The creation of the original TRIP-Br2 knockout out bred mouse strain in the 129SvJ background and the generation of a TRIP-Br2 inbred mouse strain on a C57BL/6 background by over 10 generations of backcrossing, has been previously reported21
. Mice were housed in pathogen-free facilities and maintained on a 12 hr light/dark cycle at the Foster Biomedical Research Laboratory of Brande is University in Waltham, MA. All protocols were approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and Brande is University and were in accordance with NIH guidelines.
Paired samples of visceral and subcutaneous adipose tissue were obtained from 178 Caucasian men (N=92) and women (N=86), who underwent abdominal surgery as described in detail elsewhere50
. The age ranged from 16 to 82 years and body mass index from 20.8 to 54.1 kg/m2. In these subjects, abdominal visceral and subcutaneous fat area was calculated using abdominal MRI scans or computed tomography scans at the level of L4–L5. Percentage body fat was measured by dual-energy X-ray absorptiometry (DEXA). Individual assigned to all three cohorts (lean: BMI<25; visceral obesity: visceral/subcutaneous fat area > 0.4) fulfilled the following inclusion criteria: 1) Absence of any acute or chronic inflammatory disease as determined by a leukocyte count > 7000 Gpt/l, C-reactive protein (CRP) > 5.0 mg/dl or clinical signs of infection, 2) Undetectable antibodies against glutamic acid decarboxylase (GAD), 3) No medical history of hypertension, i.e. systolic blood pressure (SBP) was < 140mmHg and diastolic blood pressure (DBP) was < 85mmHg, 4) No clinical evidence of either cardiovascular or peripheral artery disease, 5) No thyroid dysfunction, 6) No alcohol or drug abuse, 7) No pregnancy. All subjects had a stable weight with fluctuations smaller than 2 % of the body weight for at least 3 months before surgery. Normal glucose tolerance (NGT) was defined as fasting plasma glucose <6.0mmol/l and a 120 min OGTT plasma glucose <7.8mmol/l. Impaired glucose tolerance (IGT) was defined as fasting plasma glucose <6.0mmol/l and a 120 min OGTT plasma glucose >7.8mmol/l and <11.1mmol/l. Type 2 diabetes was defined as fasting plasma glucose > 7.0mmol/l and/or a 120 min OGTT glucose >11.1mmol/l. The study was approved by the ethics committee of the University of Leipzig. All subjects gave written informed consent before taking part in the study.
Fasting plasma insulin was measured with an enzyme immunometric assay for the IMMULITE automated analyzer (Diagnostic Products Corporation). Plasma leptin levels were assessed by radioimmunoassay (Millipore). The OGTT was performed after an overnight fast with 75 g standardized glucose solution (Glucodex Solution 75g; Merieux) and insulin sensitivity was assessed with the euglycemic-hyperinsulinemic clamp method as described51
TRIP-Br2 mRNA expression in human visceral and subcutaneous adipose tissue
Human TRIP-Br2 mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler, and fluorescence was detected on an ABI PRISM7000 sequence detector (Applied Biosystems). Total RNA was isolated from paired subcutaneous and omentaladipose tissue samples using TRIzol (Life Technologies), and 1 μg RNA was reverse transcribed with standard reagents (Life Technologies). TRIP-Br2 mRNA expression was determined by a premixed assay on demand: Hs00207372_m1 SERTA domain containing 2 (Applied Biosystems). Samples were incubated in the ABI PRISM 7000 sequence detector for an initial denaturation at 95°C for 10 min, followed by 40 PCR cycles, each cycle consisting of 95°C for 15 s, 60°C for 1 min, and 72°Cfor 1 min. TRIP-Br2 mRNA expression was calculated relative to the mRNA expression of hypoxanthine phosphoribosyltransferase 1 (HPRT1), determined by a premixed assay on demand for human HPRT1 (Applied Biosystems). Amplification of specific transcripts was confirmed by melting curve profiles(cooling the sample to 68°C and heating slowly to 95°Cwith measurement of fluorescence) at the end of each PCR. The specificity of the PCR was further verified by subjecting the amplification products to agarose gel electrophoresis.
