SUPPLEMENTARY DATA Materials GSK0660 and GW501516 Were Purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Tocris Bioscience (Bristol, UK), Respectively

SUPPLEMENTARY DATA Materials GSK0660 and GW501516 were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Tocris Bioscience (Bristol, UK), respectively. Insulin, puromycin, isopropyl β-D-1- thiogalactopyranoside (IPTG ), propidium iodide (PI), mouse anti-Flag monoclonal antibody, rabbit anti- β-actin polyclonal antibody, and HRP-conjugated goat anti-rabbit IgG antibody were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Recombinant human IL-6 was obtained from R&D Systems (Minneapolis, MN, USA). A monoclonal anti-HA antibody and polyclonal antibodies specific for phospho-Akt (Ser4473), Akt, and TCPTP45 were obtained from Cell Signaling (Beverly, MA, USA). Monoclonal antibodies specific for PTP1B, TCPTP, phospho-STAT3 (Tyr705), STAT3, and a polyclonal rabbit anti-SOCS3 antibody were obtained from Abcam (Cambridge, MA, USA). Polyclonal antibodies specific for phospho-IRS1 (Tyr612), IRS, and Glut4 were obtained from Millipore (Bedford, MA, USA). A rabbit anti-phospho-IR (Tyr1162/1163) polyclonal antibody was obtained from Invitrogen (San Diego, CA, USA). Polyclonal antibodies specific for PPAR α, PPAR δ, and PPAR γ, and monoclonal antibodies specific for α-tubulin, lamin B, and GST, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ND (4.3% fat, 67.3% carbohydrate, and 19.2% protein) were purchased from Altromin Spezialfutter GmbH & Co. (Lage, North Rhine-Westphalia, Germany) and a HFD (24% fat, 41% carbohydrate, and 24% protein (206.8 g/kg lard)) were purchased from Research Diets Inc. (New Brunswick, NJ, USA).

Cell culture and differentiation The human hepatoblastoma-derived HepG2 and HEK293T cells, and mouse 3T3-L1 preadipocytes, were obtained from the Korean Cell Line Bank (Seoul, Korea), and mouse C2C12 myoblast cells were a gift from Dr. Eun Jung Cho (School of Pharmacy, Sungkyunkwan University, Suwon, Korea). All were maintained in Dulbecco’s modified Eagle’s medium (DMEM). CHO-IR cells were kindly provided by Dr. Deok-Bae Park (Department of Medicine, Jeju National University, Jeju, Korea) and cultured in Ham’s F-12 medium. The primary hepatocytes from mouse were isolated by collagenase perfusion method as described previously (1) and then cultured in DMEM/F-12 medium supplemented with 0.1 µM dexamethasone, 10 mM nicotinamide, 1% insulin-transferrin-selenium premix, and growth factors. All cells were maintained in medium containing antibiotics and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air. For the differentiation of C2C12 myoblasts, cells were grown to near 100% confluency in normal DMEM, as described above, and then incubated in DMEM containing 2% horse serum for 3 days to induce differentiation into myotubes, as described previously (2). To differentiate 3T3-L1 preadipocytes, cells were cultured to near 100% confluence, then incubated in DMEM containing MDI (250 nM dexamethasone, 500 µM 3-isobutyl-1-methylxanthine, and 200 nM insulin) solution for 10 days to induce differentiation into adipocytes, as described previously (3). For further analysis, the myotubes, adipocytes, and HepG2 cells were incubated in serum-free DMEM for 16 h, and then treated with the indicated reagents for the indicated periods of time.

