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1 Supporting Information Sun et al..73/pnas.4479 SI Materials and Methods High Fat iet (HF) and oxycycline (ox)-ontaining HF. For all of the HF feeding experiments, mice were fed with a diet containing 6% calories from fat (49; Research iets). The HF paste with 6 mg/kg ox also contains 6% calories from fat (S5867; io-serv). For low-dose ox treatment, the ox powder (Sigma) was mixed with HF paste (Research iets) to a final concentration of 6 mg/kg. Protein Analysis. Tissues were lysed by methods described previously (). The protein concentration was measured by a A assay bit (Pierce). Proteins were then separated with % or 4 % is-tris gels (Invitrogen) and transferred to PVF membranes (Millipore). For immunobloting, the primary antibodies for, β-actin (Santa ruz iotechology), UP- (Aam), PG-α (NOVUS iologicals), and adiponectin () were used followed by secondary antibodies labeled with infared dye emitting at 8 nm (Li-or ioscience). The blots were analyzed by Odyssey software (version.; Li-or ioscience). Serum samples were also analyzed by immunoblotting with the method above. In addition, they were measured with the following adipokine levels with ELISA kits: adiponectin (Millipore), leptin (Millipore), SAA3 (Millipore), and (R&). Liver Lipid ontent. Frozen tissues ( mg) were used for lipid extraction and measurement by the Metabolic ore Facility at the University of Texas Southwestern Medical enter. In brief, -mg pieces were snap frozen in liquid nitrogen until analysis. Lipids were extracted and the chloroform phase was adjusted to 5 ml total volume, and triplicates of 5 ml in combination with standards were dried down by the addition of ml of : chloroform Triton X- mix. Triglyceride (TG) levels were then assayed using Infinity reagent (Thermo Fisher Scientific). Histology. Tissues were excised and fixed in % formalin (PS buffered) for 4 h. Following paraffin embedding, the tissue sections were stained with hematoxylin and eosin (H&E) and Masson s trichrome using standard protocols. For immunohistochemistry, sections were stained with primary antibodies against 3 ( ioscience), receptor (R) and phosphorylated receptor (Y75; ell Signaling), F4/8 (Santa ruz), Mac- (edarlane Laboratories), and perilipin (Affinity ioreagents) and then followed by biotinylated secondary antibodies (antimouse, antirat, and antirabbit) (ako). inding of second antibodies was visualized using A chromogen A (ako) following the company s protocol. ounterstaining was performed with hematoxylin. All of the images were acquired with the oolscope microscopy (Nikon). Quantification of adipocyte areas was done on H&E stained section using ImageJ software (NIH). Indirect alorimetric Measurements. Mice were housed individually in metabolic chambers and maintained on a -h dark light cycle at to with lights on at 7: AM to 7: PM. Metabolic measurements were obtained continuously using LAMS metabolic chambers (LAMS System) in an open circuit indirect calorimetric system. All transgenic mice and their littermate control mice were provided with HF paste containing 6 mg/kg ox and water ad libitum. Quantitative Real-Time PR. Tissues were excised and snap frozen in liquid nitrogen. Total RNA were extracted from tissues in TRIzol (Invitrogen) using a TissueLyser (Qiagen) and then isolated using the RNeasy RNA extraction kit (Qiagen). The quality and quantity of the RNA were determined by absorbance at 6/8 nm. cna was prepared by reverse transcribing μgof RNA with SuperScript III reverse transcriptase and oligo (dt) (Invitrogen). qpr reactions were carried out on an AI Prism 79 HT sequence detection system (Applied iosystems). The primer sequences are listed in Table S. Results were calculated using the threshold cycle method, normalized by β-actin or hypoxanthine phosphoribosyltransferase (HPRT). ody omposition. one-free lean body mass, fat mass, and body fluid composition were measured in nonanesthetized mice using an Echo3-in- NMR (MRI) machine mini Spec (ruker). Systemic Metabolic Tests and lood hemistry. For the oral glucose tolerance tests (OGTTs), mice were fasted for 3 h before administration of glucose (.5 g/kg body weight) by gastric gavage. At the indicated time points, tail venous blood samples were collected in heparin-coated capillary tubes. Glucose levels were measured using an oxidase-peroxidase assay (Sigma-Aldrich). Mice did not have access to food throughout the experiment. For insulin tolerance tests (ITTs), mice were fasted for 3 h before i.p injection of insulin (Sigma) at mu/kg body weight. At the indicated time points, tail venous blood samples were collected for glucose level analysis. For lipid clearance assay, mice were fasted for 3 h and then gavaged with % Intralipid (axter Healthcare). At the indicated time points, tail venous blood samples were collected. Serum TG levels (Infinity; Thermo Fisher Scientific) and free fatty acid (FFA) levels (NEFA-HR) () (Wako Pure hemical Industries) were assayed following a 3-h fast. In Vivo Hypoxia etection. The mice were injected (i.p.) a Hypoxyprobe- solution (Hypoxyprobe) at a dose of 6 mg/kg body weight. Ten minutes later, the mice were anesthetized and the tissues were fixed in % formalin PS for 4 h. The detection of Hypoxyprobe- was by a Hypoxyprobe- Plus kit (Hypoxyprobe) following the supplier s protocol. Lectin-Labeling, Immunofluorescent Staining, and Imaging of AT Vasculature. Functional blood vessels were labeled in mice by injecting with. ml of mg/ml rhodamine (for HF-fed mice) or biotinylated (for ob/ob mice) Griffonia (andeiraea) simplicifolia lectin I (Vector Laboratories) ( μg/i.v. injection per mouse) through the tail vein. The injected lectin was allowed to circulate for 3 min before the animal was perfused through the left ventricle with % paraformaldehyde. AT and liver were then excised and fixed in % PS-buffered formalin overnight and then embedded in paraffin and cut into 5-μm sections. The lectin was visualized directly (with rhodamine) or using a y3-labeled streptavidin (Vector Labs), whereas nuclei were stained with API (4,6-diamidino--phenylindole) (Sigma-Aldrich). Tissue sections were mounted on slides using Vectashield mounting media for fluorescence (Vector Labs). Lectin-stained capillaries were visualized by either confocal (Leica TS SP5 cofocal microscope) or epifluorescence microscopy (Zeiss AxioObserver wide-field epifluorescence microscope).. Halberg N, et al. (9) Hypoxia-inducible factor alpha induces fibrosis and insulin resistance in white adipose tissue. Mol ell iol 9: Halberg N, et al. (9) Systemic fate of the adipocyte-derived factor adiponectin. iabetes 58: Sun et al. of8

