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Materials
and Methods
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2. MATERIALS AND METHODS
2.1 Experimental animals
Haptoglobin null mice (Hp −/−) were originally generated in Baumann’s laboratory (Lim SK et
al., 1998). Upon a material transfer agreement, founders mice were transferred to our
laboratories, where a new colony was initiated. They have been backcrossed 10 times to the C57BL/6J background. In each experiment we employed Hp −/− and Hp +/+ obtained from the same litter generated by crossing Hp +/− mice. In our experiments age-matched littermates C57BL/6J wild type (WT) mice were used as controls.
Adult male mice (2 months old) were placed on either a CFD (Chow Food Diet, Diet 2018 Teklad global diet, 18.9% protein, 5.7% fat and 57.3% g / weight carbohydrate, Harlan Teklad IN, USA) or on a HFD (High Fat Diet, Diet F3282, 19% protein, 36% fat and 35% carbohydrate g / weight, Bioserve Frenchtown, NJ, USA) for 12 weeks. Animals that did not reach 40 grams of weight at the end of the diet were excluded from further investigations. No genotype related difference was observed in the frequency of failure to become obese. Food intake was monitored by weighing the left over food every other day throughout the diet period.
Serum, epididymal (EPI), subcutaneous (SC, from the inguinal region) and perirenal (PERI) fat pads, brown adipose tissue (BAT), kidney and liver from fasted adult mice were dissected, weighed and frozen in liquid nitrogen for mRNA expression experiments or stored in formalin for histology. Blood was withdrawn by cardiac puncture and from the tail vein (in live animals). Fat % was calculated as follows: (weight of EPI WAT + weight of PERI WAT + weight of SC WAT) / Body weightper 100.
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The experimental protocols followed the Principles of Laboratory Animal Care and a specific authorization was issued by Italian Ministry of Education (114/2003-A of 16/09/2003) to CNR Neuroscience Institute, where animals were housed.
2.2 Histology, immunohistochemistry and morphometric analysis of tissues
EPI, SC fat pads and BAT of lean and obese mice were fixed in 10% formaldehyde and embedded in paraffin. Serial five-µm sections were cut by microtome, mounted on glass slides, deparaffinized in xylene and rehydrated with sequential ethanol / phosphate-buffered saline (PBS) washes. For immunohistochemistry, EPI and SC five-µm sections were treated with 0.3% (v/v) H2O2 solution for 30 min, blocked for 1 hour at Room Temperature (RT) and then
incubated with an anti-mouse F4/80 antibody (AbD Serotec, Kidlington, UK) at 10 µg / mL for 1 hour at RT. Slides were incubated with biotinylated goat-anti rat IgG antibody (1:200) (Elite ABC KIT, Vectastain, Vector Laboratories, CA, USA) for 30 min at RT and revealed by DAB (Vector Laboratories, CA, USA), followed by counter-staining with hematoxylin. BAT sections were stained with hematoxylin and eosin (H&E).
For histology, five-µm sections of paraffin-embedded samples were stained with H&E. At least 15 random optical fields per individual sample were acquired using a Nikon Eclipse i80 microscope (40X magnification). Adipocyte areas were determined using ImageJ software.
2.3 Ex-vivo cultures and primary adipocytes
Tissue explants of visceral WAT, liver and kidney from CFD WT mice (5 months old, mean body weight (BW) 26.5 g) were isolated, cut into small pieces and incubated and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen #41965) with antibiotics and 2%
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(w/v) BSA at 37°C in 5% CO2 atmosphere. Aliquots of the medium were taken at different time
points and stored at -20°C: Hp concentration was assessed by ELISA (see Section 2.10). Time point 0 was considered as the baseline and the Hp concentration obtained at this time was subtracted from values obtained afterwards.
Human SC WAT was collected from 3 subjects who underwent bariatric surgery for obesity, for which signed, fully informed consent was obtained according to institutional guidelines. Primary human adipocytes were obtained with a modification to the method of Rodbell (Rodbell M et al.,
1964; Funicello M et al., 2007). Briefly, adipose tissue was dissected, rapidly washed in PBS,
then minced and digested for 1 h at 37°C with Collagenase A (20 mg/ml from Clostridium histolyticum, Sigma-Aldrich, St Louis, MO, USA, #C9891) in Krebs Ringer Bicarbonate buffer (KRBH-buffer), containing 10mM NaHCO3, 30 mM Hepes and 1% BSA (Sigma-Aldrich, St
Louis, MO, USA) at pH 7.4. 1 ml of collagenase solution was added to 4 g of fat pad in KRBH-buffer. Fat cells were filtered through nylon mesh (100 µm) and washed twice with the same incubation buffer. After the final wash, human adipocytes were resuspended in DMEM supplemented with heat-inactivated 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C with 5% CO2and seeded in 24-well plates. Cells were treated with
human Hp (1 mg/ml) (Sigma-Aldrich, St Louis, MO, USA #H3536) for 24 h. At the end of treatment cells were collected and total RNA isolated.
