Eight to ten week old males C57BLK6/J, pp59fyn null mice and their controls were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in a facility equipped with a 12 h light/dark cycle. Animals were fed ad libitum a standard chow diet (Research Diets, New Brunswick, NJ) containing 75.9% (Kcal) carbohydrates, 14.7% protein, and 9.4% fat. All studies were approved by and performed in compliance with the guidelines of the Yeshiva University Institutional Animal Care and Use Committee (IACUC).
Whole body indirect calorimetry
Oxygen and carbon dioxide consumption were simultaneously determined by Oxymax open-circuit indirect calorimetry system (eight-cage system) (Columbus Instruments). Animals were allowed to acclimatize for two complete light and dark cycles (48 h) and SU6656 injections were performed at the beginning of the light cycle the following day. Measurements were subsequently taken 12 h following the injection. Data were analyzed as the average of 1 h measurements for each mouse. Instrument settings were as follow: gas flow rate= 0.6 l/min, sample flow rate=0.5 l/min, settle time =120 s, measure time =60 s.
Total Body Mass and Magnetic Resonance Imaging
Total body mass (g) was recorded before (T= 0) and 12 h (T= 12 h) after the SU6656 injection. To determine fat and lean mass, animals were placed in a clear plastic holder without anesthesia or sedation and inserted into the EchoMRI-3-in-1™ System from Echo Medical Systems (Houston, TX, USA). Total body fat and lean mass were measured before (T= 0) and after (T=12 h) after the injection.
Western Blot Analysis
Animals were injected with vehicle or SU6656 (4mg/kg) at the beginning of the light cycle. They were anesthetized and sacrificed by cervical dislocation 3 h after the injection. Tissues were rapidly freeze-clamped in liquid nitrogen and stored at -80°C. Protein preparation and blotting were performed as described below. Tissues were ground in liquid nitrogen and homogenized in a buffer containing 50mM Tris (pH 7.4), 1% glycerol and 1% Triton X-100 supplemented with protease inhibitor (Complete mini, Roche Pharmaceuticals, Nutley, NJ). Homogenates were centrifuged for 30 min at 14,000 rpm at 4°C, and supernatants were collected. Protein concentration was determined with the BCA™ Protein Assay (Thermo Scientific, Rockford, IL).
Protein samples (40 μg) were separated on 8 or 10% reducing polyacrylamide gels and trasnferred onto Immobilon-P polyvinylidene difluoride membranes. Immunoblots were blocked with 2% milk and 3% BSA in Tris-buffered saline for 60 min at room temperature and incubated overnight at 4°C with the indicated antibodies (Cell Signalling, Upstate, and Alpha Diagnostic international) in Tris-buffered saline and 0.05% Tween 20 (TBST) containing 1% BSA. Blots were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:30,000) for 30 min at room temperature. Membranes were washed in TBST, and antigen-antibody complexes were visualized by chemiluminescence using an ECL kit (Pierce). Alternatively, immunoblots were incubated with IRDye800CW Goat Anti Mouse (H+L) or IRDye680 Goat Anti Rabbit (H+L) secondary antibodies and signal was detected with the Odyssey® Infrared Imaging System (Li-COR Biotechnology, Lincoln, NE).
Fatty acid oxidation in isolated muscles
Animals received a single intra-peritoneal injection of SU6656 or vehicle in the morning. Mice were sacrificed by cervical dislocation 3 h after the injection. Skeletal muscles (red soleus and white gastrocnemius) were rapidly removed and pre-incubated for 10 min in oxygenated (95% O2, 5% CO2) Earle's Solution (Invitrogen, Carlsbad, CA) supplemented with 5mM D-Glucose, 250μM palmitate and 0.5% BSA plus either 10μM SU6656 or an equal volume of DMSO. Tissues were incubated for 45 min in the same buffer containing 250μM palmitate containing 1μCi/ml [1-14C] palmitate tracer (Amersham, Piscataway, NJ) bound to 0.5% BSA. Incubations were carried out under an atmosphere of 95% O2/5% CO2 at 30°C in glass vials (Kontes, Vineland, NJ) equipped with a center well filled with 200μl of 2N NaOH (trapping agent). At the end of the incubation, perchloric acid was added through the cap to a concentration of 0.6 mM and vials were incubated for 3 h at 30°C with moderate shaking. The 14CO2 produced was determined by scintillation counting of NaOH using the UniScintBD scintillation liquid (National Diagnostics, Atlanta, GO).
