Chemicals were purchased from Sigma (Deisenhofen, Germany) and cell culture reagents from Invitrogen (Karlsruhe, Germany). Rat anti-haemagglutin (HA) 3F10 and anti-MYC 9E10 antibodies were purchased from Roche (Mannheim, Germany), rabbit anti-(phospho-)AKT and anti-IRS1 antibodies from Biosource (Solingen, Germany) or Upstate/Biomol (Hamburg, Germany), anti-phosphotyrosine antibody was from Santa Cruz (Heidelberg, Germany), anti-protein–tyrosine phosphatase b (PTPN1) antibody from Merck Bioscience (Schwalbach, Germany) and anti-beta-actin antibody from Sigma. Horseradish peroxidase-coupled secondary antibodies were from Amersham (Freiburg, Germany), Alexa Fluor 488-labelled anti-rat secondary antibody was from Molecular Probes (Karlsruhe, Germany).
Animals and sample preparation
Age-matched groups of obese male ZDF rats (Genetic Models Incorporated [GMI] fa/fa) and their lean littermates (GMI+/?) were purchased from Charles River (Sulzfeld, Germany). The rats were housed in pairs at 20°C on a 12-h light/12-h dark cycle with free access to water and standard rat diet (ssniff R/M-H; ssniff Spezialdiaeten, Soest, Germany) for 1 week upon arrival to allow recovery from transport. All experimental procedures were conducted according to the German Animal Protection Law. After 2 h of starvation (24 h for the group studied at 8 weeks of age), blood samples were drawn under terminal isoflurane. Tissue probes were excised rapidly and snap-frozen in liquid nitrogen. Determination of blood metabolic parameters was performed as described previously .
Affymetrix microarray analysis
Skeletal muscle probes (gluteus maximus, 150 mg) were lysed in RLT buffer (Qiagen, Hilden, Germany) with a homogeniser (UtraTurrax; Janke and Kunkel IKA Labortechnik, Staufen, Germany). Total RNA from the tissue lysates was isolated with the Qiagen RNeasy kit, including proteinase K digestion, DNase digestion and an additional RNeasy cleanup step, as recommended by the manufacturer. First- and second-strand cDNA syntheses were performed with 10 μg of each total RNA using SuperScript SSII RT polymerase system (Invitrogen) and a T7(dT)24 primer. Double strand cDNA was extracted with phenol–chloroform then precipitated with ethanol. Biotin-UPT- and -CTP-labelled cRNA was transcribed in vitro using the Enzo BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY, USA) and purified by RNeasy cleanup and ethanol precipitation. Aliquots of each total RNA and cRNA were monitored before and after the purification steps by UV spectrophotometry, agarose gel electrophoresis and BioAnalyzer RNA chips (Agilent, Boeblingen, Germany). cRNA samples (15 μg) were fragmented at 94°C for 35 min, added to hybridisation buffer and hybridised to RG-U34 A, B and C microarrays (Affymetrix, High Wycombe, UK) for 16 h at 45°C and 60 rev/min. Microarrays were washed and double-stained with streptavidin–phycoerythrin conjugate (Molecular Probes) and anti-streptavidin antibody, using methods described by Affymetrix. After washing, the microarrays were analysed in a confocal GeneArray Scanner (HP, Boeblingen, Germany) with Microarray Suite Version 4.0 software (Affymetrix). Quality control of each chip was performed according to the Affymetrix quality criteria, including mean average difference, raw intensity and 3′/5′ ratio of the housekeeping genes beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Expression profiling data were analysed using the in-house software GECKO 2 . Briefly, this software performed a global normalisation on all microarrays using a reference chip for each group of biological replicates and the 75th percentile of the median of all transcribed genes. Biological replicates were subsequently agglomerated and a new matrix containing ratios and p-values was generated by crosswise element-by-element concatenation of all chips in one age-group, and significance was assessed with Student’s t-test. Merging of the respective biological replicates of each age (6, 7 and 12 weeks) and phenotypic group (lean and obese) and subsequent filtering for p-values lower than 0.05 and changes in expression levels between the lean and obese groups higher than twofold resulted in the sets of differentially expressed genes reported here. The procedure we describe and the choice of a ratio of changes of at least twofold have been shown to ensure limitation of false positives and good reproducibility by alternative methods, e.g. quantitative RT-PCR (qRT-PCR) and Northern blotting [19, 20].