2 Adiponec n Promoter rtta α--a +OX rtta teto promoter AT Liver wt Apn-rtTA -OX TRE- Apn-rtTA XTRE- wt Apn-rtTA +OX TRE- Apn-rtTA XTRE- α ng/ml Serum ( ELISA) HPRT Fig. S. haracterization of AT-specific ox-inducible -A Tg mice. (A) Schematic representative of adiponectin promoter-driven ox-inducible -A transgenic mice. The ox-dependent promoter driven -A is regulated by rtta, whose expression is under the control of adiponectin promoter. -A in the double transgenic mouse is induced in the presence of ox. () q-pr analysis with the transgenic-specific primers showed that ox induces the expression of the transgene only in Apn rtta and TRE -A double transgenic mice. The readings were normalized to hypoxanthine phosphoribosyltransferase. () Western blot analysis of -A levels with α- in different fat pads and liver in -Tg or littermate control mice after ox induction for 9wk(n = per group). () Serum -A levels in -A Tg and control mice tested by an ELISA kit (n = 4 in control; n = 5 in Tg). A gram w % ody Weight (wks) Fat Mass control transgenic ody Fluid ITT (Glucose) control transgenic (Mins) AT mg/dl mg/dl Fas ng Glucose Fas ng Insulin ng/ml.5 Fig. S. -A Tg mice exhibit higher density of vasculature and improved metabolic profile. (A) ody weight changes and MRI analysis of bone-free body compositions in Tg and littermate control mice after HF plus ox treated for 6 wk. (n = 5 per group). P <.5. () omparison of different fat pads from Tg mice and control littermates after ox treatment for 8 wk. The WAT in -A treated mice showed highly dense vasculature. () Glucose levels in an insulin tolerance test (ITT) with Tg and their littermate control group (n = 5 per group). P <.5; P <.. () Fasting glucose and insulin levels in Tg and their littermate control groups. The mice were fasted for h before the tail blood was collected after HF plus ox treatment for 8 wk (n = 5 per group). P <.. Sun et al. of8

