Supporting Online Material for

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1 Supporting Online Material for Is an Anti-Inflammatory Adipokine That Modulates Metabolic Dysfunction in Obesity Noriyuki Ouchi, Akiko Higuchi, Koji Ohashi, Yuichi Oshima, Noyan Gokce, Rei Shibata, Yuichi Akasaki, Akihiko Shimono, Kenneth Walsh To whom correspondence should be addressed. (K.W.) or (N.O.) This PDF file includes Materials and Methods Figs. S1 to S11 References Published 17 June 21 on Science Express DOI: /science

2 Supporting Online Material for Is an Anti-Inflammatory Adipokine That Modulates Metabolic Dysfunction in Obesity Noriyuki Ouchi, Akiko Higuchi, Koji Ohashi, Yuichi Oshima, Noyan Gokce, Rei Shibata, Yuichi Akasaki, Akihiko Shimono, Kenneth Walsh To whom correspondence should be addressed. (K.W.); (N.O.) This PDF file includes: Materials and Methods Figs. S1 to S11 References

3 Materials and Methods Materials Phospho-Akt (Ser473), phospho-jnk (Thr183/Tyr185), phospho-cjun (Ser63), Akt, and GAPDH antibodies were purchased from Cell Signaling Technology. Phospho-IRS- 1 (Ser37) antibody was purchased from Upstate biotechnology. β-actin antibody were purchased from Sigma Chemical Co. The polyclonal antibody against mouse was generated by immunizing rabbits with two synthetic peptides conjugated to KLH through the Cys via maleimide (APTRGQEYDYYGWQAEP: amino acid residues and Acetyl-VKMRIKEIKIDNGDRKLIG: amino acid residues )(21st Century Biochemicals). SP6125 was purchased from Biomol international. Recombinant mouse Wnt5a protein produced in CHO cells (endotoxin free: <1. EU/µg protein by the LAL method) and mouse Wnt5a antibody were purchased from R&D Systems. Animal model Mice lacking were backcrossed and maintained on the C57BL6/J background. -/- mice were generated by replacing the first protein coding exon with the PGKneobpAloxA cassette as described previously (1). -/- mice and littermate wildtype () C57BL6/J mice were used. To generate mice lacking both and JNK1, -/- mice and Jnk1 -/+ mice (Jackson laboratory) were inbred. Ob/ob mice were purchased from Jackson laboratory. Zucker diabetic fatty rats and their lean littermates were purchased from Charles River Laboratories. Study protocols were approved by the Boston University Institutional Animal Care and Use Committee. Mice were fed either a normal chow diet (Harlan Teklad global 18% protein rodent diet, #218) or a HF/HS diet (Bio-Serv, #F185)(2) as indicated. The composition of the HF/HS diet was 35.8% fat (primarily lard), 36.8% carbohydrate (primarily sucrose), and 2.3% protein. For the high caloric diet feeding, mice at the age of 1 weeks were maintained on a HF/HS diet for 12 or 24 weeks. Collection of human visceral fat tissue Visceral adipose tissue was collected during gastric bypass surgery in obese adults (age 21 years) with a body mass index 3 kg/m 2 as described previously (3). Patients with unstable medical conditions such as active coronary syndromes, congestive heart failure, systemic infection, malignancy, or pregnancy were excluded. The presence or absence of macrophage crown-like structures (CLS) in adipose tissue was determined by immunohistochemical stains with CD68 in a blinded manner as described previously (3). The insulin resistance marker, homeostasis model assessment of insulin resistance (HOMA-IR) were quantified from blood samples (3). All subjects gave written, informed consent, and the study was approved by the Boston University Medical Center Institutional Review Board. Cell culture Mouse 3T3-L1 cells (ATCC) were maintained in DMEM with 1% fetal bovine serum (FBS) and differentiated into adipocytes by treatment with DMEM supplemented with 5 µg/ml of insulin,.5 mm 1-methyl-3-isobutyl-xanthin, and 1 µm dexamethazone (4). Peritoneal macrophages from lean C57BL/6 mice were maintained in DMEM

