Reagents Unless otherwise stated all chemicals and reagents were obtained from SIGMA.

Human Study Population Oberkofler et al., 2004 Oberkofler H.

Linnemayr V.

Weitgasser R.

Klein K.

Xie M.

Iglseder B.

Krempler F.

Paulweber B.

Patsch W. Complex haplotypes of the PGC-1alpha gene are associated with carbohydrate metabolism and type 2 diabetes. Todoric et al., 2011 Todoric J.

Strobl B.

Jais A.

Boucheron N.

Bayer M.

Amann S.

Lindroos J.

Teperino R.

Prager G.

Bilban M.

et al. Cross-talk between interferon-γ and hedgehog signaling regulates adipogenesis. Oberkofler et al., 2010 Oberkofler H.

Pfeifenberger A.

Soyal S.

Felder T.

Hahne P.

Miller K.

Krempler F.

Patsch W. Aberrant hepatic TRIB3 gene expression in insulin-resistant obese humans. Pontiroli et al., 2002 Pontiroli A.E.

Pizzocri P.

Giacomelli M.

Marchi M.

Vedani P.

Cucchi E.

Orena C.

Folli F.

Paganelli M.

Ferla G. Ultrasound measurement of visceral and subcutaneous fat in morbidly obese patients before and after laparoscopic adjustable gastric banding: comparison with computerized tomography and with anthropometric measurements. Study subjects included 51 obese patients and 6 nonobese controls that underwent weight reducing surgery or elective surgical procedures such as cholecystectomy. Participants were included if they had fasting plasma glucose levels < 7.0 mmol/l, no history of diabetes, no medications, no weight changes > 3% during the previous 2 months, C-reactive protein (CRP) levels < 20 mg/l and normal blood leukocyte counts. All study subjects provided informed consent and study protocols were approved by the local Ethics Committee. Tissue biopsies from visceral adipose tissue, obtained during surgery, were collected in RNA-later (Life Technologies) and stored at −80°C until further processing. In a subset of 43 obese study participants liver biopsies were obtained at the beginning of the surgical intervention. Glucose, insulin, lipid parameters, high-sensitive CRP and HOMA-IR index were determined as described (). Abdominal subcutaneous and visceral fat thickness were determined by ultrasonography using the HDI 3000 CV System (ATL) ().

Quantitative PCR Todoric et al., 2011 Todoric J.

Strobl B.

Jais A.

Boucheron N.

Bayer M.

Amann S.

Lindroos J.

Teperino R.

Prager G.

Bilban M.

et al. Cross-talk between interferon-γ and hedgehog signaling regulates adipogenesis. −ΔΔCt values were calculated to obtain fold expression levels, where ΔΔCt = (ΔCt treatment - ΔCt control). Human GLUT4 and CD68 transcripts were quantified using TaqMan gene expression assays Hs00168966_1 and Hs00154355_m1 (Applied Biosystems, Foster City, CA), respectively. Other human and mouse primers used are listed in qPCR was performed as described (). In brief, total RNA and DNA were extracted from respective tissues and cells using RNA and DNA isolation kits (RNeasy, QIAGEN; TRIzol, Invitrogen). Isolated total RNA was reverse-transcribed into cDNA using commercially available kits (Applied Biosystems). qPCR reactions were performed using the iQ SYBR Green Supermix (Bio-Rad Laboratories). Postamplification melting curve analysis was performed to check for unspecific products and primer-only controls were included to ensure the absence of primer dimers. For normalization threshold cycles (Ct-values) were normalized to acidic ribosomal phosphoprotein P0 (Rplp0) within each sample to obtain sample-specific ΔCt values ( = Ct gene of interest - Ct Rplp0). 2values were calculated to obtain fold expression levels, where ΔΔCt = (ΔCt treatment - ΔCt control). Human GLUT4 and CD68 transcripts were quantified using TaqMan gene expression assays Hs00168966_1 and Hs00154355_m1 (Applied Biosystems, Foster City, CA), respectively. Other human and mouse primers used are listed in Tables S5 and S6

Immunohistochemistry Human and mouse tissues were fixed in 4% phosphate-buffered formalin and embedded into paraffin. Tissue sections were deparaffinized and rehydrated prior to antigen unmasking using Target Retrieval Solution, pH 6.0 (Dako). Endogenous peroxidase activity was quenched with incubation of 3% hydrogen peroxide for 10 min. Sections were blocked with Biotin-Blocking System (Dako) and normal serum obtained from the secondary antibody host animal. Blocked sections were incubated with anti-HO-1 (1:500; ab13243, Abcam), anti-CD68 (1:500; M0718, Dako) and anti-MAC-2 (1:500; CL8942AP, Cedarlane). Secondary antibody staining was performed using the EnVision Detection System (Dako) and 3,3′-diaminobenzidine as chromogenic substrate (Roche Molecular Biochemicals) according to the manufacturer’s instructions. HO-1 positive cells were evaluated using the particle analysis module incorporated in ImageAccess 9 (IMAGIC). HO-1 positive cells per all hepatocytes were counted on three different high-power fields (400× magnification).

