(10–20 strokes), followed by a centrifugation at 10 000 g for 25 min at 4uC. The infranatant fraction was used for caspase 3/7 activity assays and western blot analysis (SCD-1). The pellet fraction was solubilized in homogenization buffer containing 5% SDS for 30 min at 50uC, and was used for western blot analysis (PPARc). Preparation of fractions enriched in mitochondria, was performed according to Pecqueur et al [44]. Protein determination was performed using the BCA assay (Pierce). Caspase-3/7 activity assay Caspase-3/7 activity was measured using Caspase-3/7 Assay (Promega Corp) according to the manufacturer’s protocol. Infranatant fractions from periovarial WAT homogenates (50 mg protein/well) were transferred to a black 96-well plate (FLUO- TRAC 600, Nunc) for detection of caspase 3/7-activity. Fluorescence was measured using a fluorescence microtiter plate reader (Victor 2, Wallac, Perkin-Elmer). Western blot analysis Periovarial WAT was homogenized and 25–50 mg of total protein were resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes (HyBond-c extra, Amersham Pharmacia Biotech). Primary antibodies used were peroxisome proliferator activated receptor gamma (PPARc) (1:200), stearoyl-CoA desatur- ase-1 (SCD-1) (1:200), cytochrome-c (Cyt-C) (1:1000) (Santa Cruz Biotechnology), UCP-1 (1:2500) (a kind gift from Prof. Barbara Cannon, Stockholm University, Stockholm, Sweden), b-Actin (1:5000) and a-tubulin (1:2000) (Sigma). Actin, tubulin and cytochrome-C were used as loading controls. Secondary antibod- ies were horseradish peroxidase-conjugated anti-rabbit IgG (Cyt- C, PPARc and UCP-1), anti-mouse IgG (a-tubulin and b-Actin) (Amersham) or anti-goat IgG (SCD-1) (Santa Cruz Biotechnolo- gy). Western blot analysis was performed using a chemilumines- cence system (Luminol) and detection was made using a CCD- camera (LAS 1000, Fuji). Relative protein levels were calculated after normalization to the loading control. Oxygen consumption and uncoupling in WAT pieces Periovarial WAT was collected from female wildtype and HSL mice from both feeding categories. Pieces of tissue (,20– 30 mg each) were incubated in Krebs-Ringer bicarbonate buffer pH 7.4 (KRH, 25 mM hepes, 10 mM glucose, 10 mM fructose, 25 mM NaHCO3 and 4% fatty acid free BSA (Roche)) [45]. Immediately before each assay the tissue was further cut (25 times). O2 consumption was measured polarographically by an electrode (Oxygraph, Hansatech instruments) in an air-sealed chamber containing 400 ml of buffer, under agitation. The calibration of the electrode was performed as described in [46]. After recording of the basal respiration rate, uncoupled respiration was measured by the addition of a chemical uncoupler FCCP (Carbonyl cyanide p- trifluoromethoxyphenylhydrazone, Sigma), to a final concentra- tion of 50 mM. Measurement was stopped after 36 min. As a measure of repeatability, the average value of duplicate measure- ments of the same fat pad, from a total of 10 individuals, gave a value of 160.18 nmol/mg tissue/min. The reproducibility, calculated from the SD values, was 160.21 nmol/mg tissue/min (n = 11) for wildtype samples and 160.46 nmol/mg tissue/min (n = 11) for HSL samples. RNA preparation and Real-time quantitative PCR (rtPCR) Total RNA was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer’s recommendations. Total RNA (1 mg) was treated with DNase I (DNase I amplification HSL Null Mice grade, Invitrogen) and then reversely transcribed using random hexamers (Amersham Biosciences) and SuperScriptTMII RNaseH reverse transcriptase (Invitrogen Life Technologies) according to the manufacturer’s recommendations. rtPCR was performed using an ABI 7900 system (Applied Biosystems), using either Taqman probe and primers (pRb, RIP140, PGC-1a and TBP, assays-on- demand, Applied Biosystems) or SYBR green reagent (PPARc, C/ EBPa, UCP-1 and CPT1). Primer sequences, designed using the software Primer Express 1.5 (Applied Biosystems), are listed below. Relative abundance of mRNA was calculated after normalization to TATA box binding protein (TBP). Primers used in real-time quantitative PCR experiments List of assays using Taqman probes (Applied Biosystems): pRb (Assays-on-demand, Mm00485586_m1) RIP140 (Assays-on-demand, Mm00476537_s1) PGC-1a (Assays-on-demand, Mm00447183_m1) TBP (Assays-on-demand, Mm00446973_m1) List of primers for gene expression analysis using SYBR green reagent: PPARc: (forward primer: 59-CTG TTT TAT GCT GTT ATG GGT GAA A-39 and reverse primer: 59-GCA CCA TGC TCT GGG TCA A-39 C/EBPa: (forward primer: 59-ATA GAC ATC AGC GCC TAC ATC GA-39 and reverse primer: 59-CTG TCG GCT GTG CTG GAA-39) mUCP-1: (forward primer: 59-CCT GCC TCT CTC GGA-39 and reverse primer: 59-TGT AGG CTG CCC AAT-39) CPT1: (forward primer: 59-CCA AAA CAG TAT CCC-39 and reverse primer: 59-AAG AGA CCC CGT AGC-39) Preparation of isolated white adipocytes Adipocytes from periovarial WAT were isolated after incuba- tion in Krebs-Ringers solution (pH 7.4) supplemented with 3.5% BSA, 2 mM glucose, 200 nM adenosine and collagenase (1 mg/ ml; Sigma) in a shaking incubator at 37uC for ,60 min according to a modification of the Rodbell method [47]. The digested tissue was filtered and the isolated cells were washed twice in Krebs- Ringer buffer (pH 7.4) with 1% BSA, 2 mM glucose and 200 nM adenosine by allowing the isolated adipocytes to float to the surface and then aspirate the underlying buffer. Palmitate oxidation in isolated white adipocytes Palmitate oxidation in isolated adipocytes of perigonadal WAT from 6 months old wildtype and HSL mice fed a normal chow diet was determined by production of 14CO2 from [1-14C]- palmitate as described [48]. Briefly, adipocytes were isolated as above, washed thoroughly and transferred into an assay buffer containing 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 25 mM NaHCO3, 2.5 mM CaCl2, 20 mM HEPES pH 7.2 and 3 mM glucose. Cell density was estimated after which cell suspensions (10%) were allowed to recover at 37uC for 30 min prior to the oxidation assay. Hundred ml of cell suspension was transferred to a sample cup together with a solution, containing 5 mM palmitate (unlabeled and 0.5 mCi [1-14C] palmitate) (Du-Pont, NEN, specific activity 55.8 mCi/ mmol) and 16.7 mM glucose. The sample cups were sealed with rubber stoppers into glass vials, and incubated at 37uC for one hour. The reaction was terminated by injection of 100 ml of 7% PLoS ONE | www.plosone.org 10 2008 | Volume 3 | Issue 3 | e1793