Statistical analysis for human subject data
All analyses were conducted with the two genders considered together as well as separately. TRIP-Br2 expression in subcutaneous and visceral fat was compared between lean and obese subjects by means of t tests. The unadjusted correlation between TRIP-Br2 expression and metabolic parameters was evaluated by means of Pearson’s correlation coefficients. Adjusted correlation coefficients were derived from partial r2 obtained from ANCOVA models that included each of the metabolic parameters and the visceral fat area as predictors of TRIP-Br2 expression. The association between quartiles of TRIP-Br2 visceral fat expression and risk of type 2 diabetes was evaluated by logistic regression with and without adjustment for visceral and subcutaneous fat areas and expressed by means of Odds Ratios and their 95% CI.
Body weight study
For diet-induced obesity, all mice were fed a chow diet (21.6% fat, 23% protein and 55.4% carbohydrate by kcal; #5020, LabDiet) until 6 weeks of age. Subsequently, mice were assigned randomly to either a low-fat (10% fat, 20% protein, and 70% carbohydrate by kcal; D12450B, Research Diets) or a high-fat diet (60% fat, 20% protein, and 20% carbohydrate by kcal; D12492, Research Diets) until the end of the experimental protocol. Body weight was measured weekly until 18 weeks of age.
Adipocyte size determination
Adipocyte cross-sectional area from caveolin (BD Biosciences) stained adipose tissue images (150-200 adipocytes/mouse, 3 mice/genotype) was calculated using CellProfiler image analysis software (Broad Institute, http://www.cellprofiler.org/
Fatty acid oxidation
Differentiated 3T3-L1 cells were treated with either vehicle or isoproterenol for 1 hr. Cells were then washed and incubated with assay media containing 2% fatty acid-free BSA, 0.30 mM L-Carnitine, and 3
H-palmitic acid (3 uCi/well) in low glucose DMEM media for 1 hr. Fatty acid oxidation was determined by measuring 3
O production as previously described and normalized with total cellular protein45
Isolated white and brown adipocytes were used to determine fatty acid oxidation by measuring 14
production from [U-14
C] palmitic acid (0.2 uCi/ml) after incubation for 1 hr at 37°C with gentle shaking as previously described before normalized with total lipid content54
Transcription Activator-Like Effector Nuclease (TALEN) plasmid DNA was designed, synthesized and tested by Cellectis Bioresearch. Exchange/integration matrix harboring the mutation was synthesized by GenScript. 3T3-L1 preadipocytes at 50-60% confluency were transfected with 1 μg of each TALEN plasmid and exchange matrix using Lipofectamine2000 (Invitrogen). Twenty-four h after transfection, cells were trypsinized and selected using puromycin. Colonies were then picked and expanded before screening with PCR and confirmed with sequencing.
Adipose tissue oxygen consumption and extracellular acidification
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined in mouse brown adipose tissue using a modified protocol55
. Briefly, freshly isolated BAT from WT and TRIP-Br2 KO mice (n
= 5 per genotype) were rinsed with unbuffered KHB medium containing 111 mM NaCl, 4.7 mM KCl, 2 mM MgSO4, 1.2 mM Na2HPO4, 0.5 mM carnitine and 2.5 mM glucose. Adipose tissue was cut into small pieces and rinsed with KHB medium, and 10 mg of tissue was placed in each well of a XF24-well Islet Flux plate (Seahorse Bioscience). Then, 450 μl of KHB medium was added to each well and samples were analyzed in an XF24 Extracellular Flux Analyzer (Seahorse Bioscience) at 37°C56
. The XF24 Analyzer mixed the media in each well three times for 2 min before measurements to allow oxygen partial pressure to equilibrate. Basal OCR and ECAR were measured in all wells three times. Five tissue replicates from 5 mice per genotype were analyzed in independent experiments and results were normalized to tissue weight.