Mouse and Human Multiple Tissue RNA Libraries Total RNA was isolated from tissues of three 6 week old male C57BL/6J mice to analyze HO-1 tissue distribution. A human multiple tissue RNA library (Life Technologies) was used to analyze the tissue distribution of human HO-1 expression in tissues corresponding to the mouse library.

Metabolically Healthy and Unhealthy Obese Mice Obese age- and body-weight-matched C57BL/6J mice were selected based on their response in oral glucose and insulin tolerance tests after 16 weeks on HFD. Epididymal fat pads and livers were removed and RNA and protein isolated to measure HO-1 expression.

Glucose and Insulin Tolerance Tests Following an overnight fast, mice were administered glucose (1 g/kg) by oral gavage, and blood samples for glucose and insulin determination were collected from the tail vein at the indicated times. Insulin tolerance was assessed after a 2 hr fast by intraperitoneal administration of human regular insulin (0.75 U/kg) and blood glucose monitoring. Low-dose insulin tolerance tests (0.1 U/kg) were performed in adenovirus injected mice to assess hepatic insulin sensitivity. Glycemia was assessed using a Accu-Chek (Roche) glucometer. Plasma insulin levels were determined using the Ultrasensitive Mouse Insulin ELISA kit (Mercodia).

Western Blot Analysis Proteins were extracted from tissues by homogenizing in RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) containing protease inhibitors (Complete Mini, Roche). The homogenate was cleared by centrifugation at 4°C for 30 min at 15,000 g and the supernatant containing the protein fraction recovered. Protein concentration in the supernatant was determined using the BCA Protein Assay Kit (Pierce). 20 μg of proteins were resolved by SDS-PAGE and transferred to PVDF membranes (GE Healthcare). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.2% Tween-20 (TBS-T), and incubated with primary antibodies at 4°C over night. The following antibodies were used (all from Cell Signaling unless indicated otherwise): anti-HO-1 (1:5,000; ab13243, Abcam), anti-phospho-Insulin receptor (pTyr1162/1163) (1:1,500; sc-25103, Santa Cruz Biotechnology), anti-insulin receptor (1:1,500; Cat.-No. 3025), anti-phospho-AKT (pSer473) (1:1,500; Cat.-No. 9271), anti-phospho-AKT (pThr308) (1:1,500; Cat.-No. 9275), anti-AKT (1:1,500; Cat.-No. 9272), anti-phospho-IkBα (pSer32/36) (1:1,000; Cat.-No. 9246), anti-ikBα (1:1,000; sc-371, Santa Cruz Biotechnology), anti-NRF2 (1:1,000; Cat.-No. 12721), anti-Histone H3 (1:1,000; Cat-No. 4499), anti-β-Actin (1:500; A5441, Sigma), anti-GAPDH (1:2,500; ab9485, Abcam), anti-HSC-70 (1:5,000; sc-7298, Santa Cruz Biotechnology). Expression levels of 5 different OXPHOS complexes (CI subunit NDUFB8, CII-30kDa, CIII-Core protein 2, CIV subunit I and CV alpha subunit) were analyzed using the MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (Abcam). Incubated membranes were washed and probed with the appropriate anti-igG-horseradish peroxidase-linked (HRP) secondary antibody (NA 934, anti-rabbit IgG, 1:20,000; NA 931, anti-mouse IgG, 1:20,000; GE Healthcare). Antigen-specific binding of antibodies was detected with SuperSignal West Femto and Pico Kits (Pierce) using a ChemiDoc XRS Imager (Bio-Rad). Image analysis was performed using Image Lab Software Version 3.0.1 (Bio-Rad).

Generation of Tissue-Specific HO-1 Knockout Mice Postic et al., 1999 Postic C.

Shiota M.

Niswender K.D.

Jetton T.L.

Chen Y.

Moates J.M.

Shelton K.D.

Lindner J.

Cherrington A.D.

Magnuson M.A. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. Clausen et al., 1999 Clausen B.E.

Burkhardt C.

Reith W.

Renkawitz R.

Förster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Brüning et al., 1998 Brüning J.C.

Michael M.D.

Winnay J.N.

Hayashi T.

Hörsch D.

Accili D.

Goodyear L.J.

Kahn C.R. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. He et al., 2003 He W.

Barak Y.

Hevener A.

Olson P.

Liao D.

Le J.

Nelson M.

Ong E.

Olefsky J.M.

Evans R.M. Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle. Postic et al., 1999 Postic C.

Shiota M.

Niswender K.D.

Jetton T.L.

Chen Y.

Moates J.M.

Shelton K.D.

Lindner J.

Cherrington A.D.

