masuda K. Frigerio F. Le Bacquer O. Um S.H. Gandin V. Topisirovic I. Sonenberg N. Thomas G. Kozma S.C. S6K1 plays a critical role in early adipocyte differentiation.Dev. Cell. 2010; 18: 763-774Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Glucose tolerance test (GTT) was performed with 8 h fasted animals. After determination of fasted blood glucose levels, each animal was injected intraperitoneally with 20% glucose (1 g/kg). The insulin tolerance test (ITT) was performed, with an initial fasting for 4 h, and subsequent intraperitoneal injection of insulin (1 U/kg). In all tests, tail blood glucose levels were measured with a glucometer (Roche Diagnostics, Mannheim, Germany) at the indicated times after injection. Formalin-fixed, paraffin-embedded sections of 5 µm were mounted on charged glass slides, deparaffinized in xylene, stained with hematoxylin, and processed for immunohistochemical detection of F4/80 according to standard immunoperoxidase procedure using VECTASTAIN Elite ABC kit (Vector Labs, Burlingame, CA) and anti-F4/80 antibody (Abcam, Cambridge, UK). Quantitative data are presented as means ± SD unless indicated otherwise. Differences between means were evaluated using Student's unpaired t-test. A P-value < 0.05 was considered statistically significant. We analyzed the expression of NRX during 3T3-L1 cells differentiation induced by a differentiation cocktail composed of IBMX, dexamethasone, and insulin. NRX transiently increased in the early stages of adipocyte differentiation at both mRNA and protein levels. Quantitative RT-PCR revealed that NRX mRNA reached a maximum 1 day after differentiation induction (Fig. 1A). Protein levels of NRX were proportional to mRNA levels (Fig. 1B). We next determined whether hyperglycemic conditions affected NRX levels in ob/ob mice. Interestingly, NRX protein was upregulated in WAT of ob/ob mice compared with that in WT mice (Fig. 1C). These results suggest that increased expression of NRX may be associated with adipogenesis and, ultimately, obesity. To further investigate the function of NRX in adipogenesis in vivo, we generated LSL-NRX mice, then crossed these mice with adiponectin-Cre mice to generate Adipo-NRX mice. To validate the specific overexpression of NRX in adipose tissues, we analyzed NRX protein levels in several tissues isolated from Adipo-NRX and WT mice. As expected, overexpression of NRX was observed only in adipose tissues, including white and brown adipose tissue (supplementary Fig. 1B). To clarify activity of the adiponectin promoter, we checked expression levels of adiponectin during adipogenesis. We confirmed that the expression level of adiponectin was very low before induction of differentiation but gradually increased after induction of differentiation (supplementary Fig. 1C), which was consistent with a previous report (25Díez J.J. Iglesias P. The role of the novel adipocyte-derived hormone adiponectin in human disease.Eur. J. Endocrinol. 2003; 148: 293-300Crossref PubMed Scopus (938) Google Scholar). We also observed that expression of adiponectin promoter-driven NRX was induced at early differentiation stage in primary adipocytes isolated from Adipo-NRX mice (supplementary Fig. 1D). Although there was a slight increase in body weight in Adipo-NRX mice compared with WT littermate controls, a comparison of organ weights revealed a notable increase in epididymal WAT in Adipo-NRX mice (Fig. 2A). Epididymal and perirenal fat masses were also significantly larger in Adipo-NRX mice than in WT mice (Fig. 2B, C). There was no difference in food intake between Adipo-NRX mice and WT littermate controls. An analysis of adipocyte cross-sectional areas showed that epididymal fat of Adipo-NRX mice contained hypertrophied adipocytes (Fig. 2D). A quantitative analysis revealed that adipose tissue expansion of Adipo-NRX mice was caused by an increase of adipocyte numbers (Fig. 2E) as well as an enlargement of adipocyte size, suggesting that hypertrophy was accompanied by hyperplasia in adipose tissue of Adipo-NRX mice. The plasma analysis revealed that blood glucose and insulin levels were higher in Adipo-NRX mice than in WT mice (supplementary Table 1). We further assessed glucose homeostasis in WT and Adipo-NRX mice via a GTT and ITT. Adipo-NRX mice showed the tendency to glucose intolerance compared with WT mice (Fig. 2F) and exhibited impaired insulin tolerance that was associated with reduced insulin sensitivity in WAT but not in liver and skeletal muscle (Fig. 2G, H). Although the adipose tissue appeared lipid accumulation, there was no significant change in lipid deposition in liver and skeletal muscle (supplementary Fig. 2). These data demonstrate that adipocyte-specific NRX overexpression increases lipid accumulation in WAT, resulting in insulin resistance. To evaluate whether overexpression of NRX facilitates adipogenic differentiation in cell culture system, we infected 3T3-L1 preadipocytes with a lentivirus expressing GFP-tagged mouse NRX or a control lentivirus. Stable overexpression of NRX was monitored by immunoblotting (Fig. 3A) and fluorescence imaging. 