Insulin resistance can be evaluated in primary adipocytes, allowing the study of donors under different physiopathological contexts, such as lean versus obese, or cells from different fat depos. Primary adipocytes retain many of their intrinsic properties and can be cultured under defined conditions for a long period of time, with a tight control of cellular environmental factors. To start the differentiation process, cells must be at 80%confluence.
If differentiation starts at a lower or higher confluence, cells will differentiate less or lose their differentiation capacity. After sacrificing the animal, disinfect the mice by rubbing with 70%ethanol. Dissect the inguinal subcutaneous adipose tissue from each mouse and collect it in a 15-milliliter conical tube, containing 15 milliliters of type one collagenase solution on ice.
Cut the adipose tissue into small pieces with sterile surgical scissors and digest the samples by incubation with type one collagenase solution at 37 degrees Celsius in an orbital shaker at 150 RPM for 30 minutes. Check the digestion every 10 minutes to ensure it works, and to prevent over digestion. Filter using a 200-micrometer mesh syringe to eliminate the tissue not digested with collagenase, and pass the filter over the edge of the tube to drain as many cells as possible into the solution.
Add 15 milliliters of cold DMEM 1%BSA to stop digestion, and centrifuge at 400 G for 10 minutes at four degrees Celsius. Aspirate the top layer containing mature adipocytes and most of the liquid layer. Add 20 milliliters of cold PBS 2%FBS to it and resuspend the pellet.
After centrifuging again for five minutes, remove the supernatant by aspirating the upper layer to eliminate the remaining adipocytes and fat. Resuspend the pellet in one milliliter of ACK lysing buffer. Incubate on ice for five minutes.
Add 10 milliliters of PBS 2%FBS and mix it. After centrifuging for five minutes and aspirating the supernatant, resuspend the pellet in 200 microliters of anti-FC solution. Incubate on ice for five minutes and transfer the cell suspension to a five-milliliter tube that fits into the pre-chilled racks of a magnetic cell separator.
After centrifuging for five minutes and eliminating the supernatant, add 200 microliters of the mixture of CD 31 monoclonal antibody biotin and CD 45 monoclonal antibody biotin to it. Mix well and incubate on ice for 15 minutes. Then add 400 microliters of PBS 2%FBS and centrifuge again at 400 G for five minutes at four degrees Celsius.
Aspirate the supernatant and incubate it in 100 microliters of anti-biotin microbeads for 15 minutes on ice. Add 400 microliters of PBS 2%FBS and centrifuge again at 400 G for five minutes at four degrees Celsius, as shown previously. After discarding the supernatant, resuspend the pellet in 350 microliters of PBS 2%FBS.
To remove the cell aggregates, or large particles, from the single cell-suspensions with a 70-micrometer pre-separation filter, activate the filter with 100 microliters of PBS 2%FBS. Pass the cell suspension through the filter, and collect it in a clean tube. Then wash the filter with 100 microliters of PBS 2%FBS.
To perform magnetic separation of cells using the negative separation strategy, place the sample in position A of the chilled rack and two empty tubes in positions B and C to recover the non-labeled and labeled cells. Then load the washing buffer and running buffer into the corresponding bottles. In the separation section, select the number of samples to be separated and select the deplete protocol.
Press run and start the separation. At the end of the program, recover the unlabeled cells. Next, coat a 12-well plate with basement membrane matrix by adding 400 microliters of 2.5%basement membrane matrix to cover the entire surface of each well.
Remove the excess solution and let the plate dry inside the laminar flow hood for at least one hour. After centrifuging for five minutes and discarding the supernatant, resuspend the pellet in 500 microliters of growth medium. Seed the adipocyte precursor cells, or APCs, in one well of the 12-well plate previously coated with 2.5%basement membrane matrix.
Incubate at 37 degrees Celsius in a 5%carbon dioxide atmosphere, and change the medium every 48 hours until the cells reach 80%confluence. After removing the medium completely, wash the cells with 350 microliters of PBS 2%FBS. Harvest the cells with 350 microliters of 0.05%tripsin EDTA for two minutes at 37 degrees Celsius, and add two milliliters of growth medium.
Collect the cells in a new 50-milliliter conical tube and centrifuge at 400 G for five minutes. Passage the APCs to 12-well plates, previously coated with 2.5%basement membrane matrix, and incubate at 37 degrees Celsius with 5%carbon dioxide. Change the medium every 48 hours until cells reach 80%confluence.
At 80%confluence, aspirate off any growth medium and replace it with 500 microliters of differentiation medium, containing 3.3 nanomolar bone morphogenetic protein in each well. After 48 hours, replace the medium with 500 microliters per well differentiation medium, and the differentiation cocktail. Remove the medium 72 hours later and add 100 nanomolar of insulin in 500 microliters of fresh differentiation medium.
48 hours later, induce insulin resistance with four nanograms per milliliter of tumor necrosis factor alpha, or TNF alpha. Aspirate off any differentiation medium and replace it with 500 microliters of simple medium 2%FBS with TNF alpha. After incubating for 24 hours, add four nanograms per milliliter of TNF alpha in a simple medium 0%FBS.
Activate the insulin signaling pathway 24 hours later by adding 100 nanomolar insulin and incubate for 15 minutes at 37 degrees Celsius in a 5%carbon dioxide atmosphere, as shown earlier. After incubation, remove the medium and wash with 500 microliters of PBS, include a control well without the insulin treatment. Extract protein and measure the phosphorylation of insulin-signaling markers by Western blot.
Subcutaneous adipocyte precursor cells at 80%confluence prior to inducing differentiation in the 12-well culture plates, and primary adipocytes after seven days of inducing differentiation are shown in this figure. The insulin resistance induced by TNF alpha in subcutaneous primary adipocytes was demonstrated by decreased insulin-induced phosphorylation of insulin receptor, insulin receptor substrate one, and protein kinase B or AKT. The membrane was probed with anti-phosphorylated insulin receptor, anti-phosphorylated insulin receptor substrate one, anti-phosphorylated AKT, and anti-beta tubulin was used as a loading control.
The signal was visualized with chemiluminescence detection. The representative blots and the quantification from three independent experiments are shown here. When attempting this procedure, one thing that needs to be taken care of is that the induction of insulin resistance with TNF alpha is with 2%FBS for 24 hours and with 0%FBS for the last 24 hours.