Magnuson M.A. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. fl/+;Cre+/− X female HO-1fl/+;Cre−/−) were used to generate Lhoko and Macho animals, muscle (Muhoko), adipose (Fahoko) and β-cell (Bhoko) HO-1 knockout mice as well as their respective wild-type (HO-1+/+;Cre−/−), conditional (HO-1fl/fl;Cre−/−) and Cre control littermates (HO-1+/+;Cre+/−). Offsprings were found to be born at expected Mendelian ratios and no difference in survival rates was observed. Animals were kept on a 12 hr light/dark cycle with free access to food and water and housed in accordance with international guidelines. Dietary interventions started at 6 weeks of age using standardized low- and high-fat diets which contained 10% and 60% calories of fat, respectively (Research Diets Inc., D12450B and D12492). Animal studies were approved by Austrian and German governments. Mice with a conditional HO-1 allele were generated by homologous recombination. The targeting vector was designed to introduce two LoxP sites flanking exon 2 of the HO-1 gene locus. Elimination of exon 2 leads to a consecutive frameshift in exon 3, an early stop codon, thus resulting in a truncated peptide consisting of 9 amino acids. The targeting construct was electroporated into C57BL/6N embryonic stem cells. The primary ES cells were screened and potentially targeted clones were expanded. Southern blot confirmation was used to select correctly targeted cells that were subjected to removal of the Frt-flanked neomycin cassette by Flp-recombinase, and subsequently injected into blastocysts. Offsprings were tested for germline transmission and two correctly targeted mouse lines were established. C57BL/6N founder mice were backcrossed onto the more insulin resistance prone C57BL/6J background using speed congenics (purity > 99.9%). Hepatocyte-specific HO-1 knockout (Lhoko) mice were generated by crossing the conditional HO-1 line with Alb-Cre transgenic mice expressing Cre recombinase under the control of the albumin promoter (). Mice with a targeted deletion in macrophages (Macho) were generated by crossing the conditional HO-1 line with LysM-Cre knockin mice having the Cre recombinase inserted into the first coding ATG of the lysozyme 2 gene (). Muscle-, adipose- and beta-cell targeted HO-1 knockout mice were generated using Mck-cre (), Fabp4/aP2-cre () and Rip-cre () knockin mice, respectively. Heterozygous breeding schemes (male HO-1;CreX female HO-1;Cre) were used to generate Lhoko and Macho animals, muscle (Muhoko), adipose (Fahoko) and β-cell (Bhoko) HO-1 knockout mice as well as their respective wild-type (HO-1;Cre), conditional (HO-1;Cre) and Cre control littermates (HO-1;Cre). Offsprings were found to be born at expected Mendelian ratios and no difference in survival rates was observed. Animals were kept on a 12 hr light/dark cycle with free access to food and water and housed in accordance with international guidelines. Dietary interventions started at 6 weeks of age using standardized low- and high-fat diets which contained 10% and 60% calories of fat, respectively (Research Diets Inc., D12450B and D12492). Animal studies were approved by Austrian and German governments.

Heme Oxygenase Activity Kozlov et al., 2010 Kozlov A.V.

van Griensven M.

Haindl S.

Kehrer I.

Duvigneau J.C.

Hartl R.T.

Ebel T.

Jafarmadar M.

Calzia E.

Gnaiger E.

et al. Peritoneal inflammation in pigs is associated with early mitochondrial dysfunction in liver and kidney. McCoubrey, 2001 McCoubrey Jr., W.K. Detection of heme oxygenase 1 and 2 proteins and bilirubin formation. HO activity was determined as reported elsewhere (). In brief, aliquots of frozen liver (Lhoko) or cell pellets (Macho) were homogenized using a Elvehjem potter with PTFE pestle in a buffer 1:10 (wt/vol) containing 300 mM sucrose, 20 mM Tris, and 2 mM EDTA at pH 7.4. Protein concentrations were determined using Bradford reagent and BSA as standard. For the activity assay, 100 μl of homogenate (containing about 1 mg of protein) was added to a reaction mixture containing 500 nmol NADPH in a 100 mM potassium phosphate buffer (pH 7.4) supplemented with 1 mM EDTA and 20 nmol of hemin. 30 μl rat kidney cytosolic (RKC) fraction was added to the reaction to provide sufficient biliverdin reductase activity. RKC was prepared as described elsewhere (). The reaction mixture was incubated under constant agitation for 30 min at 37°C in darkness. After adding 0.2 assay volumes of saturated potassium chloride, the formed bilirubin was extracted into chloroform (4 assay volumes). Samples were then centrifuged, the organic phase harvested for subsequent spectrophotometric determination of bilirubin concentrations. Samples were scanned three times to calculate the absorption difference between 450 and 520 nm. All samples were run in duplicates and corrected for the absorption measured in corresponding samples incubated on ice. Bilirubin standard curves were generated by spiking known amounts of bilirubin into sample homogenates. Heme oxygenase activity was calculated as nanomol bilirubin formed per milligram protein per 30 min.

Hemin Injection Hemin (2.5 mg/ml) was dissolved in 10% ammonium hydrochloride in 0.15 M sodium chloride and injected intraperitoneally into mice (10 μl/g body weight). Livers were harvested 12 hr postinjection to test HO-1 induction.

Mouse Laboratory Parameters and Cytokines Folch et al., 1957 Folch J.

Lees M.

Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues. Alanine transaminase (ALT), Aspartate transaminase (AST), cholinesterase (ChE), alkaline phosphatase (ALKP), triglycerides and cholesterol were quantified with tests certified for in vitro diagnostics at the Department of Laboratory Medicine of the Medical University of Vienna. Free fatty acids were measured using the nonesterified fatty acid (NEFA) kit (Wako Chemicals) according to the provided protocol. Blood samples were analyzed on an automated hematology analyzer (CELL-DYN 3500, Abbott Laboratories) and validated manually with Giemsa stained blood smears. IL-6, TNF-α, IL-1β and IL-10 ELISA kits (all from Biolegend) were used to determine the respective cytokine levels in mouse serum and supernatants derived from cultured macrophages according to the protocol provided by the manufacturer. For hepatic triglyceride and cholesterol measurements, total lipids were extracted from 50 mg liver as previously described (). Serum leptin, adiponectin and RBP4 levels were quantified using commercially available ELISA kits (Millipore, R&D Systems).

