Obesity is associated with macrophage accumulation in adipose tissue (original) (raw)

Animals and animal care. Unless otherwise noted, all mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) at 6–8 weeks of age and housed in ventilated Plexiglas cages (one to three animals per cage) within a pathogen-free barrier facility that maintained a 12-hour light/dark cycle. Mice had free access to autoclaved water and irradiated pellet food. C57BL/6J mice in which obesity was induced by a high-fat diet (diet-induced obese or DIO mice) were fed pellets that derived approximately 45% of calories from lipids (diet D12451; Research Diets Inc., New Brunswick, New Jersey, USA) for 12 weeks. All other mice were fed a standard pellet diet that derived about 5% of calories from lipids (PicoLab Rodent Diet 20; Purina Mills Inc., Brentwood, Missouri, USA). The toothless FVB/NJ Csf1op/op mice were fed a powdered form of the standard diet.

Mice were sacrificed by CO2 asphyxiation at 20–21 weeks of age during the second and third hour of the light cycle. Animals were weighed and adipose tissues (epididymal or parametrial, perirenal, mesenteric, and inguinal subcutaneous depots), liver, and extensor digitalis longus muscle were removed. Tissues to be analyzed by FACS were processed immediately; other samples were frozen in liquid nitrogen and stored at –75°C prior to RNA extraction and immunohistochemical analysis. All procedures were approved by Columbia University’s Institutional Animal Care and Use Committee.

A total of 24 mice were included in the microarray expression study, four from each of the following groups: C57BL/6J males, C57BL/6J females, high fat–fed C57BL/6J males, B6.Cg Ay/+ females, B6.V Lepob/ob males, and B6.V Lepob/ob females. In B6.Cg Ay/+ mice, ectopic overexpression of the agouti transcript leads to a moderate increase in the mass of adipose tissue and a hyperleptinemic form of obesity (35). Feeding male C57BL/6J mice a high-fat diet also leads to hyperleptinemic moderate obesity. B6.V Lepob/ob mice are leptin-deficient and severely obese (36, 37). A pair of 2-month-old macrophage-deficient (FVB/NJ Csf1op/op) and control (FVB/NJ Csf1+/+) female mice were a gift of E. Richard Stanley (Albert Einstein College of Medicine, New York, New York, USA). Macrophage-deficient mice are osteopetrotic and toothless; these mice were maintained on a powdered chow diet (∼5% fat content). For transplant experiments, CD45.2+ (C57BL/6J) recipient and CD45.1+ (B6.SJL Ptprca Pep3b/BoyJ) donor mice were purchased from The Jackson Laboratory. These mice are homozygous for antigenically distinct forms of the CD45 protein, a protein expressed on all leukocytes.

Human subjects and materials. Healthy lean, overweight, and obese subjects were admitted to the Clinical Research Center at Columbia Presbyterian Medical Center (New York, New York, USA) as part of a longitudinal study of the metabolic effects of weight perturbation. The details of this study have been described previously (38–40). The study protocol was approved by the Institutional Review Board of Columbia University, and written informed consent was obtained from each subject. Subjects were fed a liquid formula diet of 40% fat (corn oil), 45% carbohydrate (glucose polymer), and 15% protein (casein hydrolysate) supplemented with 5.0 g iodized NaCl, 1.9 g K, and 2.5 g calcium carbonate per day, 1 mg of folic acid twice weekly, and 36 mg ferrous iron every other day. Daily formula intake was adjusted until weight stability — defined as a slope of < 0.01 kg/d in a 14-day plot of weight versus days — was achieved. Subjects were fed the liquid formula diet for 5–8 weeks before adipose tissue aspiration was performed. Subjects in the postabsorptive state underwent needle aspirations of abdominal subcutaneous adipose tissue at the level of the umbilicus. Local anesthesia was achieved with 1% xylocaine, and 2–4 g subcutaneous adipose tissue was aspirated using a 15-gauge needle. A sample of tissue was frozen immediately in liquid nitrogen and stored at –80°C. These samples were used in the studies of tissue morphology and gene expression described here.