Ex vivo lipolysis and lipogenesis
Isolated adipocytes were incubated with or without increasing doses of isoproterenol (10−6
M), isoproterenol (5μM), forskolin (20μM) or IBMX (0.2μM) for 90 min. Extracellular glycerol release was extracted and measured using Free Glycerol Reagent (Sigma) as an indicator of lipolysis57
. For the assessment of lipogenesis, isolated adipocytes were incubated with [14
C] deoxyglucose in the presence or absence of increasing concentrations of insulin (0.1-100 nM) for 90 min. Incorporated radiolabeled glucose was extracted and measured using liquid scintillation counting58
Mouse 3T3-L1 preadipocytes were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Two days after confluence, adipocyte differentiation was initiated with the addition of 1.7 μM insulin, 0.5 mM isobutylmethylxanthine (IBMX) and 1 μM dexamethasone in DMEM media supplemented with 10% FBS for 2 days, followed by 2 days in medium supplemented with insulin, and finally cultured for 4 days in normal growth medium.
Tissue triglyceride analysis
Lipids from tissues were extracted with Folch solution consisting of a mixture of 2:1 (vol/vol) chloroform/methanol as previously described59
. Lipids were solubilized in 1% Triton X-100 before evaporation under nitrogen gas. Triglyceride content was determined using Triglyceride Determination Kit (Sigma).
Complete ORF of TRIP-Br2 was amplified from 3T3-L1 preadipocytes cDNA with PCR using oligos carrying flag epitope-tag at the C-terminal from IDT and cloned into pBabe-puro retroviral or pCDNA3.1 vector60
. Mouse hormone sensitive lipase
or β3-adrenergic receptor
) promoter and respective deletion constructs were amplified from 3T3-L1 preadipocyte genomic DNA with PCR using oligos from IDT and cloned into pGL3 or pGL3-min-promoter luciferase vector respectively (Promega). All plasmids were sequenced verified. Expression plasmids for E2F1, E2F4 and DP1 were gifts from L. Fajas (Metabolism and Cancer Laboratory, INSERM, France)19
3T3-L1 preadipocyte cell lines stably over-expressing TRIP-Br2-Flag were established by infecting 3T3-L1 cells using retrovirus containing pBabe-puro-TRIP-Br2-flag construct, followed by puromycin selection. The stable cell lines used in the experiments were mixed clones from at least three independent viral productions, infections, and selections.
Rabbit polyclonal anti-TRIP-Br2 was raised against the peptide with the amino acid sequence TRIP-Br2-25-39: DGPSKVSYTLQRQT and affinity purified with the antigen (YenZym Antibody). Chicken polyclonal anti-HSL, anti-Adrb3, rabbit anti-perilipin A and mouse anti-α-tubulin were obtained from Abcam. pHSL, pAkt, Akt, pERK, ERK, ATGL antibodies were from Cell Signaling. PPARγ, CGI-58 and G0S2 antibodies were from Santa Cruz.
Food intake, energy expenditure, physical activity and body composition
Food intake, physical activity, oxygen consumption (VO2), carbon dioxide (VCO2) and heat production was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments). The respiratory exchange ratio (VCO2/VO2) was calculated from the gas exchange data and all data were normalized to lean body mass. Body composition (fat and lean mass) was assessed by the Dual-Energy X-Ray Absorptiometry (DEXA).
Body temperature and cold exposure
Body temperatures were assessed in 18-week-old male mice using a RET-3 rectal probe for mice (Physitemp). Mice were exposed to an ambient temperature of 4°C in a cold room until their core body temperature dropped to 25°C. Body temperatures were measured at 30 min intervals using a digital thermometer.
Plasma insulin was measured with an ELISA kit (Millipore). NEFA, TG and cholesterol concentration in serum were measured with NEFA-C and Triglyceride E tests (Wako), respectively. Serum adiponectin and leptin levels were measured with ELISA kits from R&D Systems (Joslin DERC Assay Core).