Oil Red O Staining For staining of neutral lipids, liver cryosections were stained with oil red O (1% w/v isopropanol, diluted 3:2 in PBS) for 1 hr at room temperature according to standard procedures.

In Vivo Insulin Signaling For examination of in vivo insulin signaling, mice were fasted for 2 hr, anesthetized and injected with human regular insulin (1 U/kg, Novo Nordisk). Liver, muscle and adipose biopsies were removed at the indicated times, flash-frozen in liquid nitrogen and stored at −80°C until further processing.

Indirect Calorimetry Pospisilik et al., 2010 Pospisilik J.A.

Schramek D.

Schnidar H.

Cronin S.J.

Nehme N.T.

Zhang X.

Knauf C.

Cani P.D.

Aumayr K.

Todoric J.

et al. Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. Indirect calorimetry and activity measurements were performed as reported elsewhere (). In brief, body-weight-matched mice were placed in metabolic cages connected to an open-circuit, indirect calorimetry system combined with the determination of spontaneous activity by beam breaking (Oxylet, Panlab-Bioseb). The animals were accustomed to the apparatus during the first 24 hr, followed by measurements. Oxygen consumption and carbon dioxide production were recorded using a computer-assisted data acquisition program (Metabolism 2.2.01, Panlab Harvard Apparatus).

Generation of Bone-Marrow-Derived Macrophages Weischenfeldt and Porse, 2008 Weischenfeldt J.

Porse B. Bone Marrow-Derived Macrophages (BMM): Isolation and Applications. Zanoni et al., 2009 Zanoni I.

Ostuni R.

Capuano G.

Collini M.

Caccia M.

Ronchi A.E.

Rocchetti M.

Mingozzi F.

Foti M.

Chirico G.

et al. CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Bone marrow cells were obtained from femurs and tibias of 6-10 week old control and Macho mice. Bone-marrow-derived macrophages (BMDMs) were generated in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 10% supernatant derived from M-CSF transduced L929 confluent cells as described (). At day 7 postinduction macrophages were collected and cultured in the presence or absence of stimuli in RPMI-1640 medium supplemented with 10% FBS. BMDMs were polarized for 24 hr with 10 ng/ml LPS and 20 ng/ml Interferon-γ (R&D Systems) to generate classically activated M1 macrophages. Alternatively activated M2 macrophages were generated using IL-4/IL-13 (20 ng/ml each; R&D Systems). For NF-κB signaling experiments macrophages were treated with 10 ng/ml TNF-α (Peprotech) for the indicated times.

Histology, Adipocyte Size, and Number For tissue sections, hematoxylin and eosin (H&E) staining was performed on 5 μm paraffin sections of tissues fixed for 16 hr in 4% phosphate-buffered formalin at 4°C. Adipocyte size distribution was determined by semi-automated morphometry. In brief, three fields of view of 3 different sections (200 μm intervals) per animal were quantified. Epididymal fat pads from 4 animals per group were analyzed. Semi-automated morphometry (Axiovision morphometry software, Zeiss) was used to define and quantify fat cells based on shape, size and presence of a lipid droplet.

Whole-Mount Immunofluorescence Lumeng et al., 2007 Lumeng C.N.

Bodzin J.L.

Saltiel A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. Todoric et al., 2011 Todoric J.

Strobl B.

Jais A.

Boucheron N.

Bayer M.

Amann S.

Lindroos J.

Teperino R.

Prager G.

Bilban M.

et al. Cross-talk between interferon-γ and hedgehog signaling regulates adipogenesis. Whole-mount epididymal fat samples were processed and stained as described (). In brief, mice (three per group) were perfused with 1% paraformaldehyde for fixation before dissecting fat pads. Samples were incubated for 1 hr at room temperature with 5% normal goat serum (Dako) in 0.3% PBS-T to block unspecific binding sites. After blocking, tissues were incubated overnight at 4°C with primary and secondary antibodies diluted in PBS containing 5% BSA. Anti-perilipin (Cat.-Nr. 9349, Cell Signaling Technology) and anti-MAC-2 antibodies (Cat.-Nr. CL8942AP, Cedarlane Laboratories) were used to stain adipocyte lipid droplets and macrophages, respectively. The following secondary antibodies were used for immunofluorescence staining: fluorescein anti-rat IgG (Cat.-Nr. FI-4001, VECTOR Laboratories), Alexa Fluor-594 goat anti-rabbit IgG (Cat.-Nr. A11037, Life Technologies). For control experiments, the primary antibody was substituted with preimmune serum. Stained tissues were mounted in chamber slides and subjected to confocal imaging analysis on a LSM 700 Laser Scanning Microscope (Zeiss).

Flow Cytometry Lumeng et al., 2007 Lumeng C.N.

Bodzin J.L.

Saltiel A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. Pospisilik et al., 2010 Pospisilik J.A.

Schramek D.

