All cell culture reagents were from Gibco. LipofectAMINE 2000™ and SDS-PAGE reagents were from Invitrogen and FuGENE-6™ was from Roche. Hybond™-C nitrocellulose membrane and enhanced chemiluminescence (ECL) was from Amersham Life Science. Anti-phosphotyrosine antibodies were from Santa Cruz. All other reagents were from Sigma and were analytical grade.
Cell Culture and Treatment with Transfection Reagents
Chinese hamster ovary cells over-expressing the human insulin receptor (CHO-hIR cells) were grown in F-12K Nutrient Mixture (Kaighn' Modification) supplemented with 10% heat inactivated fetal bovine serum (FBS) in 75 cm2 cell culture flasks. CHO-hIR cells were cultured at 37°C in a humidified 5% CO2 atmosphere. Two days before treatment with LipofectAMINE™ 2000 (LF2000), the cells were plated out in two 6-well plates. After 24 hours, cells were starved by culturing overnight in serum-free medium. Cells were then treated in duplicate with increasing dilutions of LF2000, which were prepared in advance and allowed to sit at room temperature for 30 minutes. (0.5, 1, 2, 5, 10 μl diluted in 1 ml F-12K Nutrient Mixture (Kaighn' Modification) lacking FBS). Cells were incubated for 4 hours in 37°C in a humidified 5% CO2 atmosphere. Alternatively, cells were incubated with single concentrations of LF2000 or FuGENE as indicated in the figure legends. After washing, cells were lysed in 1ml buffer comprising 20mM Hepes pH 7.6, 20% (v/v) Glycerol, 10mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.1% NP40 (v/v), 25mM NaF, 25mM β-glycerolphosphate, 1mM DTT, 1mM Na vanadate and protease inhibitors (Boehringer). The cells were flash frozen in liquid nitrogen and then stored at -70°C until protein concentration was determined (Bradford assay) and analysis by Western blot. Where indicated, cells were stimulated with 100nM insulin for the indicated periods as controls.
L6 myocytes were maintained in minimum essential medium-alpha (α-MEM) supplemented with 10% foetal bovine serum (FBS) and 100 IU/ml penicillin-streptomycin at 37°C in 5% CO2. Cells were seeded into 96-well plates and the medium was replaced with α MEM containing 2% FCS to induce differentiation. The medium was changed every other day and cytidine (0.24 mg/ml medium) was added to the cultures after a week to suspend cycling cells. The cells were used in experiments after over night serum starvation after day 10.
3T3L1 preadipocytes were cultured in DMEM/F12 containing 10% serum until they became confluent. Differentiation to adipocytes was achieved by sequential culture in DMEM/F12/10% serum containing IBMX 0,5mM, IGF-I 20ng/ml, Dexamethasone 1 μM and with or without 1 μM Rosiglitazone for 4 days followed by DMEM/F12/10% serum and IGF-1 alone for four days. Medium was then reverted to culture medium and cells were used after 14 days once full differentiation was achieved.
3T3L1 cells, L6 cells and SHSY5Y cells were incubated 18 hours in serum free α-MEM supplemented with 0.25% (v/w) fat free albumin (50 ml DMEM + 125 mg albumin). The medium was aspirated, cells were washed with glucose-free α-MEM, and the cells were incubated for 30 minutes in glucose-free α-MEM supplemented with 100 nM insulin (when indicated) for 30 min. The medium was aspirated and glucose-free α-MEM containing the tracer was added. Glucose uptake rates were carried out for 8 minutes. The medium was aspirated and the cells were washed twice with cold PBS. Subsequently the cells were solubilized in 50 μl 0.5M NaOH over night at RT, scintillation liquid was added (100 μl/well), plates were shaken on the vortex at RT for 30 minutes and radioactivity was counted in μ-Beta counter.
Samples were matched for protein concentration and heated at 70°C for 10 minutes with 20 μl 4× concentrated sample buffer. Samples were resolved on 4–12% gradient gels and blotted onto nitrocellulose membranes. Membranes were probed for the presence of phosphotyrosine using a mouse monoclonal anti-phosphotyrosine antibody and developed using enhanced chemoluminescence. Phosphotyrosine content of the insulin receptor was then quantified by densitometric analysis.
Flow cytometric analysis of green fluorescent protein expression
Cells were transfected with pGREEN LANTERN as above and analysed using an EPICS® XL-MCL flow cytometer (Beckman Coulter). For each sample, 30 000 events were collected by list-mode data, which consisted of side scatter, forward scatter, and fluorescence emissions centered at 530 nm (FL1), 580 nm (FL2), and 610 nm (FL3). pGREEN LANTERN-1 fluorescence occurs primarily in FL1 but is also detectable in FL2 and FL3. However, we chose only to present FL1 in this work. The gates were drawn along a line of maximum detected pGREEN LANTERN-1 intensity for control samples. The gates were held unchanged through the analysis of all measurements in all experiments.