2.4 Culture, differentiation and treatment of 3T3-L1 pre-adipocytes
Murine 3T3-L1 pre-adipocytes were grown in DMEM (Invitrogen, #41965) supplemented with heat-inactivated 10% FBS and antibiotics (100 U/ml penicillin/ and 100 µg/ml streptomycin) in a humidified 5% CO2 atmosphere at 37°C. The differentiation process into adipocytes was induced
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cocktail containing 167 nM insulin (Sigma-Aldrich, St Louis, MO, USA #I-5500) 1 µM dexamethasone (DEXA, Sigma-Aldrich, St Louis, MO, USA #D1756), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, St Louis, MO, USA, #I7018). After 48 h (day 2), the medium was replaced with complete DMEM containing only 167 nM insulin and the cells were then fed every 2 days with complete DMEM. Cytoplasmic triglyceride droplets were visible already at day 4, and cells were fully differentiated by day 8-10. Following differentiation, 3T3-L1 adipocytes were treated with either BSA or Hp at different concentrations for 24 h and finally collected. 3T3-L1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).
2.5 MEFs isolation, culture,
proliferation rate and differentiation process
Primary mouse embryonic fibroblasts (MEFs) were isolated from embryos of WT and Hp−/− mice at 15 days post coitum (p.c.). Embryos were surgically removed and separated from maternal tissue and yolk sack. The bodies were finely minced, washed in PBS and digested in a solution of 0.1% trypsin-EDTA with shaking at 37°C for 10 min. The solution was allowed to settle for 2 min and the supernatant was centrifuged for 3 min at 1200 rpm at 37°C. Cells were resuspended in DMEM medium (Invitrogen, #41965) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin and plated at 104 cells/cm2 at 37°C in 5% CO2. All differentiation experiments were carried out using cells at passage 3. Cell proliferation
was measured using a commercial colorimetric BrdU incorporation based immunoassay (Roche diagnostics, #11647229001) following manufacturer’s instructions.
For differentiation, cells were allowed to reach confluence, and two days after (day 0) differentiation was induced by addition in the culture medium of an induction cocktail containing
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1.67 µM insulin (Sigma-Aldrich, St Louis, MO, USA #I-5500), 5 µM DEXA (Sigma-Aldrich, St Louis, MO, USA #D1756), 0.3 mM IBMX (Sigma-Aldrich, St Louis, MO, USA #I7018) and 1 µM rosiglitazone (BRL-49653, kindly provided by Glaxo Smith Kline). From day 2 post induction, cells were cultured with DMEM supplemented with only 1.67 µM insulin and 1 µM BRL, that was renewed every 2 days until terminal differentiation (day 10). Pictures of differentiated cells were taken using a Leica microscope (40X magnification).
2.6 Oil Red O staining
Oil Red O staining was performed on frozen liver tissue sections and at the end of MEFs differentiation process (day 10) to visualize the fat droplet accumulation. Cells were washed twice with PBS and fixed with 3.7% formaldehyde (Sigma-Aldrich, St Louis, MO, USA #F8775) in PBS for 1 h at RT. Cells were then washed 3 times with deionized water and stained with filtered Oil Red O working solution [6 parts Oil Red O stock solution (0.5% w/v in isopropyl alcohol, Sigma-Aldrich, St Louis, MO, USA #O-0625): 4 parts water] for 1 h at RT. Excess stain was removed by washing with water.
2.7 Assessment of triglyceride content and liver triglyceride content
To assess triglyceride (TG) content, liver samples were homogenized in 5% Triton-X100 in water. Samples were heated at 92°C for 5 min, cooled to RT and spun at 14000 rpm for 5 min at RT. Supernatants were used to assay triglyceride concentration using the Triglyceride Quantification Kit (BioVision, Inc., Mountain View, CA, USA). Cellular TG content was evaluated after Oil Red O staining by spectrophotometric quantification, dissolving the stained
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lipid droplets in 100% 2-propanol for 10 min. Optical density of the extracts was then measured with a spectrophotometer Bio-Rad (iMark, Microplate Absorbance Reader) at 500 nm.
2.8 Isolation of total RNA and Real-Time PCR
Total RNA from frozen tissues (WAT, liver and BAT) or from cells was extracted with Isol-RNA Lysis Reagent (5 PRIME, #2302700) according to manufacturer’s instructions and its integrity was evaluated on a formaldehyde denaturing agarose gel. RNA was treated with Rnase-free Dnase (Roche Diagnostics Corporation) to remove any contaminating genomic DNA. First-strand cDNA synthesis was performed using oligo hexamers (GE Healthcare, Milan, Italy).
Taq-Man quantitative PCR (50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95°C for 15s, 60°C for 1 min) was performed to amplify samples for adipose fatty acid binding protein-4 (FABP-4/aP2), fatty acid synthase (FAS), lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARɣ), CCAAT/enhancer binding protein alpha (C/EBPα), leptin, mitochondrial glycerol phosphate acyltransferase (mtGPAT), sterol regulatory element binding protein (SREBP), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), acetyl-CoA carboxylase ß (ACAb), cell death-inducing DNA fragmentation effector α (CIDEA), F4/80 and CD68, adiponectin, perilipin, hormone sensitive lipase (HSL), c-Myc, c-Fos, c-Jun and Jun B.