Schnidar H.

Cronin S.J.

Nehme N.T.

Zhang X.

Knauf C.

Cani P.D.

Aumayr K.

Todoric J.

et al. Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. 6 cells/ml. Isolated cells were incubated with Fc Block (BD Biosciences) prior to staining with conjugated antibodies for 30 min at 4°C followed by 2 washes in sorting buffer. Cells were resuspended in sorting buffer supplemented with Fixable Viability Dye eFluor (eBioscience) and subjected to FACS analysis (LSR Fortessa, BD Biosciences). The following antibodies were used for staining adipose tissue macrophages: anti-F4/80 PE/Cy7 (Cat.-Nr. 123113, BioLegend), anti-CD11b Percp/Cy5.5 (Cat.-Nr. 101228, BioLegend), anti-CD11c Alexa Fluor700 (Cat.-Nr. 56-0114-82, eBioscience), anti-CCR2 APC (Cat.-Nr. FAB5538A, R&D Systems) and anti-MGL Alexa Fluor 647 (Cat.-Nr. MCA2392A647T, AbD Serotec). Viable cells were gated and analyzed using FlowJo (Tree Star). Stromal vascular cells (SVCs) were obtained by collagenase digestion from epididymal adipose tissue as described previously (). In brief, minced fat depots were digested at 37°C for 30 min in a shaking water bath with a cocktail consisting of 1 mg/ml collagenase II (Worthington) in DMEM containing 3% fatty-acid-free BSA and DNase I (100 Units/ml). After digestion, the slurry was passed through a 100 μm cell strainer (Becton Dickinson) and centrifuged at 200 g for 5 min to separate SVCs and adipocyte fractions. Adipocyte fractions were examined by microscopy to detect adherent cells. Digestion was continued for another 15 min until adipocyte fractions were free of adherent cells to guarantee isolation of most adipose macrophages. Cells were then pelleted by centrifugation at 200 g for 5 min, resuspended in 0.5 ml red blood cell lysis buffer and incubated for 5 min prior to resuspension in sorting buffer (PBS with 2% FBS) at a concentration of 10cells/ml. Isolated cells were incubated with Fc Block (BD Biosciences) prior to staining with conjugated antibodies for 30 min at 4°C followed by 2 washes in sorting buffer. Cells were resuspended in sorting buffer supplemented with Fixable Viability Dye eFluor (eBioscience) and subjected to FACS analysis (LSR Fortessa, BD Biosciences). The following antibodies were used for staining adipose tissue macrophages: anti-F4/80 PE/Cy7 (Cat.-Nr. 123113, BioLegend), anti-CD11b Percp/Cy5.5 (Cat.-Nr. 101228, BioLegend), anti-CD11c Alexa Fluor700 (Cat.-Nr. 56-0114-82, eBioscience), anti-CCR2 APC (Cat.-Nr. FAB5538A, R&D Systems) and anti-MGL Alexa Fluor 647 (Cat.-Nr. MCA2392A647T, AbD Serotec). Viable cells were gated and analyzed using FlowJo (Tree Star).

Nuclear Translocation Assay To assess nuclear RelA/p65 translocation naive BMDMs were seeded in 8-well chamber slides (BD Biosciences). Cells were either stimulated with 10 ng/ml TNF-α or medium only as control for 30 and 60 min, fixed in ice-cold methanol for 10 min, washed and stained overnight at 4°C with anti-RelA/p65 antibody (sc-372, Santa Cruz Biotechnology). After several washes cells were incubated with a fluorescent secondary antibody (Alexa Fluor 488, Life Technologies) for 1 hr at room temperature. Nuclei were counterstained with DAPI. Images were obtained using a Zeiss Axio Imager A1 microscope and evaluated in a double-blinded manner by two different researchers.

Electrophoretic Mobility Shift Assay Nuclear extracts were prepared from 1x107 BMDMs. Adherent cells were washed with ice-cold PBS, transferred and washed three times in 1 ml of ice-cold hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl) to induce swelling. Thereafter, the pellet was resuspended in hypotonic buffer supplemented with 0.1% Nonidet P-40 and incubated on ice for 5 min to release nuclei. Subsequently, samples were centrifuged at 4°C to pellet nuclei and the cytoplasmic fraction was discarded. Nuclei were resuspended in 50 μl of ice-cold, high-salt buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 420 mM NaCl, 25% glycerol) and incubated for 15 min at 4°C. Disrupted nuclei were centrifuged at full speed in a table top centrifuge for 15 min at 4°C to isolate the nuclear protein fraction. EMSA was performed using the NF-κB EMSA kit and an IRDye 700 labeled double-stranded probe harboring the NF-κB consensus site (LI-COR Biosciences) according to the manufacturer’s instructions. For the binding assay, 5 μg of nuclear extracts were added to binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5) together with 50 ng poly (dI:dC), 2.5 mM DTT/0.25% Tween-20 and 0.5 pmol IRDye labeled probe in a total volume of 20 μl. For supershift assays 0.2 μg of the following antibodies were used: anti-RelA/p65 (sc-372, Santa Cruz Biotechnology), anti-p50 (06-886, Millipore) and anti-rabbit IgG (S-5000, Vector Laboratories). Unlabeled double-stranded NF-κB probe was used at a 100-fold molar excess to test specific binding. Samples were incubated at room temperature for 15 min and separated on a prerun nondenaturing polyacrylamide gel in TGE buffer (25 mM Tris-HCl pH 8.0, 190 mM glycine, 1 mM EDTA) for 3 hr at 100 V. NF-κB DNA binding was visualized using the Odyssey Imaging System (LI-COR Biosciences).

