mice (C57BL/6J background) were generated as described [8
]. For weight and survival curve studies, mice were housed in a conventional room. For all other studies, mice were housed in a pathogen-free barrier-type facility (both with a 12-h light/12-h dark cycle). Female mice were used for all studies and were fed a standard chow diet (5053 PicoLab Diet; Purina, St. Louis, MO). For the short-term calorie restriction study, we compared three groups of 15-16-month-old mice: WT-calorie restricted (WTCR), WT-ad libitum (WTAL) and Dgat1−/−
AL). Mice were individually housed and given free access to water. After a baseline assessment of average daily calorie consumption, WTCR mice were switched from ~106 kcal/d to 80 kcal/d of CR diet for 2 weeks, followed by 53 kcal/d for 2 weeks. For fecundity studies, timed matings were performed with young 8-12-week-old virgin female WT and Dgat1−/−
mice and proven male breeders. Because Dgat1−/−
mice do not lactate, embryos were harvested at day 18 of pregnancy, and the number of offspring was recorded.
Mice were fasted for 4 h and anesthetized, and their body compositions were analyzed by dual energy X-ray absorptiometry (DEXA) with a PixiMus2 scanner (GE Healthcare Lunar, Madison, WI).
Food intake (powdered standard chow) and oxygen consumption (VO2) were measured simultaneously using the Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH). Animals were acclimated to the cages on day one, and data were recorded on days two and three. Both food intake and VO2 were normalized to lean body mass, as measured by DEXA scanning on the day before calorimetry studies.
Tissue TG were analyzed as described [33
]. Briefly, lipids were extracted from heart, skeletal muscle and liver homogenates in CHCl3
:MeOH [2:1(v/v)]and separated by thin-layer chromatography with hexane:ethyl ether:acetic acid [80:20:1(v/v)]on silica gel G-60 TLC plates.
Total leukocytes and macrophages were assessed from pooled inguinal fat pads of 2 virgin female mice (4 pads per sample) fed a chow diet. Fat pads were minced with a razor blade, digested for 2 h at 37°C with 1 mg/ml type III collagenase (Worthington Biochemicals) and 100 U/ml DNase in DMEM/F12 containing 3% fatty acid-free BSA, and washed in BSA-containing medium. The cellular pellet was collected, incubated for 10 min at RT in ACK lysis buffer and washed again in staining buffer. Total leukocytes were counted at this point. To determine the proportion of macrophages in the isolated leukocyte population, cells were plated in 96-well V-bottom culture plates and incubated with rat anti-mouse CD16/CD32 (1:500 in staining buffer; BD Pharmingen, San Diego, CA) for 30 min at 4°C to block Fc receptors. Cells were then washed once with staining buffer, incubated for 30 min at 4°C with PE-conjugated F4/80 (BD), washed twice with staining buffer and fixed in 1% paraformaldehyde in PBS at 4°C until analysis with a FACS Calibur flow cytometer (BD Biosciences). Data were acquired with CellQuest software (BD Biosciences) and analyzed with FlowJo software (TreeStar, Mountain View, CA). F4/80 positive cells were separated into two populations, dim and bright. The F4/80 bright population is predicted to be the resident macrophages and the F4/80 dim population the recruited macrophages [34
Serum insulin (Linco, St. Louis, MO) and IGF1 (Diagnostics Systems Laboratories, Inc. Webster, TX) and leptin (R&D Systems, Minneapolis, MN) levels were measure by ELISA according to the manufacturer's instructions.
Liver tissues were collected from 14 month old mice and mitochondrial content was measured. Briefly, the liver was homogenized in Tris buffer [50 mM Tris-HCl (pH 7.5) containing 10 mM EDTA, 250 mM sucrose, pH 7.5]. The homogenate was centrifuged at 1000g for 5 min at 4°C, thereby pelleting genomic DNA. The supernatant was then centrifuged at 10,000g for 30 min at 4°C to pellet the mitochondria. This pellet was resuspended in lysis buffer [10 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.5% Triton X-100)] and placed on ice for 20 min. Protein K (14 mg/ml) and RNase (10 mg/ml) were added to each sample and incubated overnight at 50°C. Mitochondrial DNA was then extracted using an equal volume of phenol/chloroform and 1/5 volume of 5 mM NaCl, and precipitated in an equal volume of isopropanol at −20°C overnight. After centrifugation at 12,000g at room temperature, the resulting pellet of mtDNA was washed with 70% ethanol and then dried. The pellet was resuspended in 10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA and 20 mg/ml RNase. Genomic DNA content did not differ among groups.
Citrate synthase activity was measured by monitoring the conversion of acetyl-CoA and oxaloacetate to citrate, using the CoA thiol reaction with 5,5'-dithio-2-nitrobenzoate (DNTB) as described [35
Gene expression analyses
mRNA levels were quantified as described [33
]. Oligonucleotide primers were designed using Primer Bank (Supplemental Table 2
]. For microarray studies, three mice per group at 15-16 months of age were sacrificed, and their livers were flash frozen. Total RNA was isolated from approximately 100 mg of homogenized liver and prepared for hybridization to Mouse Affymetrix Gene 1.0 ST arrays, according to the manufacturer's protocol. The gene expression data were deposited in the Gene Expression Omnibus database (GSE26267). Expression values were obtained using RMA [37
] and associated non-adjusted t-test p-values with the limma R package [38
]. Expression clustering was performed using HOPACH [39
]. Pathway analysis and visualization were performed with the software GenMAPP-CS (http://www.genmapp.org/beta/genmappcs
After an overnight fast, mice were injected intraperitoneally with glucose (2 g/kg body weight), and blood glucose was measured at 0, 15, 30, 60, and 120 min with a One-Touch UltraSmart glucose monitoring system (Lifescan, Milpitas, CA).
Data are presented as mean ± SEM. Survival curves were analyzed using the Kaplan-Meier method. Means were compared with a Mann-Whitney rank-sum test or by analysis of variance followed by a Student Newman-Keuls multiple comparisons test. Weight curves were compared with a repeated measures ANOVA test followed by a Newman-Keuls test.