Plasmid construction and transfection To express epitope-tagged proteins, Flag- or HA-tagged pcDNA3.1 vector was used (Stratagene, La Jolla, CA, USA). First-strand synthesis of full-length cDNAs for human PPAR δ (GenBank accession no. NM_006238.4), mouse PPAR δ (GenBank accession no. NM_011145.3), mouse PPAR α (GenBank accession no. NM_011144.6), mouse PPAR γ (GenBank accession no. NM_001127330.2), human TCPTP48 (GenBank accession no. NM_002828.3), human TCPTP45 (GenBank accession no. NM_080422.2), and mouse TCPTP45 (GenBank accession no. NM_008977.3) was performed using 1 µg of total RNA and TOPscript RT DryMIX kit (Enzynomics, Seoul, Korea). Reverse transcription was followed by 25 cycles of PCR amplification using the primers 5 ʹ - CTGAAGCTTATGGAGCAGCCACAGGAG-3ʹ and 5 ʹ -AGTTCTAGATTAGTACATGTCCTTGTA- 3ʹ (human PPAR δ), 5 ʹ -GCCAAGCTTATGGAACAGCCACAGGAG-3ʹ and 5 ʹ -

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA GCCGATATCTTAGTACATGTCCTTGTAG-3ʹ (mouse PPAR δ), 5 ʹ - GCCAAGCTTATGGTGGACACAGAGAG-3ʹ and 5 ʹ -GCCGATATCTCAGTACATGTCTCTGTAG- 3ʹ (mouse PPAR α), 5ʹ -GCCAAGCTTATGGTGGACACAGAGAG-3ʹ and 5 ʹ - GCCGATATCTCAGTACATGTCTCTGTAG-3ʹ (mouse PPAR γ), 5ʹ - CTGGGATCCATGCCCACCACCATCGAG-3ʹ and 5 ʹ -AGTCTCGAGTTATAGGGCATTTTGCTG- 3ʹ (human TCPTP48), 5 ʹ -CTGGGATCCATGCCCACCACCATCGAG-3ʹ and 5 ʹ - AGTGATATCTTAGGTGTCTGTCAATCT-3ʹ (human TCPTP45), and 5ʹ - CTGGGATCCATGTCGGCAACCATCGAG-3ʹ and 5 ʹ -AGTCTCGAGTTAGGTGTCTGTCAATCT- 3ʹ (mouse TCPTP45). Each PCR product was digested with appropriate restriction enzymes, and ligated into the similarly digested pcDNA3.1/Flag or pcDNA3.1/HA vector to yield the expression vectors pcDNA3.1-Flag-hPPAR δ, pcDNA3.1-Flag-mPPAR δ, pcDNA3.1-Flag-mPPAR α, pcDNA3.1- Flag-mPPAR γ, pcDNA3.1-HA-hTCPTP48, pcDNA3.1-HA-hTCPTP45, and pcDNA3.1-HA- mTCPTP45, respectively. The pMT2-hTCPTP45 D182A plasmid was a gift from Dr. Nicholas K. Tonks (Cold Spring Harbor Laboratory, New York, USA). For GST-fusion proteins, full-length hTCPTP45 cDNA was cloned into the Bam HI and Xho I sites of the pGEX4T-1 vector (GE Healthcare Life Sciences, PA, USA) to yield the GST-fusion vector pGEX4T-1-hTCPTP45. Deletion mutants of GST-hTCPTP45 were constructed by PCR amplification of fragments from pGEX4T-1-hTCPTP45. These fragments were digested with Bam HI/ Xho I and ligated into the similarly digested pGEX4T-1 vector, generating GST-hTCPTP45 (D1) and GST-hTCPTP45 (D2). Site-directed mutants of GST-hTCPTP45, GST- hTCPTP45 350-A3 (R350A, K351A, and R351A), GST-hTCPTP45 378-A3 (K378A, R379A, and K380A), and GST-hTCPTP45 350/378-A3 (R350A, K351A, R351A, K378A, R379A, and K380A) were created using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) and pGEX4T-1-hTCPTP45 plasmid. HA- tagged hTCPTP45 350/378-A3 was generated by PCR amplification of GST-hTCPTP45 350/378-A3 using the primers 5 ʹ - CTTAAGCTTATGAGCGATAACGATGAC-3ʹ and 5 ʹ - CCATCTAGATCAGCTCTCGCTTTCCCCTT-3ʹ . The PCR product was digested with Hind III and Xba I, and ligated into the similarly digested pcDNA3.1/HA vector to yield the expression vector pcDNA3.1-HA-hTCPTP45 350/378-A3 . For the localization assay, GFP-hPPAR δ, DsRed-hTCPTP45 D182A , and DsRed-hTCPTP48 D182A were generated using pEGFP-C1 and pDsRed-Express-C1 (Clontech Laboratories, Inc., CA, USA). All recombinant plasmids were sequenced and verified. HepG2 and HEK293T cells were transfected with the indicated plasmids using Genefectin (Genetrone Biotech, Gwangmyeong, Korea) for 6 h, and the existing medium was then replaced with fresh medium. After incubation for an additional 48 h, the cells were stimulated with the indicated reagents for the specified period of time.

