For analysis of body weight, body composition, and food intake, mice were kept at an ambient temperature of 23 ± 2°C with constant humidity and a 12-hour light/12-hour dark cycle. Mice had free access to water and were fed ad libitum with either a regular standard chow diet (5.6% fat; LM-485, Teklad) or a HFD (58% kcal fat; Research Diets Inc.). Measurements of energy expenditure were performed using a customized indirect calorimetric system (TSE Systems Gmbh.). After adaptation for 24 hours, recordings were collected over the following 118 hours.
Gene expression analysis.
Gene expression of tissue samples (n = 7–9 mice per group) was profiled with quantitative PCR–based (qPCR-based) techniques using SYBR green, TaqMan Single Probes, or TaqMan Low Density Arrays (Applied Biosystems). TaqMan Low Density Arrays were performed in n = 4 mice of each genotype. The relative expression of the selected genes was measured using the 7900HT Fast Real-Time PCR System (Applied Biosystems). For low-density arrays, the PCR reactions took place on a 384 well reaction card preloaded with the specific primers and probes by the manufacturer. The sequences of primers and probes were designed and validated by Applied Biosystems and were taken from the Assay-on-Demand mouse library. The relative expression levels of each gene were normalized to the housekeeping gene 18S, HPRT, or RPL32.
Cell extracts for Western blotting were prepared in radioimmunoprecipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors). Lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to Nitrocellulose-ECL membranes (GE Healthcare), where the immune complex was detected by chemiluminescence (GE Healthcare). Antibodies were purchased from Santa Cruz Biotechnology Inc. (Gapdh, CytC, p38, p-p38, Ppargc1a), Abcam plc (Ucp1), Progen Biotechnik (p62), and Cell Signaling Technology Inc. (Creb, P-Creb, Mapk1, P-Mapk1, Lipe, P-Lipe, P-Atf2).
Cell culturing and analysis of BAT primary cells and HIB1B cells.
Culturing of adipocytes was performed according to a protocol provided by C. Ronald Kahn (Joslin Diabetes Center). For isolation of BAT primary cells, the interscapular BAT of 1-day-old global p62–/– and WT mice was dissected and digested for 20 minutes in isolation buffer containing 61.5 mM NaCl, 2.5 mM KCL, 0.65 mM CaCl2, 2.5 mM glucose, 50 mM HEPES, 2% BSA Fraction V, and 1.5 mg/ml collagenase A (Roche Diagnostics Gmbh). Cells were cultured for 2 days in DMEM High-Glucose (Fisher Thermo Scientific) containing 20% FBS and 1% penicillin/streptomycin. After reaching confluency, differentiation was induced by switching to DMEM High-Glucose containing 10% FBS, 20 nM insulin, 1 nM T3, 0.125 mM indomethacin, 0.5 mM IBMX, and 5.1 μM dexamethasone. Two days after start of differentiation, medium was switched to DMEM High-Glucose containing 10% FBS, 20 nM insulin, and 1 nM T3 until start of the experiments. The HIB1B cells were provided from Bruce Spiegelman (Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School).
Depletion of macrophages from BAT primary cells.
Separation of macrophages from BAT primary cells was performed by magnetic immunoaffinity isolation using anti-Cd11b antibodies conjugated to magnetic beads (MACS Cell Separation System; Miltenyi Biotec). Following BAT digestion, Itgam-positive (Cd11b) cells were separated using positive selection columns (LD columns; Miltenyi Biotec) according to the manufacturer’s instructions. For validity of cell separation, cell eluates were taken before and after depletion of Cd11b-positive cells as well as from the retained cell fraction. Successful depletion of macrophages was confirmed by flow cytometry and qPCR analysis.
Flow cytometry to determine macrophage frequencies in BAT primary cells.
Dissociated BAT primary cells were suspended in PBS/10% FCS and preincubated with 5 μg/ml anti-Cd16/32 (FcγRII/III block, clone 2.4G2; BD Biosciences). Cells were then stained with FITC-conjugated anti-CD45 (clone 30-F11; eBioscience) and APC-conjugated anti-F4/80 (clone BM8; eBioscience) on ice for 20 minutes. Propidium iodide (1 μg/ml) was used to exclude dead cells. Flow cytometry was performed on a FACS Aria III (BD Biosciences), and results were analyzed using FACS Diva software (BD Biosciences).
Bioenergetics of BAT primary cells.
Primary cells of litters were pooled and independently differentiated and measured. Measurement of oxygen consumption rate (OCR) was performed using an extracellular flux analyzer (XF24, Seahorse Bioscience; Billerica). After differentiation for 2 days on a XF24 well plate, cells were washed in 1× PBS and incubated in 675 μl of DMEM (no. 5030; Sigma-Aldrich) in a non-CO2 incubator for 1 hour. Untreated oxygen consumption was recorded for 20 minutes followed by isoproterenol injection (0.5 μM) and subsequent OCR measurement. OCR measurement in macrophage-depleted primary cells was performed after differentiating for 7 days. Untreated oxygen consumption was recorded for 20 minutes followed by measurement of OCR after injection of isoproterenol (0.5 μM, 35 minutes), oligomycin (2 μg/ml, 21 minutes), FCCP (1 μM, 21 minutes), and rotenon/antimycin A (2.5 μM each, 14 minutes).
Cox activity of interscapular BAT was assessed polarographically using a Clark type electrode (RANK Brothers) and a Powerlab for data processing (ADInstruments). Tissue was weighted and homogenized mechanically in tissue buffer (100 mM potassium phosphate, 2 mM EDTA, 10 mM glutathione, pH 7.2, at 37°C) using QIAGEN TissueLyser. Homogenates were treated with detergent (0.1% n-dodecyl-β-D-maltoside) and subjected to a temperature-controlled reaction chamber containing 130 μM cytochrome c from horse heart (Sigma-Aldrich) and 18 mM ascorbate in measuring buffer (100 mM potassium phosphate, 5 mM EDTA, pH 7.2, at 37°C ).
Measurement of p38α.
Protein levels of total and phosphorylated p38α were assessed from BAT tissue using an InstantOne ELISA (eBioscience Inc.). BAT samples were disrupted using a TissueLyser (Qiagen) and lysed and analyzed according to the manufacturer’s instructions.
Transmission electron microscopy.
Tissue samples were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 (Science Services), postfixed in 2% aqueous osmium tetraoxide, dehydrated in gradual ethanol (30%–100%) and propylene oxide, embedded in Epon (Merck), and cured for 24 hours at 60°C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids and stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus; Carl Zeiss NTS GmbH). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions).
Statistical analyses were performed using the statistical tools implemented in Graph Pad Prism (GraphPad Software). Differences between treatment groups were assessed by 2-way ANOVA followed by Dunnett’s or Bonferroni’s post hoc test, 1-way ANOVA, or Student’s 2-tailed t test. All results are given as mean ± SEM. P < 0.05 was considered statistically significant.
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and were performed in accordance with the NIH principles of laboratory animal care.