Microarray gene expression. Total RNA was extracted from the perigonadal (epididymal or parametrial) adipose tissue of individual mice using a commercially available acid-phenol reagent (TRIzol; Invitrogen Corp., Carlsbad, California, USA). RNA concentration was assessed by absorbance spectroscopy, and RNA integrity was confirmed by nondenaturing agarose gel electrophoresis. Twenty micrograms of RNA from each sample was further purified to remove contaminating organics and non-RNA species using a silica resin (RNeasy; Qiagen Inc., Valencia, California, USA) protocol according to the manufacturer’s instructions. Total RNA from single animals was individually converted into biotinylated, fragmented cRNA using protocols recommended by the microarray manufacturer (Affymetrix Inc., Santa Clara, California, USA). Samples of cRNA derived from single animals were hybridized in recommended buffer to microarrays (Murine Genome Array U74Av2, Affymetrix Inc.) at 45°C for 16 hours. The samples were stained and washed according to the manufacturer’s protocol on a Fluidics Station 400 (Affymetrix Inc.) and scanned on a GeneArray Scanner (Affymetrix Inc.). Primary data extraction was performed with Microarray Suite 5.0 (Affymetrix Inc.), and signal normalization across samples was carried out using all probe sets with a mean expression value of 500.

Statistical analyses. Without any computational prefiltering of genes, the normalized expression signal from each microarray probe set was examined to identify genes that were significantly associated with body mass among the 24 mice in this study. We used Kendall’s τ statistic, a rank-based test of correlation, to detect transcripts whose expression levels were significantly correlated with body mass. Each analysis was performed by allowing the false discovery rate to be no more than 0.03, where the false discovery rate was defined as the expected proportion of falsely rejected hypotheses (“false positives”) as described by Benjamini and Hochberg (41). We used a false discovery rate rather than standard significance measures to avoid an inflated false-positive rate as a consequence of the large number of hypothesis tests (42). All statistical analyses were implemented in S-Plus (http://www.insightful.com) or R (http://www.r-project.org).

Immunohistochemistry. Adipose tissue, muscle, and liver samples were fixed for 12–16 hours at room temperature in zinc-formalin fixative (Anatech Ltd., Battle Creek, Michigan, USA) and embedded in paraffin. Five-micron sections cut at 50-μm intervals were mounted on charged glass slides, deparaffinized in xylene, and stained for expression of F4/80 as described by Cecchini et al. (43) with an anti-F4/80 monoclonal antibody provided by E. Richard Stanley (Albert Einstein College of Medicine), or for expression of CD68 with the commercially available monoclonal antibody PG-M1 according to the manufacturer’s instructions (Dako CytoMation, Carpinteria, California, USA). For each individual mouse adipose depot, four different high-power fields from each of four different sections were analyzed. The total number of nuclei and the number of nuclei of F4/80-expressing cells were counted for each field. The fraction of F4/80-expressing cells for each sample was calculated as the sum of the number of nuclei of F4/80-expressing cells divided by the total number of nuclei in sections of a sample. The same procedure was used to measure the fraction of CD68-expressing cells in human tissues. Adipocyte cross-sectional area was determined for each adipocyte in each field analyzed using image analysis software (SPOT version 3.3; Diagnostic Instruments Inc., Sterling Heights, Michigan, USA). Average adipocyte cross-sectional area was calculated for each animal using Microsoft Excel (Microsoft Corp., Redmond, Washington, USA).

Isolation of adipose tissue macrophages, adipocytes, and SVCs. Adipose tissue was isolated from mice immediately after CO2 asphyxiation. Tissues were handled using sterile techniques and minced into fine (<10 mg) pieces. Minced samples were placed in HEPES-buffered DMEM (Invitrogen Corp.) supplemented with 10 mg/ml fatty acid–poor BSA (FAP-BSA; Sigma-Aldrich, St. Louis, Missouri, USA) and centrifuged at 1,000 g for 10 minutes at room temperature to pellet erythrocytes and other blood cells. An LPS-depleted collagenase cocktail (Liberase 3; Roche Applied Science, Indianapolis, Indiana, USA) at a concentration of 0.03 mg/ml and 50 U/ml DNase I (Sigma-Aldrich) was added to the tissue suspension and the samples were incubated at 37°C on an orbital shaker (215 Hz) for 45–60 minutes. Once digestion was complete, samples were passed through a sterile 250-μm nylon mesh (Sefar America Inc., Depew, New York, USA). The suspension was centrifuged at 1,000 g for 10 minutes. The pelleted cells were collected as the SVCs, and the floating cells were collected as the adipocyte-enriched fraction. The adipocyte fraction was further digested for 1 hour, washed twice with DMEM, and centrifuged as above until there was no further cell/debris pellet. The SVCs were resuspended in erythrocyte lysis buffer and incubated at room temperature for 5 minutes. The erythrocyte-depleted SVCs were centrifuged at 500 g for 5 minutes, and the pellet was resuspended in FACS buffer (PBS containing 5 mM EDTA and 0.2% [wt/vol] FAP-BSA).