Bacterial expression of GST-fusion proteins and GST pull-down assay The binding of PPAR δ to TCPTP45 was assessed using bacterially expressed GST-fusion proteins essentially as previously described (4). Briefly, mouse full-length GST-hTCPTP45 (FL), GST- hTCPTP45 (D1), GST-hTCPTP45 (D2), GST-hTCPTP45 350-A3 , GST-hTCPTP45 378-A3 , and GST- hTCPTP45 350/378-A3 cloned into the pGEX4T-1 vector were transformed into BL21 competent cells. Clones were cultured in LB medium containing 50 µg/ml ampicillin, and the expression of GST or GST- fused hTCPTP45 was initiated with 0.5 mM IPTG at an OD 600 of approximately 0.6 for 18 h at 18 °C. The bacterial pellets were collected by centrifugation at 10,000 × g for 15 min and then resuspended in lysis buffer (30 mM Tris-Cl, 1% NP-40, protease inhibitors, 1 mM DTT, 0.1 mM NaCl, pH 7.5). The bacterial lysates were sonicated for 3 × 1 min, and the soluble fraction was obtained by centrifugation at 12,000 rpm for 30 min. Because the GST-fusion proteins were largely in the soluble fraction, the supernatants were mixed with glutathione-Sepharose 4B beads (GE Healthcare Life Sciences) for 4 h to immobilize the GST-fusion proteins. To identify binding of PPAR δ to TCPTP45, the immobilized GST- fusion proteins were mixed with cell lysates overexpressing PPAR δ in the presence or absence of GW501516 overnight at 4 °C. Following extensive washing with phosphate-buffered saline (PBS), the

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA interaction between PPAR δ and TCPTP45 was evaluated by immunoblotting.

Co-immunoprecipitation and immunoblot analysis Cell and tissue lysates were prepared using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) and then pre-cleared with protein G Sepharose™ 4 Fast Flow (GE Healthcare Life Sciences, Buckinghamshire, UK). Pre-cleared lysates were mixed with the specified antibodies (1 µg) or relevant IgG overnight at 4 °C, and then mixed with protein G Sepharose for 4 h. After washing with PBS, proteins were separated from the Sepharose beads by boiling in 2 ×SDS gel- loading buffer and resolved on 10% SDS-polyacrylamide gels. The immunoprecipitates and total lysates (input) were subjected to immunoblot analysis with the specified antibodies, and immunoreactive bands were detected using West-ZOL Plus (iNtRON Biotechnology), as previously described (4). Two percent of each whole-cell lysate was used as the input.

Creation of HepG2 cells stably expressing shRNA targeting PPAR δδδ PPAR δ-silenced HepG2 cells were created by infection with lentiviral particles expressing PPAR δ (TRCN0000350974, MISSION Lentiviral Transduction Particles, Sigma-Aldrich) or a control containing a puromycin resistance gene (pLKO.1-puro Non-Target shRNA Control Transduction Particles, Sigma-Aldrich). The viral particles were used to infect HepG2 cells according to the manufacturer’s instructions, and positive cells were selected using 2 µg/ml puromycin. The efficacy of PPAR δ silencing was confirmed by immunoblot analysis.