4 supplemented 1% FBS and placed in DMEM with.5% FBS for 6 h for serum starvation. Macrophages were incubated in the conditioned media from 3T3-L1 adipocytes transduced with adenoviral vectors in the presence of Wnt5a protein (2 ng/ml) or vehicle for 3 min or 24 h. In some experiments, cells were pretreated with SP6125 (15 µm) or vehicle for 1 h followed by treatment with Wnt5a protein. Construction of adenoviral vectors For transduction experiments, tetracycline-regulated adenovirus (Ad) vectors were used (5). The Ad vectors expressing β-galactosidase (β-gal) or were constructed under the control of seven consecutive tetracycline-responsive elements (TRE) and a CMV minimal promoter (AdTRE-β-gal or AdTRE-). Co-transfection of AdTRE vector with Ad vectors encoding tetracycline transactivator (tta, a fusion of TRE-binding protein and VP16 transactivation domain) under the control of the CMV promoter/enhancer (AdCMV-tTA) results in activation of transgene. Ad vectors expressing adiponectin (Ad- APN) under control of the CMV promoter was constructed as previously described (6, 7). For in vivo gene transfer, AdTRE-β-gal or AdTRE- along with AdCMV-tTA (2.5x1 8 pfu for each adenovirus) was injected into the jugular vein of mice. For some experiments, Ad-APN (5.x1 8 pfu total) was intravenously administered to mice. For in vitro transduction, differentiated 3T3-L1 adipocytes were transfected with AdTRE-β-gal or AdTRE- in the presence of AdCMV-tTA (125 MOI for each adenovirus) for 24 h. The media was then replaced with fresh DMEM, and cells were incubated for additional 24 h. Cells were treated with or without recombinant Wnt5a protein (2 ng/ml) for indicated length of time. For detection of in media, protein was concentrated 3- fold with a Microcon column. Luciferase reporter assays 3T3-L1 adipocytes were transduced with adenoviral vectors for 24 h and co-transfected with a TOPflash (Upstate) construct and a Renilla luciferase control plasmid (prl-sv4, Promega) to normalize for transfection efficiency. After transfection, cells were treated with Wnt5a protein (2 ng/ml) or vehicle. Cells were lysed and analyzed on a luminometer by using dual luciferase assay kit (Promega). Metabolic measurements Serum insulin levels were determined by ELISA (Crystal Chemical Inc.). Serum glucose levels were measured with enzymatic kits (Wako Chemicals). Glucose tolerance testing was performed on 16 h-fasted mice injected intraperitoneally with D-glucose (1 g/kg body weight)(2). Blood glucose levels were determined immediately before and 3, 6, 9, and 12 min after injection as determined by an Accu-Chek glucose monitor (Roche Diagnostics Corp.). Insulin tolerance testing was performed on 6 h-fasted mice injected intraperitoneally with human insulin (Humulin R, Eli Lilly) at U/kg body weight for lean mice or at 4.5 U/kg body weight for obese mice. Blood glucose levels were determined immediately before and 15, 3, and 6 min after injection. Insulin signaling in adipose tissues was determined by measurement of Akt phosphorylation following insulin administration. Mice were fasted overnight and treated with insulin (4.5 U/kg body weight) or saline via the inferior vena cava. Epididymal fat was isolated after 4 min, and immunoblot analysis was preformed. To determine triglyceride content of liver,