Immunophenotyping and FACS. SVCs isolated from adipose tissue samples were cooled on ice and counted using a hemocytometer. Cell survival rates ranged from 70% to 90%. After counting the cells, we centrifuged them at 500 g for 5 minutes and resuspended in FACS buffer at a concentration of 7 × 106 cells/ml. Cells were incubated in the dark at 4°C on a bidirectional shaker for 30 minutes in FcBlock (20 μg/ml) (BD Pharmingen, San Jose, California, USA), then for an additional 50 minutes with fluorophore-conjugated primary antibodies or isotype control antibodies. Antibodies used in these studies included: CD11b-phycoerythrin (CD11b-PE) (2 μg/ml), CD45.1-PE (5 μg/ml), CD45.2-PE (5 μg/ml; eBioscience, San Diego, California, USA), and F4/80-APC (5 μg/ml; Caltag Laboratories Inc., Burlingame, California, USA). Following incubation with primary antibodies, 1 ml FACS buffer was added to the cells. Cells were centrifuged at 500 g for 5 minutes and resuspended in 1 ml FACS buffer. The wash was repeated twice. Cells were analyzed on a FACSCalibur and analysis was performed using CellQuest software (Becton, Dickinson and Co., Franklin Lakes, New Jersey, USA). Macrophages stained with F4/80-APC were separated from F4/80– cells using a FACSAria cell sorter (BD Biosciences Immunocytometry Systems Inc., San Jose, California, USA). F4/80+ and F4/80– cells were collected into cooled FACS buffer, centrifuged at 500 g for 5 minutes, and immediately frozen for gene expression analysis.

Quantitative real-time PCR. Total RNA was extracted from frozen adipose tissue (100 mg), FACS-isolated cells (>105), or cultured cells (60-mm confluent plate) using a commercially available acid-phenol reagent (TRIzol; Invitrogen Corp.). For tissue samples, first-strand cDNA was synthesized using SuperScript II reverse transcriptase and random hexamer primers as described in the manufacturer’s protocol (Invitrogen Corp.). Samples of cDNA were diluted 1:25 in nuclease-free water (Qiagen Inc.). For isolated cell populations, SuperScript III was used to generate cDNA. Samples from each cDNA pool were diluted 1:10, 1:30, 1:90, and 1:270 in order to create a standard curve for calculation of relative gene expression levels. PCR amplification mixtures (20 μl) contained 10 μl of 2× PCR SYBR Green I QuantiTect Master Mix (Qiagen Inc.), 0.4 μl of a mixture of 25 μM reverse and forward primers, and 11.6 μl diluted cDNA template. Real-time quantitative PCR was carried out using either the LightCycler (human samples; Roche Applied Science) or DNA Engine Opticon instruments (mouse samples; MJ Research Inc., Waltham, Massachusetts, USA) with the following cycling parameters: polymerase activation for 15 minutes at 95°C and amplification for 40 cycles of 15 seconds at 94°C, 10 seconds at 58°C, and 10 seconds at 72°C. After amplification, melting curve analysis was performed as described in the manufacturer’s protocol (Qiagen Inc.). Relative expression values were calculated based on the standard curve using LightCycler software (version 3.5; Roche Applied Science).

Determination of relative expression values. The expression rates of three macrophage-specific genes (Emr1, Cd68, and Csf1) that correlated with body mass in our microarray studies, an adipocyte-specific gene (Acrp30), and proinflammatory genes (Tnfa, Nos2, Il6) were determined by quantitative RT-PCR. To normalize expression data, we used multiple internal control genes as described by Vandesompele et al. (44). Internal control genes were selected from our murine microarray data set and a separate human data set (A.W. Ferrante and D. McCann, unpublished results). For each species, we chose control genes with high expression levels and little sample-to-sample variability. For human controls these were Excision repair cross-complementing group 3 (ERCC3), Amplified in osteosarcoma (OS-9), and Casein kinase-1d (CSNK1D). For each gene to be assayed, intron-spanning primers were designed using publicly available genomic contig sequences obtained through LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/index.html), the public domain primer design software Primer3 (http://www-genome.wi.mit.edu/genome\_software/other/primer3.html), and the DNA analysis software Vector NTI Suite, version 7 (Informax Inc., Bethesda, Maryland, USA) (45). Human primer sequences were as follows: hCD68 forward (5′-GCTACATGGCGGTGGAGTACAA-3′), hCD68 reverse (5′-ATGATGAGAGGCAGCAAGATGG-3′); hERCC3 forward (5′-ACATTGACCTAAAGCCCACAGC-3′), hERCC3 reverse (5′-AGTTGCCCAGCACCAGACAG-3′); hOS-9 forward (5′-TAAACGCTACCACAGCCAGACC-3′), hOS-9 reverse (5′-AGCCGAGGAGTGCGAATG-3′); hCSNK1D forward (5′-AGGAGAAGAGGTTGCCATCAAG-3′), hCSNK1D reverse (5′-TCCATCACCATGACGTTGTAGTC-3′).

