The overall goal of this procedure is to measure the insulin stimulated glucose uptake in human primary muscle cells. This method can help answer the key questions in the muscle metabolism field, such as muscle cell insulin sensitivity. The main advantage of this technique is that it quantifies the effective muscle cell biological effect in response to hormonal stimulation.
To begin the experiment, prepare proliferation medium, or PM, by supplementing Ham's F-10 medium with 2%fetal calf serum, or FCS, 2%serum substitute, 2 millimoles of glutamine, and 5 micrograms per milliliter of penicillin streptomycin. Prepare a differentiation medium, or DM, by supplementing Dulbecco's Modified Eagle Medium, or DMEM, with 2%FCS, 2 millimoles of glutamine, and 5 micrograms per milliliter of penicillin streptomycin. To prepare X Dulbecco's Phosphate Buffered Saline, or XDPBS, make a solution of DBPS containing 0.2%weight per volume bovine serum albumen.
Filter the solution through a 0.2 micrometer filter. Then, store the XDPBS at 4 degrees Celsius. To prepare cold 2DG solution, weigh 16.4 milligrams of 2DG, insolubalize it in 10 milliliters of distilled water to obtain a 10 millimolar solution.
Store the solution at 4 degrees Celsius. Next, add 600 microliters of cold 2DG and 6 microliters of radiolabeled 2DG to 5, 400 microliters of XDPBS to obtain the radiolabeled 2DG solution. Set aside a 20 microliter aliquot of radiolabeled 2DG solution.
For the cytochalasin B mixture, add 2 microliters of 20 millimolar cytochalasin B to 2 milliliters of radiolabeled 2DG solution. For the DMSO mixture, add 4 microliters of DMSO to the remaining 4 milliliters of radiolabeled 2DG solution. Rapidly thaw frozen vials of human muscle satellite cells in pre-warmed water until only a small ice block remains in the vial.
Next, pour the cells directly into a 50 milliliter plastic tube containing 10 milliliters of pre-warmed PM.Centrifuge the tube for five minutes at 500 times G.Then, discard the supernatant. Add 18 milliliters of pre-warmed PM to the cell pellet and gently re-suspend the cells. Distribute 3 milliliters in each well of a six-well plate.
Incubate the cells at 37 degrees Celsius and 5%CO2 for 48 to 72 hours until cells reach 90%confluence. After 48 to 72 hours, remove the PM and replace it with 3 milliliters of pre-warmed DM per well. Incubate the cells in standard conditions.
Replace the DM medium every two days. It is important that cultured muscle cells reach the fully differentiated state for optimal insulin response and differentiated cells will have a expression inverse weak or no insulin stimulation of the glucose uptake. Wash differentiated muscle cells twice with 2 milliliters of PBS.
Then, remove the PBS carefully and incubate the cells in 3 milliliters of DM without FCS for serum depletion. Next, replace the media in all wells with 3 milliliters of DM without FCS. Add 100 nanomoles of insulin to wells five and six.
Then, incubate the human myotube culture. After one hour of insulin stimulation, wash the wells twice with 1 milliliter of XDPBS. Add 1 milliliter of cytochalasin B mixture to wells one and two and 1 milliliter of DMSO mixture to wells three to six.
Incubate for 15 minutes at 37 degrees Celsius and 5%CO2. At the end of the incubation, immediately place the plate on ice. Wash the cells twice with 1 milliliter of ice cold PBS.
Lyse the cells in each well with 600 microliters of 50 millimolar sodium hydroxide. Incubate the cells on ice for five minutes and mix them gently with slow orbital rotation. Using a pipette, re-suspend and collect the cell lysate.
Add 400 microliters of each cell lysate to a liquid scintillation counting vial. Prepare a negative control vial with 400 microliters of 50 millimolar sodium hydroxide and a positive control vial with 20 microliters of TC20. Next, add 4 milliliters of liquid scintillation solution to each vial.
Close the cap and mix each vial thoroughly for one to two seconds. Insert each vial in a liquid scintillation counter and measure the radioactivity according to the manufacturer's instructions. Record counts per minute for each scintillation vial for 10 minutes.
Optical microscope images taken on day three reveal that myoblasts reach confluence and are typically mononucleated. After the medium is changed and on day eight, differentiation is complete and myotubes are aligned and typically polynucleated. The glucose uptake rate was calculated in the control condition and the treatment condition.
Insulin significantly increases glucose transport in the BSA condition. When the human myotube cell response to insulin is expressed as fold change, it is clear that the response to insulin decreased in myotubes treated with palmitate. After watching this video, you should have a good understanding of how to investigate insulin sensitivity in muscle cell through the determination of glucose uptake rates.