5 lipid was extracted using the Bligh-Dyer method (8). Liver tissues were homogenized in chloroform/meoh/h 2 O (1:2:.8) and centrifuged. Supernatants were collected, and equal amounts of chloroform and H 2 O were added. After vortexing and centrifugation, the chloroform layer was collected, dried completely and resuspended in isopropanol containing 1% Triton-X. Triglyceride levels were measured with enzymatic kits (Wako Chemicals). Histology The sections of epididymal adipose tissue were fixed in 1% formalin, dehydrated, and embedded in paraffin. Adipose tissue sections were stained with hematoxylin and eosin to examine the morphology and with anti-f4/8 antibody (Santa Cruz) to detect macrophages. Adipocyte cross-sectional areas were measured in 2 cells per mouse using Image J software. Macrophage accumulation was quantified by measuring the number of F4/8-positive cells per mm 2 in 2 randomly chosen microscopic fields. Liver tissues were embedded in OCT compound (Sakura Finetech USA Inc.) and snap frozen in liquid nitrogen and stained with oil red O for lipid deposition by standard methods (2). Isolation of mouse adipocyte and stromal vascular (SV) fraction Epididymal fat pads from male wild-type C57BL/6 mice fed a normal diet were excised, minced in PBS and digested with 1 mg/ml collagenase Type 1 (Worthington Chemical Corporation) at 37 C for 3 min. The digested fat tissue was filtered through a mesh and centrifuged at 1 rpm for 5 min to separate floating adipocytes from the SV fraction. Measurement of mrna levels Gene expression level was quantified by real-time PCR. Total RNA was prepared with the use of a Qiagen or Ambion kit. cdna was produced using ThermoScript RT-PCR Systems (Invitrogen). PCR was performed on icycler iq Real-Time PCR Detection System (Bio-Rad) using SYBR Green 1 as a double standard DNA specific dye (Applied Biosystems). Primers were: 5 -GAAAGTTGATTGGAGCCCAGAA-3 and 5 - GCCCGTCAGGTTGTCTAACTGT-3 for mouse, 5'- CAGCCAGATGCAGTTAACGC-3' and 5'-GCCTACTCATTGGGATCATCTTG-3' for mouse MCP-1, 5 -CTTTGGCTATGGGCTTCCAGTC-3 and 5 - GCAAGGAGGACAGAGTTTATCGTG-3 for mouse F4/8, 5 - CTTCTGCTGTGGAAATGCAA-3 and 5 -AGAGGGGCTGGTAGGTTGAT-3 for mouse CD68, 5 -TGCCATCCATGCGGAAA-3 and 5 -AGCGGGAAGAACTCCTCTTC-3 for mouse cyclind1, 5 -ATACAGGTGCCAGGAAGGTG-3 and 5 - CAAGGGCAGAAAGTTGGTGT-3 for mouse WISP2, 5'- TTGGGTCAGCACTGGCTCTG-3' and 5'-TGGCGGTGTGCAGTGCTATC-3' for mouse gp91 phox, 5'-GATGTTCCCCATTGAGGCCG-3' and 5'-GTTTCAGGTCATCAGGCCGC-3' for mouse P47 phox, 5'-ACCTATTCCTGCGTCGGTGT-3' and 5'- GCATCGAAGACCGTGTTCTC-3' for mouse GRP78, 5'- GTCCTGTCCTCAGATGAAATTGG-3' and 5'-GCAGGGTCAAGAGTAGTGAAGGTT-3' for mouse CHOP 5 -AATCAAGAACGAAAGTCGGAGG-3 and 5 - GCGGGTCATGGGAATAACG-3 for 18S, 5 -GAAGCTGGTTCTGCACATGA-3 and 5 - TTCTTGTCCCAGCGGTAGAC-3 for rat, 5 -

6 CATCTTCTCAAAACTCGAGTGACAA-3 and 5 - TGGGAGTAGATAAGGTACAGCCC- 3 for rat TNFα, 5 -CCGCTGGGACAAGAAGAATA-3 and 5 - GTAGAGGGAGCAGGGGTAGG-3 for human, 5 -CGAGTGACAAGCCTGTAGC- 3 and 5 - GGTGTGGGTGAGGAGCACAT-3 for human TNFα, 5'- CGACCTGGAAGTCCAACTAC-3' and 5'-ATCTGCTGCATCTGCTTG-3' for human 36B4 and 5'-GCTCCAAGCAGATGCAGCA-3' and 5'-CCGGATGTGAGGCAGCAG-3' for mouse or rat 36B4. Primers for mouse TNFα and mouse IL-6 were purchased from Qiagen. Western blot analysis Tissue and cell samples were homogenized, and equal amounts of proteins were separated with denaturing SDS 4-15% polyacrylamide gels. Following transfer to membranes, immunoblot analysis was performed with the indicated antibodies followed by incubation with secondary antibody conjugated with horseradish peroxidase at a 1:5 dilution. ECL Western Blotting Detection kit (Amersham Pharmacia Biotech) or ECL Advance Western Blotting Detection kit (Amersham Pharmacia Biotech) was used for detection. Relative phosphorylation or protein levels were quantified by Image J program. Statistical analysis All data are expressed as means ± SEM. Differences were analyzed by Student s unpaired t test or analysis of variance (ANOVA) for multiple comparisons. A value of P<.5 was accepted as statistically significant