Taq-Man quantitative PCR was performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA), using TATA-binding protein as an endogenous control for equal loading. Predesigned mouse primers and probes were obtained from Applied Biosystems Inc. (Foster City, CA,USA). Data were analyzed using the delta delta-ct method.
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2.9 Isolation of genomic DNA
50 mg of frozen tissues (EPI and SC from CFD and HFD mice) were lysed using 200 µl of lysis buffer (10 mM Tris-HCl pH 8.8, 60 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, Proteinase K 1mg/ml). After incubation at 50°C for 4 hours, samples were spun at 14000 for 10 min, supernatants collected, phenol-chloroform-isoamyl alcohol extracted. Genomic DNA was finally precipitated with 3M sodium acetate and 2.5 volumes of ethanol. Quantification was performed using a BioRad spectrophotometer at 260 nm (Bio-Rad Laboratories, Hercules, CA, USA).
2.10 Glucose tolerance, insulin sensitivity and ELISAs
Glucose tolerance was measured in fasted (16 h) mice after intraperitoneal (i.p.) injection (intraperitoneal glucose tolerance test, ipGTT) of 1 g glucose/kg BW. Insulin sensitivity was measured in fasted (6 h) animals after i.p. injection (intraperitoneal insulin tolerance test, ipITT) of 1.2 units insulin/kg BW. Glucose-stimulated insulin secretion was performed as described by Nguyen et al. (Nguyen KT et al., 2006). Plasma glucose was measured using a glucometer (LifeScan, Inc., Milpitas, CA). ELISA kits were used to assess plasma levels of insulin (Millipore, Billerica, MA), leptin (R&D Systems, Inc., Minneapolis, MN), adiponectin and Hp (R&D Systems, Inc.; Immunology Consultants Laboratory, Inc., Newberg, OR).
2.11 Protein isolation
Proteins were isolated from frozen adipose tissue samples by homogenization in ice-cold lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.25 % sodium deoxycholate, and 0.1% protease inhibitors (Sigma-Aldrich, St Louis, MO,
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USA, 8340), phosphatase 1 and 2 inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA, #P-2850 and #P-5726 respectively). Lysates were sonicated, incubated on ice for 2 h and then centrifuged for 10 min at 10000 × g at 4°C. The protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).
2.12 In vivo insulin signaling
For insuling signaling 6 h fasted HFD WT and Hp−/− mice were injected with insulin (10 units/kg BW) and sacrificed 10 min thereafter. EPI WAT, gastrocnemius muscle and liver were then promptly collected and immediately processed for protein extraction. After homogenization in ice-cold lysis buffer (see Protein isolation), lysates were centrifuged at 14000 × g for 20 min at 4 °C. Supernatants were resolved by SDS-PAGE (Criterion, Bio-Rad Laboratories, Hercules, CA, USA). Phosphorylated and total proteins were identified by immunoblotting using the following primary antibodies: anti- Akt polyclonal and anti-phospho Akt (Ser (P) 473) (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-actin (Sigma-Aldrich, St Louis, MO, USA). Immunoblots were developed using Immun-Star HRP kit (Bio-Rad Laboratories, Hercules, CA, USA), signals were acquired by Molecular Imager Chemidoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis of their intensity was performed by using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
2.13 Microarray analysis
The gene expression profiling was performed on the liver of HFD WT and Hp−/− mice using the One-Color Microarray Agilent Platform (Agilent Technologies, Santa Clara, CA) according to
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the Agilent protocol (Agilent 8 X 60 K whole mouse genome oligonucleotide microarrays, G4852A). Data extraction from the Agilent scanner images was accomplished by Feature Extraction software. Data filtering and analysis were performed using Agilent GeneSpring GX 11.0 and Microsoft Excel (Microsoft Corp., Redmond, WA). Filtered data were quantile-normalized. Differentially expressed genes were selected by Mann-Whitney U nonparametric test. Hierarchic clustering analysis was done using MeV 4.4 (Saeed AI et al., 2006).
2.14 Statistical analysis
All values are expressed as means ± SEM. The number of mice in each experimental group is indicated in the figure legends. Pairwise comparisons of quantitative phenotypes between different groups (e.g., HFD WT vs HFD Hp−/−) were assessed by two-tailed Student’s t test. When specified, one- or two-way ANOVA followed by Bonferroni post hoc tests for selected comparisons was used. The chi-square test was employed to evaluate differences and calculate P values for cell size distributions; standardized residuals for each cell were calculated in order to determine the major contributors of global significance. The area values have been logarithmic transformed so as to correct the Skewness of the distribution. Preliminary tests were also conducted to check for data normality and homogeneity of variances using the Kolmogorov-Smirnov and the Levene’s tests, respectively. A significance limit of P<0.05 was set.