For expression analysis in mice, we used microarray data as described above to select two internal control genes, cyclophilin B (Cphn2) and ribosomal protein S3 (Rps3). Mouse primer sequences were as follows: F4/80 forward (5′-CTTTGGCTATGGGCTTCCAGTC-3′), F4/80 reverse (5′-GCAAGGAGGACAGAGTTTATCGTG-3′); CD68 forward (5′-CTTCCCACAGGCAGCACAG-3′), CD68 reverse (5′-AATGATGAGAGGCAGCAAGAGG-3′); CSF1-R forward (5′-GCATACAGCATTACAACTGGACCTACC-3′), CSF1-R reverse (5′-CAGGACATCAGAGCCATTCACAG-3′); TNF-α forward (5′-CCAGACCCTCACTAGATCA-3′), TNF-α reverse (5′ CACTTGGTGGTTTGCTACGAC-3′); IL-6 forward (5′-CCAGAGATACAAAGAAATGATGG-3′), IL-6 reverse (5′- ACTCCAGAAGACCAGAGGAAAT-3′); RPS3 forward (5′-ATCAGAGAGTTGACCGCAGTTG-3′), RPS3 reverse (5′-AATGAACCGAAGCACACCATAG-3′); iNOS forward (5′-AATCTTGGAGCGAGTTGTGG-3′), iNOS reverse (5′-CAGGAAGTAGGTGAGGGCTTG-3′); PEX11a forward (5′-GACTTTTCAGAGCCACTCAGCAC-3′), PEX11a reverse (5′-GCCCACCTTTGCCATTTCTC-3′); ACRP30 forward (5′-GCTCCTGCTTTGGTCCCTCCAC-3′), ACRP30 reverse (5′-GCCCTTCAGCTCCTGTCATTCC-3′).

For each cDNA and standard curve sample, quantitative PCR reactions were performed to assay the expression of each internal control gene. To verify that the relative expression values of the control genes provided an accurate reflection of cDNA loading, we correlated the relative expression values of the control genes with one another and with the geometric mean of the three values. The Pearson correlation coefficients were consistently greater than or equal to 0.97, strongly suggesting that expression of the three control genes provided a reasonably accurate reflection of cDNA loading. To calculate the normalized relative expression levels of each gene assayed in each sample, we divided the relative gene expression value for that sample by the geometric mean of the relative expression values of the control genes. Separate analyses in which relative expression values were normalized with the relative expression values of each control gene yielded similar results.

Adipocyte differentiation of preadipocytes. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, Virginia, USA) were grown to confluence in DMEM-FBS at 37°C in a 5% CO2 incubator. Two days after reaching confluence (day 0), the culture medium was changed to DMEM-FBS supplemented with insulin (50 μg/ml), 3-isobutyl-1-methylxanthine (0.4 mM), and dexamethasone (1 μM). After 2 days, the medium was again changed to DMEM-FBS supplemented with insulin (50 μg/ml). Finally, 3 days later, the medium was changed to DMEM-FBS. Medium was replaced with fresh DMEM-FBS every 3 days thereafter.

Bone marrow transplantation. Six-week-old male CD45.2+ (C57BL/6J) recipient mice and syngeneic CD45.1+ (B6.SJL Ptprca Pep3b/BoyJ) donor mice were purchased from The Jackson Laboratory. At 8 weeks of age, recipient mice received a dose of 12 Gy from a Cs137 source (Gammacell-40; Atomic Energy of Canada Ltd., Mississauga, Ontario, Canada). The γ radiation was administered as two 6-Gy doses separated by a 3- to 4-hour interval in order to minimize radiation toxicity. Bone marrow was collected from the femurs and tibias of sex-matched donor mice into HBSS. Donor marrow cells (5 × 106) were injected into the recipient’s lateral tail vein 4 hours after irradiation. The recipient mice were maintained in a pathogen-free facility and were fed a standard chow diet and water supplemented with 0.5 ml/l of 10% Baytril (Bayer Corp., Shawnee Mission, Kansas, USA). After 4 weeks, blood was collected from recipient mice and peripheral engraftment was confirmed via FACS analysis based on presence of the CD45.1 antigen and absence of the CD45.2 antigen. Ninety-five percent of mice survived transplant and more than 95% of peripheral blood leukocytes in recipients were donor-derived CD45.1+ cells. Once engraftment was confirmed, recipient mice were placed on a high-fat diet containing about 60% of calories from lipids (D12492; Research Diets Inc.) for 6 weeks. Mice were sacrificed by CO2 asphyxiation, and SVCs from perigonadal adipose tissue were collected for FACS analysis.