Gene silencing with siRNA Gene silencing with specific siRNAs was performed essentially as previously described (5). Briefly, HepG2 cells were transfected with transfection-ready control siRNA (Ambion, Austin, TX, USA), human PPAR δ-targeting siRNA (Ambion), and human TCPTP-targeting siRNA designed against the sequences 5ʹ -GGUCCACUUCCUAACACAUGCdTdT-3ʹ and 5 ʹ - GCAUGUGUUAGGAAGUGGACCdTdT-3ʹ of the PTPN2 mRNA, and human PTP1B-targeting siRNA designed against the sequences 5ʹ -UAGGUACAGAGACGUCAGUdTdT-3ʹ and 5 ʹ - ACUGACGUCUCUGUACCUAdTdT-3ʹ of the PTPN1 mRNA, in serum-free DMEM. The siRNAs against TCPTP and PTP-1B were synthesized by Bioneer (Daejeon, Korea). Following incubation for 6 h, the medium was replaced with fresh medium containing 10% FBS and the cells were incubated for an additional 24 h. The cells were then serum-starved for 18 h and treated with the specified reagents for the indicated period of time, after which the effects of gene silencing were assessed.

Fluorescence confocal laser microscopy Fluorescence images were analyzed as described previously (4). Briefly, 1 × 10 4 cells were seeded on cover-glasses in 35 mm dishes (SPL Life Sciences, Seoul, Korea), and then transfected with GFP- hPPAR δ and DsRed-hTCPTP45 D182A or DsRed-hTCPTP48 D182A using Genefectin (Genetrone Biotech). Forty-eight hours after transfection, cells were treated with insulin, GW501516, and/or GSK0660 for the indicated periods of time. The cells were then incubated with 2 µg/ml DAPI (4′,6-diamidino-2- phenylindole) solution for 10 min at room temperature. Following staining, the cover-glasses were mounted and examined using an Olympus FV-1000 confocal laser fluorescence microscope (Olympus, Tokyo, Japan). The line intensity profiles were obtained using Image J 1.5 (imagej.nih.gov/ij/) software.

Glut4 translocation assay C2C12 myotubes and 3T3-L1 adipocytes seeded on 35 mm cover-glasses in dishes (SPL Life Sciences) were incubated in serum-free DMEM for 16 h and then stimulated with 100 nM insulin in the presence or absence of GW501516. After treatment for 30 min or 6 h, the cells were fixed using neutral buffered 4% formaldehyde solution for 7 min. After permeabilization with PBS containing 0.1% Tween-20 for 3

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA min, the fixed cells were incubated overnight at 4 °C with 1:200 anti-Glut4 antibody. Secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Sigma) was applied for 2 h at room temperature, and then the cells were incubated for 10 min in PI solution. The subcellular localization of Glut4 was evaluated in single cells using an Olympus FV-1000 confocal laser fluorescence microscope.

Hepatocyte glucose production assay The glucose content of media was measured according to a previously described method (1). Briefly, HepG2 cells seeded on a 96-well plate were incubated in serum-free DMEM for 16 h and then the medium was replaced with glucose production medium (glucose- and phenol red-free DMEM supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate). HepG2 cells were treated with 100 nM GW501516 or vehicle for 30 min, and then exposed to 100 nM insulin for 8 h. The glucose concentration in the conditioned medium was measured using a QuantiChrom Glucose assay kit (Bioassay Systems, CA, USA) and normalized to total protein content.

Animal study Male ICR mice (4-week-old, 15–20 g) were purchased from Orient Bio (Seongnam, Korea) and maintained in pathogen-free environmental conditions on a 12 h light-dark cycle at 22 ± 2 °C. All procedures were approved by the Institutional Animal Care and Use Committee of Konkuk University (approval number: KU16144). After 1 week of acclimation, the mice were divided into two groups, and were fed either a ND (Altromin Spezialfutter GmbH & Co., Lage, Germany) or a HFD (Research Diets, Inc. NJ, USA) for 10 weeks, during which water and diet were available ad libitum . The ND contained 4.3% fat, 67.3% carbohydrate, and 19.2% protein, whereas the HFD was composed of 24% fat, 41% carbohydrate, and 24% protein (206.8 g/kg lard). Body mass was measured once a week.