Chromatin Immunoprecipitation Assay 6 cells were crosslinked for 10 min at 37°C by adding formaldehyde (methanol-free, Thermo Scientific) into the culturing media to a final concentration of 1%. Reactions were stopped by adding glycine to a final concentration of 0.125 M. Cells were harvested, washed twice with ice-cold PBS and lysed in 100 μl lysis buffer containing protease inhibitors. Cell lysates were transferred to microTUBEs (Covaris) and subjected to shearing on the Covaris S2 sonicator (4 cycles; cycle time: 60 s, peak power: 140, duty factor: 5, cycles/burst: 200). Dynabeads (Life Technologies) were precoupled with anti-RelA/p65 (sc-372, Santa Cruz Biotechnology) or rabbit IgG according to the protocol provided. 3 μg of antibody was used for each ChIP experiment. Sheared chromatin DNA was diluted to 200,000 cells per ChIP reaction and incubated with antibody-coupled beads at 4°C with constant rotation for 4 hr. Input controls were set aside for each IP sample. Bound chromatin was washed with a series of washing buffers included in the kit and formaldehyde crosslinking was reversed using Proteinase K. DNA was purified using DNA purification magnetic beads provided in the kit and eluted in 150 μl of DNA Elution Buffer. Precipitated DNA was subjected to quantitative PCR using primers 5′-CCCCAGATTGCCACAGAATC-3′ and 5′-CCAGTGAGTGAAAGGGACAG-3′ for the Tnf promoter (NF-κB site 1) ( Kuwata et al., 2006 Kuwata H.

Matsumoto M.

Atarashi K.

Morishita H.

Hirotani T.

Koga R.

Takeda K. IkappaBNS inhibits induction of a subset of Toll-like receptor-dependent genes and limits inflammation. Guo et al., 2008 Guo H.

Mi Z.

Kuo P.C. Characterization of short range DNA looping in endotoxin-mediated transcription of the murine inducible nitric-oxide synthase (iNOS) gene. Chromatin immunoprecipitation (ChIP) assays were performed using the MAGnify ChIP Kit (Life Technologies) as detailed by the manufacturer’s instruction. In brief, 2 × 10cells were crosslinked for 10 min at 37°C by adding formaldehyde (methanol-free, Thermo Scientific) into the culturing media to a final concentration of 1%. Reactions were stopped by adding glycine to a final concentration of 0.125 M. Cells were harvested, washed twice with ice-cold PBS and lysed in 100 μl lysis buffer containing protease inhibitors. Cell lysates were transferred to microTUBEs (Covaris) and subjected to shearing on the Covaris S2 sonicator (4 cycles; cycle time: 60 s, peak power: 140, duty factor: 5, cycles/burst: 200). Dynabeads (Life Technologies) were precoupled with anti-RelA/p65 (sc-372, Santa Cruz Biotechnology) or rabbit IgG according to the protocol provided. 3 μg of antibody was used for each ChIP experiment. Sheared chromatin DNA was diluted to 200,000 cells per ChIP reaction and incubated with antibody-coupled beads at 4°C with constant rotation for 4 hr. Input controls were set aside for each IP sample. Bound chromatin was washed with a series of washing buffers included in the kit and formaldehyde crosslinking was reversed using Proteinase K. DNA was purified using DNA purification magnetic beads provided in the kit and eluted in 150 μl of DNA Elution Buffer. Precipitated DNA was subjected to quantitative PCR using primers 5′-CCCCAGATTGCCACAGAATC-3′ and 5′-CCAGTGAGTGAAAGGGACAG-3′ for the Tnf promoter (NF-κB site 1) () and 5′-CCTAGTGAGTCCCAGTTTTGAAGT-3′ and 5′-CATCAGGTATTTATACCCCTCCAG-3′ for the proximal NF-κB site in the Nos2/iNos promoter (). Data were analyzed according to the MAGnify ChIP Kit (Life Technologies) manual.

Oxygen Consumption Assay and Bioenergetic Profile Haschemi et al., 2012 Haschemi A.

Kosma P.

Gille L.

Evans C.R.

Burant C.F.

Starkl P.

Knapp B.

Haas R.

Schmid J.A.

Jandl C.

et al. The sedoheptulose kinase CARKL directs macrophage polarization through control of glucose metabolism. Teperino et al., 2012 Teperino R.

Amann S.

Bayer M.

McGee S.L.

Loipetzberger A.

Connor T.

Jaeger C.

Kammerer B.

Winter L.