3 mg/dl Lipid learance (Hrs) Serum NEFA mg/g meq/ug Liver holesterol Liver NEFA mg/g Liver TG meq/l.5 Tg E Rela ve value LPL(q-PR ) LPL(q-PR heart) 4 Rela ve value Fig. S3. -A Tg mice have increased lipid clearance and decreased HF-induced hepatic steatosis. (A) Serum triglyceride levels measured in a lipid clearance test on -A Tg and control littermates after HF plus ox treatment for 8 wk. The differences at all of the time points are significant by Student s t test with P <.. () Serum NEFA in Tg and control littermates after HF plus ox treatment for 8 wk (n = 4 in control; n = 5 in -A Tg). P <.. () Liver cholesterol, triglyceride, and NEFA in -A Tg and their control littermates after HF plus ox treatment for 8 wk (n = 4 in control; n =5in -A Tg). P <.5; P <.. () H&E staining of the liver tissue in -A Tg and control littermates. (Left) HF-treated liver have more and bigger lipid droplets than (Right) -A Tg liver. (E) q-pr analysis of lipoprotein lipase (LPL) in Tg mice and their littermate controls (n = 4 in each group) in (Left) and heart tissue (Right). The readings were normalized with the HPRT gene. P <.5; P <.. Sun et al. 3of8

4 5 Fat Size 3 Fat Size Area (μm ) 5 PPARγ (q-pr) ZFP43 (q-pr) rela ve value PGα (Quan fica on for W).7 fold 6 fold UP (Quan fica on for W) 6.fold 34.8 fold Fig. S4. Local overexpression of -A in AT decreases adipocyte size and enhances a AT-like phenotype in WAT. (A) Quantitative measurement of the fat size in Tg and their littermate control groups after HF plus ox treatment for 8 wk (H&E staining is shown in Fig. 3A). P <.. () q-pr analysis of adipogenic genes PPARγ and ZFP43 in Tg mice and their littermate controls. The readings were normalized with HPRT gene (n = 4 in control, n =5in Tg). () Quantitative measurements of the immunobloting bands for PG-α and UP- in Fig. 3 by ImageJ software from NIH. Sun et al. 4of8

5 rela ve value rela ve value Lep n (q-pr ) Apn (QPR ) Serum Levels ng/ml Lep n (Serum ELISA) α-adiponec n Tissue Lysates AT Liver AT Liver α-adiponec n 5 serum AT Fig. S5. -A Tg mice have lower serum leptin and adiponectin levels. (A) q-pr analysis of leptin mrna levels in of Tg and their littermate control mice after HF plus ox treatment for 8 wk (Left). P <.5; and ELISA analysis of circulating leptin levels in both groups (n = 4 in control; n = 5 in Tg, Right). P <.. () q-pr analysis of adiponectin mrna levels in in Tg and their littermate groups (n = 4 in control; n = 5 in Tg). () Western blot analysis for serum adiponectin levels (Upper) and tissue levels (Lower) with α-adiponectin (n = 4 for control, n = 5 for Tg) after HF plus ox treatment for 8 wk. () Quantitative measurements of the bands in with ImageJ software. P <.. Sun et al. 5of8

6 SAA3 (ELISA) Trichrome staining 5 Quan fica on of crown-like structures in WAT % 5 Fig. S6. -A Tg mice decreases the abnormal EM development and local inflammation in WAT. (A) irculating SAA3 levels analyzed by ELISA in -A Tg and their littermate controls after HF plus ox treatment for 8 wk (n = 4 in control; n = 5 in Tg). P <.5. () Trichrome staining of WAT in -A Tg mice and littermate controls. The positive staining (blue color) is indicated by the arrows in the control HF-fed mice. () Quantitative measurements of the numbers of crowns for Fig. 4 and for fat pads (image data not shown). A 35 IgG ody Weight IgG IgG g Relative value (ays) (q-pr) IgG IgG p=.5 5 Quan fica on of the W p=.6 IH with an -Mac IgG IgG IgG An -Apn AT IWAT Liver Fig. S7. lockade of angiogenesis during early stage of fat expansion impairs adiponectin secretion. (A) ody weight changes during and control IgG treatment under HF challenge (n = 5 per group). () q-pr analysis of 3 in fat tissues and liver (n = 4 per group). () Western blot analysis of the serum from - and control IgG-treated groups with antiadiponectin (Upper, n = 4 in control; n = 5 in Tg) and quantitative measurements of the immunoblot band density by ImageJ software (Lower). () Histoimmunochemical staining of Mac, a macrophage marker in in and control IgG mice after antibody treatment for 7 wk. Red arrows indicate the crown-like structures. Sun et al. 6of8