7 S1 A mrna levels GAPDH WAT BAT Brain Heart Lung Liver Pancreas Kidney S. Muscle.2 B mrna levels P<.1 Adipocyte SV Figure S1: expression in various tissues in wild-type () mice. (A) Tissue distribution of mrna and protein in mice fed normal diet. mrna levels were analyzed by quantitative real-time PCR (QRT-PCR) and expressed relative to 18S levels (n=3).equal amounts of proteins were loaded, and and GAPDH protein levels were determined by immunoblot analysis. WAT, white adipose tissue; BAT, brown adipose tissue; S. Muscle, skeletal muscle. (B) transcript levels in adipocyte and stromal vascular (SV) fractions isolated from white adipose tissues of mice fed a normal diet as measured by QRT-PCR analysis and expressed relative to 36B4 (n=3).

8 S2 A 4 P<.1 2 P<.1 2. P<.1 Body weight (g) 3 2 Glucose (mg/dl) Insulin (ng/ml) Lean ZDF Lean ZDF Lean ZDF B P<.5 mrna levels TNFα mrna levels Lean ZDF P<.1 Lean ZDF Wnt5a/ protein ratio Wnt5a β-actin 4 2 Lean ZDF Lean ZDF Figure S2: Metabolic parameters and expression of and Wnt5a in fat tissue in obese Zucker diabetic fatty (ZDF) rats and lean littermates at the age of 12 weeks. (A) Body weight, serum glucose and serum insulin levels in lean and ZDF rats (mean ± SEM, n=4). (B) Expression of and Wnt5a in epididymal fat tissue in lean and ZDF rats. and TNFα transcript levels were measured by QRT-PCR analysis and expressed relative to 36B4 (mean ± SEM, n=4). Protein expression of and Wnt5a was determined by immunoblot analysis. Wnt5a/ protein ratio was determined using Image J program.

9 S3 A B mrna levels Body weight (g) P<.1 ND HF/HS (12W) P<.1 P<.1 Glucose (mg/dl) Wnt5a β-actin P<.1 ND HF/HS (12W) P<.5 Insulin (ng/ml) Wnt5a/ protein ratio P< P<.5 ND HF/HS (12W) ND HF/HS/12W HF/HS/24W C mrna levels P<.1 P<.5 TNFα P<.5 P<.5 P<.5 P<.5 gp91 phox P47 phox P<.5 P<.5 P<.1 P<.1 F4/8 CD68 P<.5 GRP78 ND HF/HS/12W HF/HS/24W P<.5 P<.5 P<.1 CHOP Figure S3: Expression of and Wnt5a and metabolic parameters in lean and obese mice. (A) Expression of and Wnt5a in epididymal fat tissue in wild-type () mice fed normal diet (ND) or high-fat/high sucrose (HF/HS) diet for 12 weeks. transcript levels were measured by QRT-PCR analysis and expressed relative to 36B4 (n=6-7). Protein expression of and Wnt5a was determined by immunoblot analysis. (B) Body weight, serum glucose and serum insulin levels in lean and obese mice (mean ± SEM, n=5-6). (C) Transcript levels of TNFα, oxidative stress markers (gp91 phox and P47 phox), macrophage markers (F4/8 and CD68) and ER stress markers (GRP78 and CHOP) in epididymal fat tissue in lean and obese mice. mice at the age of 1 week were fed ND for 12 weeks or HF/HS diet for 12 or 24 weeks. Gene expression levels were measured by QRT-PCR analysis and expressed relative to 36B4 (mean ± SEM, n=5-6).

10 S4 Crown-like structure (-) Crown-like structure (+) mrna levels P<.5 TNFα mrna levels P<.5 HOMA-IR P<.1 Figure S4: transcript levels in visceral adipose tissue in human subjects. Expression of and TNFα, and homeostasis model assessment of insulin resistance (HOMA-IR) were assessed in obese subjects with or without macrophage crown-like structures in visceral fat tissue as determined by immunohistochemical stains with CD68. Transcript levels of and TNFα were measured by QRT-PCR analysis and expressed relative to 36B4 (mean ± SEM, n=18).