Serum analysis Serum levels of insulin, glucose, and IL-6 were analyzed in circulating blood samples obtained from ND or HFD-fed mice that had been starved for 14 h and treated with 10 mg/kg GW501516 or DMSO for 30 min. Blood was collected, allowed to clot for 2 h at room temperature, and centrifuged for 20 min at 4,000 rpm, as described previously (4). Serum glucose was determined using an OneTouch automatic glucose monitor (LifeScan, Inc.). Serum levels of insulin and IL-6 were measured using a Ultra Sensitive Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science, Inc., Yokohama, Japan) and a Mouse IL-6 Platinum ELISA Kit (eBioscience, San Diego, CA, USA), respectively.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 1. Ligand-dependent interaction of PPAR δ with TCPTP45. A: HepG2 cells were transfected with control shRNA or shRNA targeting PPAR δ, and the stable transfectants were selected in the presence of 2 µg/ml hygromycin. The transfectants were lysed and immunoblotted to determine the expression levels of PPAR δ and β-actin. B: HEK293T cells co-transfected with Flag-hPPAR δ and/or HA-hTCPTP45 for 48 h were treated with 100 nM GW501516 or DMSO for the indicated periods of time, and then whole-cell lysates were prepared and immunoprecipitated with anti-Flag antibody. Immunoblot analysis was performed using the immunoprecipitates and total lysates (input). C: HEK293T cells co-transfected with pcDNA-Flag, pcDNA-Flag-mPPAR α, pcDNA-Flag-mPPAR δ, or pcDNA-Flag-mPPAR γ in the presence of pcDNA-HA-mTCPTP45 for 48 h were treated with 10 µM WY-14643 (PPAR α activator), 100 nM GW501516 (PPAR δ activator), or 10 µM rosiglitazone (PPAR γ activator) for 45 min, and then whole-cell lysates were prepared and immunoprecipitated with anti-Flag antibody. Immunoblot analysis was performed using the immunoprecipitates and total lysates (input). Two percent of each whole-cell lysate was used as the input.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 2. Interaction of PPAR δ with TCPTP in subcellular fractions. A: HepG2 cells were treated with 100 nM GW501516 or DMSO for 45 min, and then whole-cell lysates were prepared and immunoprecipitated with IgG or anti-PPAR δ antibody. The immunoprecipitates and total lysates (input) were subjected to immunoblot analysis. B and C: HepG2 cells were treated with 100 nM GW501516 or DMSO for 45 min, and then separated into nuclear ( B) and cytoplasmic ( C) fractions. Each subcellular fraction was immunoprecipitated with anti-PPAR δ antibody. D: Schematic illustration of the C-terminal bipartite NLS region of TCPTP45 replaced with canonical SV40 large T antigen NLS (PKKKRKV) by site-directed mutation to yield mutant TCPTP45 350/378-SV40 . E: HepG2 cells were transfected with HA-hTCPTP45 350/378-SV40 for 48 h and then separated into nuclear (N) and cytoplasmic (C) fractions. Each fraction was analyzed by immunoblot. F: HepG2 cells co-transfected with Flag- hPPAR δ and HA-hTCPTP45 350/378-SV40 for 48 h were treated with 100 nM GW501516 or DMSO for 30 min. Following exposure to 100 nM insulin for 30 min, the cells were lysed and immunoprecipitated with anti-Flag antibody. The immunoprecipitates and total lysates (input) were subjected to immunoblot analysis. Two percent of each whole-cell lysate was used as the input.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 3. Localization of PPAR δ and TCPTP45 or TCPTP48 complexes. A and B: CHO-IR cells co-transfected with GFP-hPPAR δ and DsRed-hTCPTP45 D182A (A) or DsRed- hTCPTP48 D182A (B) for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin or vehicle. After a further incubation for 30 min, the fluorescence of each fusion protein was visualized using confocal laser fluorescence microscopy. The co-localization of PPAR δ and TCPTP45 or TCPTP48 is indicated by the presence of white in the merged image. Representative fluorescence intensity profiles across the cell were obtained from the merged images through the white arrows. Scale bar = 20 µm.