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1 Supporting Information Herrero et al..73/pnas.9537 SI Materials and Methods Mice ap2-nsrp-c transgenic males (strain 3393, 57L/6J x SJL) were purchased from Jackson Laboratories and crossed to P8, using WT 57L/6J females to avoid potential consequences of maternal diabetes; offspring were genotyped by PR following established protocols. Study mice had free access to water and chow (Purina 58). In selected experiments -wk-old 57L/6J mice were fed for 6 weeks with either standard chow (Research iets 245, % kcal fat) or H (Research iets 2492, 6% kcal fat); -wk-old ob/ob and control littermates (Jackson Laboratories) were maintained on standard chow for 2 weeks in our facility to acclimatize prior the experiments. Sodium salicylate (4 g/kg) was incorporated into Purina 58 chow by Harlan Teklad. Lipopolysaccharide (4 pg/g body weight,. coli :4, SIGMA L32) was injected intraperitoneally and mice were killed 2 h later. LyzM-re and Ikkβ lox/lox mice on 57L/6J backgrounds were crossed to generate Ikkβ Δmye mice with myeloid-specific deletion of Ikkβ (). ap2-nsrp-c transgenic males were crossed with Ikkβ Δmye females to generate /Ikkβ Δmye mice and control /Ikkβ lox/lox and WT/Ikkβ lox/lox littermates. All mice were housed under alternating 2 h light and dark cycles, and experiments were conducted in accordance with the National Institutes of Health guidelines under protocols approved by the Joslin Institutional Animal are and Use ommittee. irculating Metabolites lood samples were obtained from the tails of unanesthetized mice after an overnight 6 h fast. lood glucose concentrations were measured using a Glucometer lite (ayer). Serum insulin (rystal hem), TNα and IL-6 (Invitrogen, iosource), and IL- (R&) were measured by LISA in the Joslin R Assay ore. Serum MP-, leptin, and resistin were measured by Luminex (Linco). Quantitative Real-Time RT-PR Total RNA was extracted from frozen, pulverized mouse tissue using RNeasy (QIAGN); cna was synthesized using hexamer primers with the Advantage RT-for-PR kit ( iosciences). PR amplifications using SYR Green or TaqMan Universal PR Master Mix (Applied iosystems) were normalized against 8S and TATA box binding protein (Tbp). Primers from Applied iosystems include 8S (43893), TNα (Mm m), IL-6 (Mm4469-m), XL (Mm m), IL-2α (Mm43465-m), IL-Rα (Mm m), IL-Rβ (Mm m), TGβ (Mm44724-m), and IL- (Mm43966-m). orward/reverse primers for other genes: Adiponectin: 5 -TAAAGGGAAT-3 /5 -TGT- ATTAAATTTGTT-3 Arginase : 5 -TAAGAAAGTTTAGAG-3 /5 - AGGAGTGTATTAGGGAAT-3 4/8(mr): 5 -TTTTGTGTTTT-3 /5 -- GTTTGTATTAA-3 Iκα: 5 -GGGATGGTAAGAAGGA-3 /5 - TGAAG- TGAGGAAGA-3 IL-β: 5 -GATTTGTGATAT-3 /5 -AGGA- AGGTATTTTGTG-3 Leptin: 5 -TAAGAGTGTATAGA-3 /5 -AAG- AGGAATGAAGTA-3 MP: 5 -TAATGAGTAGGTGGAG-3 /5 -AAGTG- TTGAGGTGGTTGTG-3 Mgl: 5 -TGAGAAAGGTTTAAGAATGGG-3 /5 -GA- ATGTAGTGATGTGGG-3 Mgl2: 5 -TTAGAATGTGTTAGTGG-3 /5 -GGT- AATTTTGAAAT-3 Mrc2: 5 -TAAGTAGTATGGATT-3 /5 -ATT- AGTTGAGGTAT-3 Nos2: 5 -AGTGGGTGTAAAATT-3 /5 -ATTGG- AAGTGAAGGTTTG-3 PU.: 5 -GGATGTGTTTTATAAA-3 /5 -TGA- TTTTTATGTGT-3 Resistin: 5 -TTTTTTTTTTTTTG-3 /5 -AG- ATGTGTGTTTGGG-3 Tbp: 5 -ATTAAATGATTATG-3 /5 -TGAT- GAGAAATGTTGG-3 UP-: 5 -ATAGGATTGGTTAG-3 /5 -GGGG- TTTGATATGAGA-3 Ym/hi3l3: 5 -AGAAGGGAGTTTAAATGGT-3 /5 - GTTTGTATGTGTGTAAGTGA-3 Immunohistochemistry Immunohistochemistry was performed using 4 μm thick formalinfixed, paraffin-embedded tissue sections done at the Histology ore from the Joslin iabetes enter. Immunostaining was performed at the Longwood Specialized Histopathology ore at the righam and Women's Hospital. riefly, slides soaked in xylene were passed through graded alcohols and into distilled water. Slides were then pretreated with mm TA, ph 8. for 3 or with mm citrate, ph 6. for Mac2, 22 and aspase3 in a steam pressure cooker (ecloaking hamber, io- are Medical) followed by a distilled water wash as instructed by the supplier (Zymed). Subsequent steps were performed at room temperature in a hydrated chamber. Slides were pretreated with Peroxidase lock (AKO) for 5 min to quench endogenous peroxidase activity. ach of the following steps was conducted in AKO diluent for h. or macrophage staining: monoclonal rat anti-murine Mac2 (clone M3/38, edarlane) at :2,; 2 rabbit anti-rat Ig at :75. or T cells staining: polyclonal rabbit anti-murine 3 antibody (M363, ell Marque) at :,5. or cells staining: monoclonal rat anti-murine 22 (clone RA3-62, Pharmingen) at :2; 2 rabbit anti-rat Ig at :75. or apoptosis: monoclonal rabbit anti-murine aspase3 antibody (ell Signaling #9664) at :. or perilipin, guinea pig anti-perilipin (RI ivision of itzgerald, oncord, MA #RI- PROGP29) at :. Slides were washed in 5 mm Tris-l, ph 7.4, and detected with anti-rabbit nvision+ (AKO). After further washing, brown immunoperoxidase staining was developed using a A chromogen (AKO) and counterstained with hematoxylin (blue). or mast cells:.% toluidine blue (lectron Microscopy Sciences, #225) was applied for 2 seconds followed by washing steps with water and 95% ethanol. low ytometry pididymal WAT, inguinal s.c. WAT, and interscapular AT were excised and minced into ml of KR solution containing 2.5 mm hepes ph 7.4, 2 mm Nal, 6 mm Kl,.2 mm MgSO 4, mm al 2, 2% SA, and 2.5 mm glucose. ollagenase II (Sigma 6885; mg/ml) and Nase I (Sigma N25;.2 mg/ml) were added and incubated at 37 for 2 min with shaking.. M TA was added 5 min before the end of the incubation. Larger particles were removed using 25 μm nylon Herrero et al. of7