Quantitative real-time PCR
Total RNA (1 μg) was reverse-transcribed with the AMV-RT first-strand cDNA synthesis kit (Roche) in a 20 μl reaction volume. Reverse-transcribed single strand cDNA (2 μl) was used as a template for amplification in a Lightcycler using FastStart DNA Master Sybr Green according to the instructions of the manufacturer (Roche). Primers used were 5′-TGCTATGTTATCCTTTCTCTTG-3′ and 5′-GGACACTTAATACACGATGTT G-3′ (rat SCD1), 5′-CTTCTTCATCTTCACCTTCTTA-3′, 5′-GCCCTAAGTATTCAAGTTCTGT-3′ (rat solute carrier family 2, member 4 [SLC2A4, also known as glucose transporter 4, GLUT4]), 5′-AAGTCCCTCACCCTCCCAAAAG-3′ and 5′-CCTCAACACCTCAAACCACTCC-3′ (rat beta-actin). The correct sizes of the resulting fragments (121, 136 and 268 base pairs, respectively) were monitored by agarose gel electrophoresis. Total RNA contents were calculated using a concentration standard curve of the respective amplified fragments and normalised to expression levels of the housekeeping gene beta-actin. Confidence of the microarray data was assessed by TaqMan qRT-PCR of selected genes (covering a range of fold changes between +10.63 and −2.19), using the Applera TaqMan Universal PCR Master Mix and Pre-Developed TaqMan Assay Reagents on an ABI Prism 7900 device according to the instructions of the manufacturer (Applera, Norwalk, CT, USA). Endogenous RNA control 18S rRNA (Applera; 4310893E) was used for normalisation as an internal control. Table S1 of the Electronic Supplementary Material (ESM) summarises the design of primer and probe sequences and sequence identifiers of the commercially available primer and probe sets from Applera.
HPLC analysis of long-chain acyl-CoA species
HPLC analysis from freshly prepared KH2PO4 cell lysates was performed using a Waters Alliance 2690 system (2487 detector; Millennium 2010 chromatographic manager; Waters, Eschborn, Germany) as described elsewhere .
Cloning and plasmids
The complete open reading frame of human SCD1 was amplified from human skeletal muscle cDNA (Invitrogen) by PCR using primers 5′-CGGGATCCCGCCACCATGCCGGCCCACTTGCTGCAG-3′ and 5′-CCGCTCGAGCGGTCAAGCGTAGTCTGGGACGTCGTATGGGTACATGCCACTCTTGTAGTTTCC-3′. The 3′ primer introduced a sequence encoding a carboxy-terminal in-frame HA epitope tag. The resulting fragment and pcDNA3.1(+) hygro vector (Invitrogen) were digested with BamHI/XhoI and ligated to obtain pcDNA3.1(+) hygro-SCD1-HA. The identity of the construct was confirmed by sequencing.