Wiche G.

et al. Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat. 2 -free incubator and maintained at 37°C for 1 hr before starting the assay. Following instrument calibration, cells were transferred to the XF24 Flux Analyzer to record cellular oxygen consumption rates. The measurement protocol consisted of 2 min mixture, 2 min wait and 4 min OCR measurement times (macrophages), and 4 min mixture, 0 min wait and 3 min OCR measurement times (hepatocytes). For the mitochondrial stress test ATP synthase was inhibited by injection of 1 μM oligomycin, followed by 3 μM FCCP-induced mitochondrial uncoupling to determine the spare/maximal respiratory capacity. Nonmitochondrial respiration was determined after rotenone/antimycin A injection (1 μM each). At the end of the assay, the medium was carefully aspirated and cellular DNA measured using CyQuant (Life Technologies) to adjust for potential differences in cell densities. For acute ROS-quenching experiments, 10 μM buffered N-acetyl-cysteine (NAC) was added to naive BMDMs or primary hepatocytes 1 hr prior to mitochondrial stress tests and OCR measurements. For adenoviral rescue experiments, primary hepatocytes isolated from Lhoko animals were transduced with Adeno-LacZ (AdLacZ) or Adeno-HO-1 (AdHO-1) virus particles (10 pfu/cell). Mitochondrial stress tests and OCR measurements were initiated 48 hr after transduction. Analysis of oxygen consumption rates (OCR) was performed using the XF24 Flux Analyzer (Seahorse Bioscience) as reported previously (). In brief, naive bone-marrow-derived macrophages and primary hepatocytes were seeded into XF 24-well cell culture microplates and allowed to recover for 24 hr. A final volume of 600 μl of buffer-free Assay Medium (Seahorse Bioscience) was added to each well prior to the experimental protocol. Cells were then transferred to a CO-free incubator and maintained at 37°C for 1 hr before starting the assay. Following instrument calibration, cells were transferred to the XF24 Flux Analyzer to record cellular oxygen consumption rates. The measurement protocol consisted of 2 min mixture, 2 min wait and 4 min OCR measurement times (macrophages), and 4 min mixture, 0 min wait and 3 min OCR measurement times (hepatocytes). For the mitochondrial stress test ATP synthase was inhibited by injection of 1 μM oligomycin, followed by 3 μM FCCP-induced mitochondrial uncoupling to determine the spare/maximal respiratory capacity. Nonmitochondrial respiration was determined after rotenone/antimycin A injection (1 μM each). At the end of the assay, the medium was carefully aspirated and cellular DNA measured using CyQuant (Life Technologies) to adjust for potential differences in cell densities. For acute ROS-quenching experiments, 10 μM buffered N-acetyl-cysteine (NAC) was added to naive BMDMs or primary hepatocytes 1 hr prior to mitochondrial stress tests and OCR measurements. For adenoviral rescue experiments, primary hepatocytes isolated from Lhoko animals were transduced with Adeno-LacZ (AdLacZ) or Adeno-HO-1 (AdHO-1) virus particles (10 pfu/cell). Mitochondrial stress tests and OCR measurements were initiated 48 hr after transduction.

Primary Hepatocyte Isolation Hepatocytes were isolated and cultured according to the protocols provided by the manufacturer of Liver Perfusion Medium and Liver Digest Medium, respectively (Invitrogen). In brief, mice were anaesthetized by intraperitoneal injection of ketamine-xylazine (10% ketamine, 5% xylazine). The liver was perfused in situ with Liver Perfusion Medium (Invitrogen), and digested using Liver Digest Medium (Invitrogen). Digested livers were removed, minced, and filtered through a 70 μm cell strainer (BD Biosciences). Filtered hepatocytes were washed in William’s E medium supplemented with 5% FBS, purified from the nonparenchymal cells by centrifugation and plated in the respective experimental dishes. After 4 hr of incubation adherent hepatocytes were switched to serum-free RPMI-1640 and incubated over night. Experiments were performed on the following day.

PTP1B Activity Assay Samples were homogenized in immunoprecipitation buffer (20 mM Tris pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 10% glycerol, protease inhibitors, and 1% Triton X-100. Lysates were cleared by centrifugation for 15 min at 20.000 g. Liver (2 mg) and hepatocytes (1 mg) samples were precleared for 30 min at 4°C by incubation with 20 μl protein G coupled Dynabeads (Life Technologies). Precleared samples were incubated for 1 hr at 4°C with goat anti-PTP1B (Santa Cruz Biotechnology). Next, 20 μl protein G coupled Dynabeads were added to capture PTP1B and incubated for 2 hr at 4°C. Samples were washed two times with immunoprecipitation buffer and two times with phosphatase assay buffer (25 mM HEPES pH 7.2, 50 mM NaCl, 2 mM EDTA). Total PTP1B activity was measured using 50 mM of para-Nitrophenylphosphate (New England BioLabs) as a substrate according to the manufacturer’s instructions. Subsequently, immunoprecipitated PTP1B samples were boiled and subjected to immunoblotting using rabbit anti-PTP1B (Abcam). PTP1B signals were quantified by densitometry. Total PTP1B activity results were normalized to the amount of PTP1B in the precipitates to obtain specific PTP1B activity values (1 Unit = hydrolysis of 1 nmol of para-Nitrophenylphosphate in 1 min).

Hydrogen Peroxide Measurements Hydrogen peroxide (H 2 O 2 ) production was measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) according to the protocol provided by the manufacturer.