2 sieves and the filtrates were centrifuged at 3 g for 5 min to separate floating adipocytes from the stromal-vascular fraction (SV) pellets. Adipocyte fractions resuspended in 5 ml of KR were centrifuged a second time (3 g for 5 min at room temperature). The two SV pellets were combined, washed (3 g for 5 min at 4 ), resuspended in 3 μl of staining buffer (PS containing 2% S) containing clock ( iosciences), and cells were stained with the following conjugated antibodies (5 min at 4 in the dark): 45-P-y7, 4/8-biotin, 4-P, Ter9-AP, 3e-AP, 22-AP (all from -ioscience), and b-ap-y7, c-ap, and NK.-AP (- Pharmingen). iotinylated antibodies were visualized following a second incubation with SA-Alexa P-6 (invitrogen). Spleens were mashed in ml staining buffer and centrifuged (8 g for 3 min at 4 ); cells were treated with μl of AK lysis buffer (iowhitaked; min at room temperature), washed, and stained as described above. ells from adipose tissue SV or spleen were resuspended in 2 μl of staining buffer containing.2 μg/ml propidium iodide (Sigma), filtered through a μm mesh, and analyzed by AS (LSRII, iosciences). Sorted macrophages (b+4/8+) analyzed by AS (ASAria, iosciences) were collected in.5 ml of staining buffer. RNA was extracted with.5 ml TRIzol (Invitrogen) and precipitated with isopropanol. at Transplantation WAT transplantation experiments were performed as previously described (2), using 5-wk-old female recipients and 5-wk-old WT female littermate donors. riefly, recipients were anesthetized with ketamine/xylazine ( mg/kg; Henry Schein). onor parametrial fat pads (4 5 mg each, approximately 5 mg total) were transplanted s.c. through small incisions in the shaved back skin, piece per incision. Mice were housed individually for week after the surgery. To measure triglyceride content in transplanted mouse liver, pulverized frozen tissue (-2 mg) was homogenized in 5 μl PS. hloroform-extracted lipid was dried under a N 2 stream, redissolved in isopropanol, and triglyceride was quantified using the L-Type TG H kit (Wako). Adipocyte area was measured using the ImageJ software. N-κ Assay Nuclear proteins were isolated from liver and muscle. lectrophoretic mobility shift assays (MSA, Promega) and LISAs of N-k binding activity were performed as previously described (3). Oct activity was measured as a control transcriptional factor for all samples. TG ontent Liver and muscle triglycerides were extracted with chloroform and quantified (L-Type TG H kit, Wako). Statistics ata are presented as mean ± SM. Student t tests were used for statistical analysis. A probability level of P <.5 was considered to be statistically significant.. Arkan M, et al. (25) IKK-β links inflammation to obesity-induced insulin resistance. Nat Med : Gavrilova O, et al. (2) Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice. J lin Invest 5: ai, et al. (24) IKKbeta/N-kappa activation causes severe muscle wasting in mice. ell 9: wt (ng/ml).5 Adipokine.5 Leptin Resistin ig. S. irculating adipokines. irculating leptin and resistin concentrations in 36-wk-old male WT control (open bars) vs. (filled bars) littermates (n = 6). P <.5 WT vs.. Herrero et al. 2of7