Physiological studies and Histological analyses
Blood glucose was monitored with an automated glucose monitor (Glucometer Elite, Bayer). Glucose tolerance tests and insulin tolerance tests were performed 16 hr after fasting as described previously61
. Mice were anesthetized, and tissues were rapidly dissected, weighed and processed for immunohistochemistry as described previously61
In anesthetized mice, the posterior aspect around the neck and intra-scapular region were first shaved and cleaned before a T-shaped incision (1/2 inch each direction) in the mid-scapular region was made. Under a dissecting microscope, the intra-scapular fat pad was carefully separated from the surrounding skin and a drop of 1% filtered sterilized toludine blue was applied to the fat pad to allow visualization of the nerves. After careful separation of the nerves from the surrounding tissues and vasculature around the fat pad, small nerves were cut in 3 different locations and around 3mm segments of the larger nerves were removed to prevent possible re-connection. Sham-operated animals were treated similarly using the same procedure except cutting the nerves. After recovery, operated animals were placed on HFD and monitored for their body weight and temperature until the end of the experiment.
3T3-L1 preadipocytes were cotransfected by Lipofectamine 2000 reagent (Invitrogen) with HSL and Adrb3 reporter constructs, various combinations of pCDNA3.1-TRIP-Br2-Flag and mammalian expression constructs expressing E2F1, E2F4, and DP1, along with a promoterless Renilla luciferase construct. Lysates were collected 36 h after transfection, and firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter System (Promega).
TAG hydrolase assay
To prepare the substrate for the TG hydrolase activity, triolein (Sigma) and [9,10-3H] triolein (Perkin Elmer) as radioactive tracer was emulsified with phosphatidylcholine/phosphatidylinositol (Sigma) using a sonicator. The final substrate concentration was 167 nmol of triolein/assay (8000 cpm of [9,10-3H] triolein/nmol). 100 μl with or without HSL specific inhibitor were incubated with 100μl of the substrate (adipose tissue extract) in a water bath at 37°C for 60 min. The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5 before vortex twice for 30 s. After centrifugation at 3200 rpm for 15 min at room temperature, the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting.
Adipocytes derived from the Cont-3T3-L1 and TRIP-Br2-Flag-3T3-L1 cell lines were fixed at day six of their differentiation by addition of 37% formaldehyde to a final concentration of1% formaldehyde and incubated at room temperature for 10 min. Cross-linking was stopped by addition of glycine to a final concentration of 0.125 M. Cells were then scraped and samples were prepared using the EZ-Magna ChIP™
G Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. The chromatin fractions were incubated in each case with 10 μg of one of the following antibodies: anti-Flag M2 (F-1804; Sigma), anti-RNA polymerase II and Normal IgG mouse (both provided by Millipore Kit), Millipore) at 4 °C overnight with Magnetic Protein G Beads. After extensive washing and final elution, the product was treated for 4h at 65°C to reverse cross-linking. Input DNA and immunoprecipitated DNA were purified using kit column and analyzed by quantitative PCR using Maxima™
Sybr Green qPCR Master Mix (Fermentas Life Sciences) with different set of primers (both proximal and distal promoter regions) listed here. Mouse HSL
proximal promoter (F 5′-gcggaatggaaacagcgtagtgaa-3′, R 5′-tgttacctgccattgcttcggaga-3′), mouse HSL
distal promoter (F 5′-aacacagttcaagggctggagaga -3′, R 5′-caccatgtggttgctgggaattga -3′), mouse GAPDH
(F 5′-tactcgcggctttacggg-3′, R 5′-tggaacagggaggagcag-3′), mouse β3 adrenergic receptor
proximal (F 5′-cttggatggtttgggttgttcggt -3′, R 5′-agggcttctgctgcaaagaagaga -3′) and mouse β3 adrenergic receptor
distal (F 5′-ttgtcccaaccaggacagaacaga -3′, R 5′-aagaaagccaggcacgttcacaag -3′). Data were normalized by input values (for each set of promoter oligonucleotides) and binding was expressed relative to the non-specific binding of IgG immunoprecipitated DNA content.
All data are presented as mean ± SEM and analyzed by unpaired two-tailed Student’s t-test or analysis of variance (ANOVA) as appropriate. A p-value less than 0.05 is considered significant.