7 5 ob/ob mice ody weight.5 3 (q-pr) 45. g IgG Rela ve Value. 3 4 wks.5. IgG IgG Fig. S8. blocks 3 levels in ob/ob mice. (A) ody weight change in ob/ob mice treated with or control IgG via i.p. injection. The -treated mice gained less body weight starting from the second week of treatment. () q-pr analysis of 3 levels in WAT of - and control IgG-treated mice (n = 5 per group). (mg/dl) Lec n staining IgG OGTT (glucose) IgG mins Rela ve values F4/8 MP- IH with an -perilipin IgG mg/dl Glucose p=.5 mmol/l Serum FFA Fig. S9. treatment enhances insulin sensitivity to improve the metabolic phenotype in ob/ob mice. (A) Functional blood vessels in AT labeled by biotinylated lectin- in mice treated with or control IgG for 3 wk. lood vessels are shown in red, the nuclei in blue, by API staining. () Glucose levels in an OGTT in mice treated with or control IgG for 3 wk. () Fasting glucose and serum FFA levels in - or control IgG-treated mice. Mice were fasted for h before serum glucose and FFA levels analyzed (n = 5 per group). P <.5. () q-pr analysis of inflammatory factor MP- and macrophage marker F4/8 (n = 5 per group). P <.5. (E) Immunohistochemical analysis of perilipin in of - and control IgG-treated mice. Sun et al. 7of8

8 Table S. Gene name Primers for q-prs Sequences -A 3 R olα ol3α ol6α HIFα LOX F4/8+ MP- TNF-α IL-6 β-actin Nur77 PPARγ Pref- ZFP43 SAA3 FOXO HPRT F 5 -GGAGATTTGAGGAGATT-3 ; R5 -GGGATTTAGAGAGATATAAGAA-3 F 5 -ATGAAGAAATTAA-3 ; R5 -GAAGGATGGAAATAAAA-3 F 5 -TGTATATGTTT-3 ; R5 -AAGGAATATGTTG-3 F 5 -GTGTTGGTATTGTGGT-3 ; R5 -AAGGAATATGTTG-3 F 5 -GGGTTTTGGTTAAAg-3 ; R5 -TGGTTTATTTTT-3 F 5 -GATGAGGGTGAAGTGGGAGA-3 ; R5 -AGAGAAGAGGATGTAA-3 F5 -AAGATTGGGAAGAA-3 ; R5 -GGTGAGTATAAAGAAGTTT-3 F 5 -AAGATGGAGAATTA-3 ; R5 -AGTTGTTTGTGGTTA-3 F 5 -TTTGGTATGGGTTAGT-3 ; R5 -GAAGGAGGAAGAGTTTATGTG-3 F 5 - AGAAGAATTA-3 ; R5 - TTGGAATTTTTTG-3 F5 -GAGAAAGTAATTTTG-3 ; R5 - GAAGATTAGGTATATG-3 F 5 - AGAGATAAAAGAAATGATGG-3 ; R5 - ATAGAAGAAGAGGAAAT-3 F 5 -TAAAGGATTGTGATGG-3 ; R5 -TTTGATGTAGAGATTT-3 F 5 -GTTGGAATTGAA-3 ; R5 -ATAGTGTGAAGAAGATTGT-3 F5 -TGAGGAGAGAGAAAG-3 ; R5 -GAGAGAGTATTGGTATT-3 F 5 -AGTGATTGAAGGATGGTG-3 ; R5 -TTGTGTGGAGTTTTAG-3 F 5 -GAGAGAGAGAAGTGAG-3 ; R5 -GAATAGTGGAGAGGA-3 F 5 -TGGGTGTGTAAAGTAT-3 ; R5 -AAGTAGTTGTTT-3 F 5 -GAGTGAGAATGAAGGAATG-3 ; R5 -TTAGAAAATGAGAAGGGTG-3 F 5 -AGAGTAAGAAAA-3 ; R5 -TTTGGTTTTAGTTTA-3 Sun et al. 8of8