3 A Vertebra Adipose tissue Larynx Spinal cord Thymus Aorta ronchus Heart sophagus Adipose tissue Lymph node ronchus Adrenal Lymph node Aorta Kidney G H Adipose tissue ig. S2. Nose-to-tail histological examination. An intracardiac perfusion fixation with formalin was performed in 6-wk-old male WT control and littermates. (A H) Histological sections (H&) from mice from the cervical (A, ), mediastinum (, ), retroperitoneum (, ), mesentery (G) and pelvic (H) areas. The identity of the tissues in each case is specified. Herrero et al. 3of7

4 WT N-κ binding (A.U.) 2 A Liver N-κ MSA.5.5 Iκα Liver Spleen mrna fold change epi WAT Leptin Adiponectin Resistin Leptin Adiponectin Resistin sc WAT.5.5 Leptin Adiponectin Resistin UP- AT ig. S3. N-κ binding activity and adipose tissue gene expression. (A) N-κ binding activity in liver and muscle from 8-wk-old male WT control (open bars) vs. (filled bars) littermates (n = 4). ( ) mrna expression in liver, muscle, spleen, epididymal (epi) WAT, s.c. (sc) WAT and interscapular AT of 8-wk-old male WT control (open bars) vs. (filled bars) littermates (n = 6). P <.5 WT vs.. Herrero et al. 4of7

5 5 4 25K 25K 2K 2K 94.8 SS-A A 5K K 5K 2K 25K S-W 5K K 5K 97 5K K 5K 2K 25K 5K K 5K PI 25K 2K 5K K 5K Larger cells Non-aggregates PI NK.-Ter9-4/8+b+ 25K 2K 5K K 5K AP (T,, NK and R) b /8 control H or Ob/Ob Macrophages (% SV) 5 5 G H Ob/Ob ig. S4. low cytometry analyses. Stromal-vascular cells from epididymal WAT were stained with fluorescent conjugated antibodies and analyzed by flow cytometry. (A ) ells were gated according to forward and side scatter (to eliminate debris, cell fragments, and aggregates), propidium iodide- (PI-, viability), 45+ (leukocytes), 3-, 22-, NK.-, and Ter9- (to eliminate T,, NK, and red blood cells, respectively) and 4/8+b+ (macrophages). (G) Quantitation of epididymal WAT macrophage numbers from H-induced and ob/ob models of obesity. Six-wk-old 57L/6J males were fed chow (control) vs. H for 6 weeks and killed at 22 weeks old. ontrols and ob/ob male littermates were 4 weeks old (n =5,P <.5). Herrero et al. 5of7

6 ig. S5. Liver and muscle TG content. (A) Liver triglyceride content of salicylate-treated mice. WT control (open bars) and (filled bars) littermates were fed normal chow (control) or chow containing 4 g/kg sodium salicylate for 5 weeks. (, ) Liver and muscle triglyceride content in male mice with myeloid-selective deletion of IKKβ. 24-wk-old WT control (open bars), (gray bars), and /Ikkβ Δmye (black bars) littermates were fed normal chow. n =4 7, P <.5. WT epi WAT A epi WAT epi WAT sc WAT AT WT G H I WT epi WAT H ig. S6. apoptosis. (A, ) Histological sections of epididymal WAT from 2-wk-old male WT control and littermate. Sections were stained with anti-perilipin antibody followed by colorimetric detection (brown) and counterstaining with hematoxylin (blue). () indicates dying adipocytes with perilipin-negative lipid droplets. ( I) Histological sections of epididymal WAT (, ), inguinal s.c. WAT (, G) and interscapular AT (, H) from a representative WT control and littermates. Sections were stained with anti-caspase 3 antibody followed by colorimetric detection (brown) and counterstaining with hematoxylin (blue). aspase-3 staining is also shown for a 22-wk-old 57L/6J male mouse fed H for 2 weeks (I). Herrero et al. 6of7

7 pididymal WAT A T cells (Anti-3) cells (Anti-22) Mast cells (Toluidine blue) wt wt ells / HP T cells cells 6 G 8 H epi sc AT epi sc AT Mast cells I epi sc AT ig. S7. histology. (A ) Histological sections from epididymal WAT from a representative 28-wk-old male WT control and littermates stained with anti-3 (T cells), anti-22 ( cells), and toluidine blue (mast cells) followed by colorimetric detection (brown) and counterstaining with hematoxylin (blue) for the case of T and cells. (G I) ounting of cells per high power field (HP) from the above male WT control (open bars) and (filled bars) littermates ( HP counted per mouse, n = 5 mice per genotype). P <.5 WT vs.. Herrero et al. 7of7