11 S5 Body weight (g) P<.5 KO Food intake (kcal/day) N.S KO Glucose (mg/dl) P<.5 KO 2. P<.1 4 P< P<.5 Insulin (ng/ml) 1..5 Liver weight (g) Fat weight (g) KO KO KO Figure S5: Body weight, food intake, serum glucose, serum insulin, liver weight and fat weight in wild-type () and -/- (KO) mice after 12 weeks of HF/HS diet feeding (mean ± SEM, n=9-12).

12 S6 A B mrna levels KO P<.1 P<.1 F4/8 CD68 F4/8 CD68 ND HF/HS mrna levels KO P<.1 P<.1 P<.1 TNFα IL-6 MCP1TNFα IL-6 MCP1 ND HF/HS C mrna levels N.S CyclinD1 N.S WISP2 KO Figure S6: Expression of macrophage marker, pro-inflammatory mediator and canonical Wntrelated genes in fat tissues from wild-type () and -/- (KO) mice. (A) Gene expression of F4/8 and CD68 in epididymal adipose tissue from and KO mice receiving normal diet (ND) or HF/HS diet was quantified by QRT-PCR and expressed relative to 18S levels (mean ± SEM, n=6-7). (B) Gene expression of TNFα, IL-6 and MCP-1 in stromal vascular fraction from epididymal fat tissues of and KO mice when fed a normal diet (ND) or HF/HS diet for 12 weeks. Transcript levels were quantified by QRT-PCR and expressed relative to 18S levels (mean ± SEM, n=4). (C) Quantification of mrna levels of CyclinD1 and WISP2 in adipose tissues of and KO mice by QRT-PCR methods (mean ± SEM, n=9). Gene expression levels were presented relative to 18S levels.

13 S7 A β-gal Media Cell B TOPflash reporter activity Wnt5a N.S N.S C 2. P<.1 D 2. β-gal P<.1 IL-6 mrna levels 1..5 IL-6 mrna levels 1..5 Wnt5a Wnt5a β-gal Vehicle SP6125 Figure S7: Effect of on TOPFlash reporter activity and IL-6 expression in adipocytes. (A) Increased production of in cell lysate and media of 3T3-L1 adipocytes after overexpression of. Differentiated 3T3-L1 adipocytes were transduced with AdTRE-β-gal or AdTRE- along with AdCMV-tTA. After 48 h of transduction, cells and media were collected, and immunoblot analysis was performed. (B) Effect of on TOPFlash reporter activity. Differentiated 3T3-L1 adipocytes were transduced with AdTRE-β-gal or AdTRE- along with AdCMV-tTA for 24 h, and co-transfected with TOPflash and Renilla luciferase control constructs. At 48 h after transduction, cells were treated with Wnt5a (2 ng/ml) or vehicle for 24 h. Reporter activity was analyzed by using dual luciferase assay kit (mean ± SEM, n=6). (C) Effect of on Wnt5a-stimulated IL-6 expression in adipocytes. After 48 h of transduction with adenoviral vectors, cells were treated with Wnt5a (2 ng/ml) or vehicle for 24 h. Transcript levels were quantified by QRT-PCR and expressed relative to 36B4 levels (mean ± SEM, n=4). (D) Effect of JNK inhibitor on Wnt5a-induced IL-6 expression in adipocytes. Differentiated 3T3-L1 adipocytes were pretreated with SP6125 (15 µm) or vehicle and stimulated with Wnt5a (2 ng/ml) or vehicle for 24 h. Transcript levels were quantified by QRT-PCR and expressed relative to 36B4 levels (mean ± SEM, n=4).