C2C12 myoblasts were grown in Dulbecco's Modified Essential Medium (DMEM, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum. Differentiation into myotubes was initiated by switching the myoblasts to DMEM complemented with 2% horse serum for 4-6 days as described previously (Yaffe and Saxel, 1977a
). 3T3L1 preadipocytes were cultured in DMEM supplemented with 10% calf serum at 37°C. Confluent cultures were induced to differentiate into adipocytes as described previously (Min et al., 1999
pcDNA3.1-Fyn-V5 was generated by RT-PCR performed on spleen total RNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) with a pair of oligonucleotides: 5′-CACCATGGGCTGTGTGCAATGTAAGG-3′ and 5′-CAGGTTTTCACCGGGCTGAT-3′. The PCR product was separated on 2% agarose gel, and the specific single band was extracted using the QIAquick PCR purification kit (Qiagen). The purified PCR product was cloned into the pcDNA3.1D/V5-His-TOPO using the pcDNA3.1 Directional TOPO Expression Kit (Invitrogen, Carlsbad, CA). pcDNA3.1-Fyn-CA(Y527F)-V5 was obtained using the oligonucleotides: 5′-CACCATGGGCTGTGTGCAATGTAAGG-3′ and 5′-CAGGTTTTCACCGGGCTGAAACTGGGGCTCT-3′ and following by the same protocol. pcDNA3.1-Fyn-KD(K299M)-V5 was constructed by overlapping extension PCR. The gene encoding Fyn was amplified with the pair of oligonucleotides: 5′-CACCATGGGCTGTGTGCAATGTAAGG-3′ and 5′-CTGGCTTAAGGGTCATTATGGCTACTTTT-3′ and the pair of oligonucleotides: 5′-AAAAGTAGCCATAATGACCCTTAAGCCAG-3′ and 5′-CAGGTTTTCACCGGGCTGAT-3′. PCR products were extracted and purified. Each product was mixed and a second PCR was performed using the oligonucleotides: 5′-CACCATGGGCTGTGTGCAATGTAAGG-3′ and 5′-CAGGTTTTCACCGGGCTGAT-3′. Products were cloned into the pcDNA3.1D/V5-His-TOPO. The pYX-LKB1 construct was obtained from Open biosystems (Rockford, IL) and used to generate the pcDNA3.1-Flag-LKB1 construct. The gene encoding LKB1 was amplified with the oligonucleotides: 5′-ATG GAC TAC AAG GAC GAT GAC GAC AAG ATG GAC GTG GCG GAC CCC-3′ and 5′-TCACTGCTGCTTGCAGGC-3′ and cloned to pcDNA3.1D/V5-His-TOPO. pEGFPC2 and pcDNA3.1-LKB1 were digested by Hind3 and Sac2. Products were purified and ligation was performed using the DNA Ligation Kit (Takara, Shiga, Japan) to obtain the pEGFPC2-LKB1 construct. LKB1 mutants were obtained using an overlapping extension PCR with the following primers: LKB1-Y60F: 5′- AGG GCT CGT TCG GCA AGG TGA -3′, 5′- TCA CCT TGC CGA ACG AGC CCT -3′, LKB1-Y156F: 5′- AGC TCA TGG GTT CTT CCG CCA G -3′, 5′- CTG GCG GAA GAA CCC ATG AGC T -3′, LKB1-Y166F: 5′- GGC CTG GAA TTC CTA CAC AGC -3′, 5′- GCT GTG TAG GAA TTC CAG GCC -3′, LKB1-Y261F: 5′- GGG GAC AAT ATC TTC AAG CTC TTT GAG AAC -3′, 5′- GTT CTC AAA GAG CTT GAA GAT ATT GTC CCC -3′, LKB1-Y365F: 5′- GAC GGC ATT ATC TTC ACC CAG GAC TT -3′, 5′- AAG TCC TGG GTG AAG ATA ATG CCG TC -3′. LKB1-Y261/365F was obtained using primers for LKB1-Y261F and for LKB1-Y261F. The GST-AMPK α subunit and Omni-STRADα cDNAs were kind gifts from Dr. Bin Zheng, Harvard Medical School.
In vitro LKB1 phosphorylation assay
His-LKB1 fusion protein was purified using HisPur Purification kit and Slide-A-Lyzer Dialysis Cassette (Pierce, Rockford, IL). His-LKB1 protein (1μg) was incubated with the recombinant His-FynT kinase (1.8U) (Calbiochem, Gibbstown, NJ) in presence of Src Mg/ATP cocktail (Millipore, Billerica, MA) and kinase reaction was performed for 1 hour at 35°C. Samples were separated on 10% SDS-polyacrylamide gels and immunoblotting was performed with PY100 monoclonal antibody and LKB1 polyclonal antibody. Signals were detected with the Odyssey® Infrared Imaging System (Li-COR Biotechnology, Lincoln, NE).