3T3-L1 preadipocytes stably overexpressing NRX showed increased accumulation of lipid droplets following induction of adipocyte differentiation compared with control cells (Fig. 3B, C). We confirmed that the adipogenic markers, PPARγ and FABP4, were also upregulated in NRX-overexpressing cells compared with control cells (Fig. 3D). To confirm that NRX overexpression positively regulates adipocyte differentiation, we next isolated and cultured primary adipocytes from epididymal fat pads of Adipo-NRX and WT mice. Differentiation of primary adipocytes was increased in Adipo-NRX mice compared with that of WT mice (Fig. 3E). Consistent with these morphological observations, expression of the adipogenic marker, FABP4, was also increased in primary adipocytes from Adipo-NRX mice (Fig. 3F). These data strongly suggest that NRX enhances adipogenesis. We found that expression of enzymes involved in lipid catabolism, including Atgl, Mcad, and Cpt1a were downregulated, indicating that adipocytes of Adipo-NRX mice lacked the ability to burn excess fat. These results suggest that increased fat mass in Adipo-NRX mice is due to decreased lipolysis and fatty acid oxidation. Because adipokine dysregulation is a hallmark of adipocyte impairment (26Huh J.Y. Kim Y. Jeong J. Park J. Kim I. Huh K.H. Kim Y.S. Woo H.A. Rhee S.G. Lee K.J. et al.Peroxiredoxin 3 is a key molecule regulating adipocyte oxidative stress, mitochondrial biogenesis, and adipokine expression.Antioxid. Redox Signal. 2012; 16: 229-243Crossref PubMed Scopus (112) Google Scholar), we further measured adipokine expression in WAT of Adipo-NRX mice. The mRNA levels of adiponectin were downregulated, and PAI-1 was upregulated in WAT of Adipo-NRX mice. Obesity has been consistently associated with inflammation (4Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. et al.Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5185) Google Scholar, 27Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr, A.W. Obesity is associated with macrophage accumulation in adipose tissue.J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7456) Google Scholar). We examined mRNA expression levels of several inflammatory genes in WAT of WT and Adipo-NRX mice. Among the genes upregulated in Adipo-NRX mice were TNFα and Cxcl10, which encode proinflammatory cytokines; Cybb, which is involved in phagocytosis; and Emr1 and Itgax, markers of macrophage infiltration (Fig. 3G). Consistently, increased macrophage infiltration in WAT was shown by immunostaining using a macrophage surface marker, F4/80 (Fig. 3H). Obesity is associated with an overall increase in expression of several collagens that results in fibrotic state (28Halberg N. Khan T. Trujillo M.E. Wernstedt-Asterholm I. Attie A.D. Sherwani S. Wang Z.V. Landskroner-Eiger S. Dineen S. Magalang U.J. et al.Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue.Mol. Cell. Biol. 2009; 29: 4467-4483Crossref PubMed Scopus (618) Google Scholar, 29Wang Q.A. Scherer P.E. Gupta R.K. Improved methodologies for the study of adipose biology: insights gained and opportunities ahead.J. Lipid Res. 2014; 55: 605-624Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Then, we tested mRNA levels of several fibrosis-related genes, and found that Col1a1, Col3a1, Col6a1, and Elastin were upregulated (Fig. 3G) in WAT of Adipo-NRX. These suggest that adipocyte expansion is closely linked to inflammation and fibrosis in Adipo-NRX mice. To test whether endogenous NRX influences adipogenesis, we examined the effect of NRX knockdown in 3T3-L1 preadipocytes. 3T3-L1 cells were infected with a lentivirus expressing an shRNA targeting NRX (NRX shRNA) or nontargeting (scrambled) shRNA. Depletion of endogenous NRX was confirmed by immunoblot analyses (Fig. 4A). Knockdown of NRX attenuated differentiation of 3T3-L1 preadipocytes into mature adipocytes, reducing accumulation of lipid droplets compared with control 3T3-L1 cells (Fig. 4B). Moreover, expression levels of the adipocyte markers, PPARγ and FABP4, also were decreased in NRX-depleted cells compared with control cells (Fig. 4C). Next, to exclude the possibility of off-target effects of shRNA targeting the NRX coding sequence, we transfected NRX-knockdown 3T3-L1 cells with an expression plasmid for an shRNA
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