14 S8 A P<.1 2. P<.5 TNFα mrna levels IL-6 mrna levels 1..5 Wnt5a Wnt5a CM-β-gal CM- CM-β-gal CM- B TNFα mrna levels P<.1 IL-6 mrna levels P<.5 Wnt5a Wnt5a Vehicle SP6125 Vehicle SP6125 Figure S8: Role of in regulation of Wnt5a-induced expression of pro-inflammatory mediators in macrophages. (A) Effect of the conditioned media from -transfected adipocytes on Wnt5astimulated cytokine expression in macrophages. Mouse peritoneal macrophages were stimulated with Wnt5a (2 ng/ml) or vehicle for 24 h in the presence of the conditioned media from 3T3-L1 adipocyte transduced with AdTRE-β-gal or AdTRE- along with AdCMV-tTA. (B) Involvement of JNK in Wnt5a-induced expression of pro-inflammatory mediators in macrophages. Peritoneal macrophages were pretreated with SP6125 (15 µm) or vehicle and stimulated with Wnt5a (2 ng/ml) or vehicle for 24 h in the presence of the conditioned media from 3T3-L1 adipocyte transduced with AdTRE-β-gal and AdCMV-tTA. Gene expression levels were quantified by QRT-PCR and expressed relative to 36B4 levels (mean ± SEM, n=4).

15 S9 A -KO Jnk1-KO /Jnk1-DKO B -KO Jnk1-KO /Jnk1-DKO Body weight (g) P<.5 P<.1 P<.1 N.S mrna levels P<.5 P<.5 P<.1 P<.1 P<.1 P<.1 TNFα IL-6 P<.5 P<.1 P<.1 MCP-1 Figure S9: Body weight and expression of pro-inflammatory mediators of fat tissue in wild-type (), -/- (-KO), Jnk1 -/- (Jnk1-KO) and -/- Jnk1 -/- (/Jnk1-DKO) mice (A) Body weight of, -KO, Jnk1-KO and /Jnk1-DKO mice maintained on a high-fat/high sucrose (HF/HS) diet for 12 weeks (mean ± SEM, n=6-1). (B) Gene expression of TNFα, IL-6 and MCP-1 in epididymal fat tissues from, -KO, Jnk1- KO and /Jnk1-DKO mice after 12 weeks of the HF/HS diet feeding. Transcript levels were quantified by QRT-PCR and expressed relative to 18S levels (mean ± SEM, n=6-7).

16 S1 A Serum β-gal B Glucose levels (mg/dl) Time (min) +β-gal KO+β-gal + KO+ Figure S1: Effect of systemic delivery of on glucose metabolism in the HF/HS diet-fed wild-type () and -/- (KO) mice. After 1 weeks of HF/HS diet feeding, and KO mice were intravenously treated with AdTRE-β-gal (2.5x1 8 pfu total) or AdTRE- (2.5x1 8 pfu total) along with AdCMV-tTA (2.5x1 8 pfu total). (A) Detection of in serum of mice following adenovirus-mediated intravenous injection of. At 1 week after treatment of mice with adenoviral vectors (β-gal or ), serum was collected. protein level in serum (1 µl) was determined by immunoblot analysis. (B) Glucose tolerance test (left) and insulin tolerance test (right) at 2 weeks after injection of adenoviral vectors (β-gal or ) (mean ± SEM, n=6-7 in each group)., P<.1 vs. corresponding β-gal treatment. Glucose levels (% of control) Time (min)

17 S11 A mrna levels ob/ob+β-gal ob/ob+ TNFα IL-6 MCP1 F4/8 CD68 B P-JNK β-actin β-gal Figure S11: Effect of systemic delivery of on gene expression of pro-inflammatory mediators and macrophage markers (A) and phosphorylation of JNK (B) in epididymal fat tissue from ob/ob mice at 2 weeks after treatment with adenoviral vectors (β-gal or ). Gene expression was quantified by QRT-PCR analysis (n=5)., P<.1 vs. β-gal treatment.

18 Supplementary References S1. W. Satoh, M. Matsuyama, H. Takemura, S. Aizawa, A. Shimono, Genesis 46, 92 (28). S2. Y. Izumiya et al., Cell Metab 7, 159 (28). S3. C. M. Apovian et al., Arterioscler Thromb Vasc Biol 28, 1654 (28). S4. N. Maeda et al., Diabetes 5, 294 (21). S5. T. Mano et al., Hum. Gene Ther. 11, 1625 (2). S6. N. Maeda et al., Nat. Med. 8, 731 (22). S7. R. Shibata et al., Nat. Med. 1, 1384 (24). S8. E. G. Bligh, W. J. Dyer, Can J Biochem Physiol 37, 911 (1959).