Transfection of C2C12 myotubes and 3T3L1 adipocytes
C2C12 myotubes and 3T3L1 adipocytes were electroporated as previously described (Waters et al., 1995
). A suspension of 3T3L1 adipocytes was electroporated with 500μg of plasmid under low-voltage condition (0.16kV, 950μF). C2C12 myotubes were electroporated with a total of 250μg of plasmid under 0.22kV, 950μF. Adipocytes and myotubes were allowed to adhere onto collagen-coated tissue culture dishes for 30–48 h.
Transfection of skeletal muscle in vivo
Three month old wild type mice were anesthetized with isoflurane and the right tibialis anterior was injected with 125 μg of pcDNA3-Flag-LKB1-WT or pcDNA3-FlagLKB1-(Y261/365F) cDNAs and the left tibialis anterior with the pcDNA empty vector as control. Electroporation (8 shocks) was performed using the S48 SQUARE PULSE STIMULATOR (Grass Technologies, West Warwick, RI) with the following settings: train rate = 1TPS; train duration =500ms; pulse rate =1PPS; duration = 20ms, voltage = 80V. Electroporation was repeated 5 days later. Animals were sacrificed 5 days after the second set of electroporation. The tibialis anterior muscles were rapidly removed and immediately embedded into Optimal Cutting Temperature (O.C.T) compound (Sakura Finetek USA, inc., Torrance, CA). Tissues were frozen in liquid nitrogen. 10 μm frozen sections were prepared and subjected to immunofluorescence labeling as previously described, using anti-Flag Mouse mAb antibody and Alexa Fluor 488 anti-Mouse secondary antibody. Sections were washed 3 times with PBS and mounted with Prolong Gold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA) and were imaged as described above. Settings (Iris (pinhole), laser intensity, gain and offset) were fixed and identical for all samples. Muscle extracts were also used for immunoprecipitation and immunoblotting as described below.
The ratio of cytosolic and nuclear LKB1 was quantified using the Image J software (National Institutes of Health). Images of 15 representative cells were processed and results represent mean ± s.e from 3 independent experiments
Cells were homogenized in a NP-40 lysis buffer containing 25mM Hepes, pH 7.4, 10% glycerol, 50mM sodium fluoride, 10mM sodium phosphate, 137mM sodium chloride, 1mM sodium orthovanadate, 1mM PMSF, 10μg/ml aprotinin, 1μg/ml pepstatin and 5μg/ml leupeptin and rocked for 10 min at 4°C. Muscle extracts (100mg) were homogenized in the Bullet Blender (Next Advance, Inc., Averill Park, NY) using zirconium silicate beads (speed 8 for 3 min) in the buffer described above. Homogenates were centrifuged for 10-30 min at 13,000g at 4°C, and supernatants were collected. Protein concentration was determined using the BCA™ Protein assay. Cell lysates (3-4 mg) were incubated with 10μg of antibody for 2 h at 4°C. 50 μl of TrueBlot™ anti-Rabbit Ig IP Beads (eBioscience, Inc., San Diego, CA) was added and samples were rocked for 60 min at 4°C. Samples were washed three times with NP-40 lysis buffer and were resuspended in 100μl of Laemmli Buffer containing 50mM DTT. Samples were heated at 90-100°C for 10 min and centrifuged at 10,000×g for 3 min. Supernatants were collected and loaded on 10% SDS-polyacrylamide gels.
C2C12 myotubes were co-transfected with 50μg pEGFP-C2-LKB1 or pcDNA3-Flag-LKB1 or pcDNA3-Flag-LKB1 mutants and 200μg of the indicated pcDNA3-Fyn constructs. Transfected cells were washed with PBS and fixed for 10 min in PBS containing 4% PFA and 0.2% Triton X-100. Immunofluorescence was performed using a rabbit LKB1 polyclonal antibody, a rabbit Flag specific polyclonal antibody and a mouse Fyn monoclonal antibodies followed by Alexa Fluor 488 anti-rabbit IgG and Alexa Fluor 594 anti-mouse IgG. Samples were mounted on glass slides with Prolong Gold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA). Cells were imaged using a confocal fluorescence microscope (TCS SP5 confocal; Leica microsystems).
Results are expressed as mean ± standard error of the mean (SEM). Differences between animals and/or treatments were tested for statistical significance (p<0.05) using Student's unpaired t test