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 4. Differential effects of PPAR ligands on the insulin-stimulated phosphorylation of IR β. A: HepG2 cells transfected with or without control siRNA or siRNA targeting PTP1B for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin or vehicle for a further 30 min. Whole-cell lysates were analyzed by immunoblotting to detect the indicated proteins using specific antibodies. B: HepG2 cells were transfected with pcDNA- Flag or pcDNA-Flag-hPPAR δ for 48 h and then lysed and immunoblotted with anti-PPAR δ and anti-β- actin antibodies to detect PPAR δ and β-actin, respectively. C and D: HepG2 cells transfected with pcDNA-Flag ( C) or pcDNA-Flag-hPPAR δ ( D) for 48 h were treated with 100 nM GW501516 or DMSO for 30 min and then exposed to 100 nM insulin for the indicated periods of time. Immunoblot analysis was performed using whole-cell lysates to detect the indicated proteins using specific antibodies. E and F: HepG2 cells treated with 10 µM WY-14643 ( E) or 10 µM rosiglitazone ( F), specific ligands for PPAR α and PPAR γ, for 30 min were exposed to 100 nM insulin or vehicle for the indicated time periods. The cells were then lysed and analyzed by immunoblotting.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 5. Involvement of TCPTP45 in the PPAR δ-mediated potentiation of insulin signaling. A: HepG2 cells stably expressing control shRNA were transfected with pcDNA-HA or pcDNA-HA-hTCPTP45 for 48 h. The cells were then treated with 100 nM GW501516 or DMSO for 30 min, and exposed to 100 nM insulin for a further 30 min. Whole-cell lysates were prepared and subjected to immunoblot analysis using specific antibodies. B: HepG2 cells stably expressing control shRNA were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for the indicated periods of time. Whole-cell lysates were prepared and subjected to immunoblot analysis.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 6. PPAR δ-mediated modulation of insulin signaling. A and B: C2C12 myotubes (A) or 3T3-L1 adipocytes ( B) were treated with 100 nM GW501516 or DMSO for 30 min. Following exposure to 100 nM insulin for 30 min, the cells were lysed and sequentially analyzed by immunoprecipitation and immunoblotting. C: C2C12 myotubes co-transfected with Flag-hPPAR δ and HA-hTCPTP45 or HA-hTCPTP45 350/378-A3 for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for a further 30 min. Whole-cell lysates were prepared and analyzed by immunoprecipitation and immunoblotting. Two percent of each whole-cell lysate was used as the input. D: HepG2 cells transfected with siRNA were treated with or without 20 ng/ml IL-6. Following treatment for 90 min, the cells were incubated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for a further 30 min. The cells were lysed and subjected to immunoblot analysis.

©2017 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0499/-/DC1 SUPPLEMENTARY DATA Supplementary Figure 7. In vivo effects of GW501516 in mice fed a normal or high-fat diet. A: Time- dependent changes in body mass of mice fed a ND (black circles) or HFD (white circles) for 10 weeks. Data are expressed as mean ± SE (n = 6). *p < 0.01 and ** p < 0.05 vs ND group. B: Mice fed a ND or HFD for 10 weeks were starved for 14 h and then treated with 10 mg/kg GW501516 (red bars) or DMSO (blue bars) for 30 min. Circulating levels of IL-6 were measured by ELISA using sera prepared from blood collected from the carotid artery. Data are expressed as mean ± SE (n = 4). ** p < 0.05.

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