Cell culture, transfections and immunofluorescence
Rat L6GLUT4myc myoblasts expressing MYC epitope-tagged SLC2A4 (GLUT4)  were kindly provided by A. Klip (Hospital for Sick Children, Toronto, ON, Canada). Cells were maintained in alpha-MEM supplemented with 10% FCS, antibiotics (penicillin–streptomycin) and 2 μg/ml blasticidin at 37°C in 5% CO2 and 95% humidity and were subcultured twice weekly. Transfections were carried out with L6GLUT4myc cells grown in six-well plates to 60% confluence, 2.5 μg plasmid DNA and Fugene 6 reagent (Roche), as recommended by the manufacturer. For all transient transfections and for stably selected cell clones, empty pcDNA3.1(+) hygro vector served as negative control (subsequently referred to as ‘wild-type’ [WT]). Stable single-cell clones were selected with 500 μg/ml hygromycin B, picked manually and expanded. SCD1-HA expression levels were determined by SDS-PAGE and Western blotting (NuPAGE; Invitrogen) using anti-HA 3F10 monoclonal (Roche), anti-rat horseradish peroxidase secondary antibodies and chemiluminescence detection (Amersham). For fatty acid treatment, palmitate was dissolved in ethanol at 70°C to obtain a 20 mmol/l stock solution and coupled at a concentration of 200 μmol/l to 0.75% (w/v) fatty-acid free BSA in cell culture medium for 1 h at 37°C to obtain a molar ratio of 2:1. Immunofluorescence assays were performed as described , using stable L6GLUT4myc SCD1-HA cells or L6GLUT4myc cells transiently transfected with pcDNA3.1(+) hygro SCD1-HA grown on coverslips to 70% confluence. SCD1-HA expression was detected using anti-HA 3F10 antibody (Roche) and anti-rat Alexa Fluor 488 antibody (Molecular Probes). Neutral lipid staining was performed with 1 μg/ml Nile Red (Sigma) in PBS for 10 min after fixation and permeabilisation of the cells. Nuclei were visualised using 1 μmol/l ToPro iodide in PBS (Molecular Probes). Confocal images were taken with a Leica TCS SP2 confocal laser scanning microscope.
2-Deoxyglucose uptake assay and analysis of insulin signalling
Stably transfected L6GLUT4myc cells were plated in 96-well Cytostar-T scintillating microplates (Amersham) at 3.0×104 viable cells per well. After 32 h the cells were serum-starved with alpha-MEM supplemented with 2% newborn bovine serum and antibiotics (penicillin–streptomycin) for 16 h. To analyse glucose uptake, the cells were washed twice in KRB, pH 7.3 (KRB), and incubated for 25 min with the given insulin concentrations in KRB. [14C] 2-deoxyglucose (0.01 MBq per well; Amersham) was added and the cells were incubated for another 25 min in a total volume of 150 μl per well. Reactions were stopped by adding 10 μmol/l cytochalasin B; plates were sealed and scintillation was measured in a Wallac Microbeta counter (Perkin Elmer, Rodgau-Juegesheim, Germany). Unspecific uptake was determined by incubating control wells with 20 μmol/l cytochalasin B and subtracted from each value. Insulin signalling was monitored in stably transfected L6GLUT4myc cells plated in 12-well plates upon direct stimulation with 10 nmol/l insulin for 15 min and subsequent lysis in 1-TBS, 1% NP-40, 2 mmol/l sodium vanadate, 2 mmol/l Pefabloc and 1 μg/ml leupeptin. Levels of IRS1 tyrosine phosphorylation were quantified after immunoprecipitation of the proteins with anti-IRS1 antibody (Cell Signaling, Frankfurt, Germany) and immunoblotting using a phosphotyrosine antibody (Santa Cruz) and anti-IRS1 antibody for normalisation. AKT1 phosphorylation levels were determined with anti-phospho-S473 AKT1 (Biosource) and anti-AKT1 (Upstate) antibodies and direct immunoblotting of the lysates. Protein concentrations were determined with BCA protein assay (Pierce, Bonn, Germany).
Data are means±SEM of at least three independent experiments or show a representative graph of several independently performed experiments with similar results, as appropriate. Statistical significance was determined with Student’s t-test (p-values below 0.05 were considered significant) using the SigmaStat 2.03 program (SPSS, Chicago, IL, USA). Quantitative RT-PCR gene expression and phosphorylation data were calculated as fold change compared with controls and are given in arbitrary units (mean set to 1.0).