Superoxide Dismutase Assay Samples were sonicated and centrifuged at 1,500 g for 5 min at 4°C in ice-cold HEPES buffer (20 mM) containing EGTA (1 mM), mannitol (210 mM) and sucrose (70 mM). Superoxide dismutase (SOD) activity was measured using a commercially available test kit (Cayman) according to manufacturer’s instructions.

Cytosolic ROS and Mitochondrial Content Kuznetsov et al., 2011 Kuznetsov A.V.

Kehrer I.

Kozlov A.V.

Haller M.

Redl H.

Hermann M.

Grimm M.

Troppmair J. Mitochondrial ROS production under cellular stress: comparison of different detection methods. Kuznetsov et al., 2011 Kuznetsov A.V.

Kehrer I.

Kozlov A.V.

Haller M.

Redl H.

Hermann M.

Grimm M.

Troppmair J. Mitochondrial ROS production under cellular stress: comparison of different detection methods. BMDMs and primary hepatocytes were cultured in Lab-Tek II Chambered Coverglass polystyrene media chambers mounted to extrathin borosilicate cover glass for optimum high-power inverted microscopy (Nalge Nunc). Cytosolic ROS were quantified as communicated recently (). In brief, cells were loaded for 20 min with 5 μM 2′,7′-Dichlorofluorescin diacetate (DCF-DA; Invitrogen). Stained cells were imaged with an inverted confocal microscope (LSM 510, Zeiss), the AxioVision software package (Zeiss) was used to analyze recorded images as described (). Results were expressed as arbitrary fluorescence intensity units. MitoTracker Deep Red 633 (500 nM, staining time 40 min at 37°C) was used to analyze the mitochondrial content in isolated primary hepatocytes. In BMDMs mitochondrial content was determined using MitoTracker Green FM (Life Technologies) according to the manufacturer’s instructions. Stained cells were washed once with serum-free RPMI-1640 and incubated at 37°C for 30 min. Thereafter, cells were washed extensively with PBS, resuspended in FACS buffer (PBS, 2 mM EDTA, 2% FBS) and analyzed with the LSR II Flow Cytometer (Becton Dickinson). Result were analyzed using the FlowJo software package (Tree Star).

Adenovirus Experiments Dmitriev et al., 1998 Dmitriev I.

Krasnykh V.

Miller C.R.

Wang M.

Kashentseva E.

Mikheeva G.

Belousova N.

Curiel D.T. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. Hardy et al., 1997 Hardy S.

Kitamura M.

Harris-Stansil T.

Dai Y.

Phipps M.L. Construction of adenovirus vectors through Cre-lox recombination. Pospisilik et al., 2007 Pospisilik J.A.

Knauf C.

Joza N.

Benit P.

Orthofer M.

Cani P.D.

Ebersberger I.

Nakashima T.

Sarao R.

Neely G.

et al. Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes. 9 pfu/g body weight) of PBS-diluted AdHO-1 and AdLacZ particles was injected into the tail vein of recipient mice. OGTT and ITT were performed on day 5 and 7 postinjection. The macrophage tropic RGD-fiber-modified () Ad(RGD)-HO-1 and Ad(RGD)-GFP obtained commercially from Vector Biolabs were used for Macho-BMDM rescue experiments. BMDMs were infected with 100 pfu/cell on day 4 of differentiation. Experiments were performed on day 7 of differentiation, i.e., 3 days after adenoviral transduction. For adenovirus experiments targeting livers and isolated primary Lhoko hepatocytes, in vivo grade HO-1 (AdHO-1) and LacZ (AdLacZ) expressing viruses were produced according to published protocols (). In brief, Sfi I-digested Adlox plasmid DNA was cotransfected with psi5 DNA into Cre8 cells using FuGENE 6 Transfection Reagent (Roche). Three days after transfection cells were collected by centrifugation and recombined viruses extracted from cell pellets by four freeze and thaw cycles. Cell debris was removed by centrifugation. HEK293 cells (ATCC) were used to amplify adenoviral particles. Amplified AdHO-1 and AdLacZ were purified by cesium chloride density-gradient ultracentrifugation, collected from the gradient, diluted 1:1 in 2x storage buffer (10 mM Tris pH 8.0, 100 mM NaCl, 0.1% BSA, 50% glycerol) and stored in small aliquots at −20°C. Primary Lhoko hepatocytes were infected with 10 plaque forming units (pfu)/cell. Transduced cells were incubated for 48 hr before experiments were started. In vivo tail vein injections were performed as described (). In brief, a total volume of 200 μl (0.2 × 10pfu/g body weight) of PBS-diluted AdHO-1 and AdLacZ particles was injected into the tail vein of recipient mice. OGTT and ITT were performed on day 5 and 7 postinjection.

NRF2 DNA-Binding Activity The NRF2 TransAM ELISA-kit (Active Motif) was used to evaluate NRF2 DNA-binding activity according to the manufacturer’s protocol. In brief, 8 μg of nuclear extracts isolated from naive BMDMs were incubated for 1 hr in 96 well plates on which oligonucleotides containing the antioxidant response element (ARE) consensus-binding site were immobilized. After binding and washing, NRF2 was detected using an antibody specific for active, i.e., DNA-bound NRF2. Addition of a horseradish peroxidase (HRP) coupled secondary antibody was used to quantitate-binding activity by spectrophotometry.