Insulin stimulation assays

Insulin sensitization and stimulation assays of hPSC-adipocytes 

Materials and reagents

1. DMEM, low glucose, pyruvate (Thermo Fisher 11885)
2. DMEM, no glucose, no glutamine, no phenol red (Thermo Fisher A1443001)
3. Insulin solution human (Sigma-Aldrich I9278)
4. Cellastim - Recombinant human albumin (InVitria 777HSA017S)
5. Penicillin-Streptomycin (Thermo Fisher 15140122)

Sensitization medium:

1. DMEM, low glucose, pyruvate (Thermo Fisher 11885)
2. 100pM Insulin solution human (Sigma-Aldrich I9278)
3. 200ug/mL Cellastim - Recombinant human albumin (InVitria 777HSA017S)
4. 1% Penicillin-Streptomycin (Thermo Fisher 15140122)

Assay medium:

1. DMEM, no glucose, no glutamine, no phenol red (Thermo Fisher A1443001)

Procedure

Insulin sensitization 

1. Differentiate hPSCs into adipocytes as previously described.
2. Completely remove all medium.
3. Add 0.25mL/cm² sensitization medium. 
4. After 48 hours and 96 hours repeat step 2-3. 
5. 120 hours after the start of sensitization, 24 hours after the last medium change, cells are sensitized and ready for functional assays. 

Note: For hyperinsulinemia-induced insulin resistance sensitization medium can be modified to include 3nM insulin. 

Insulin stimulation assays

1. After insulin sensitization, hPSC-adipocytes can be prepared for functional assays. 
2. Fully remove medium and rinse cells with assay medium. 
3. Fully remove medium and add 0.25mL/cm² assay medium to the cells. 
4. Place cells in an incubator at 37°C for 30 minutes. 
5. Fully remove medium. 
6. Add assay medium ±insulin as appropriate for the assay. 
7. Place cells in an incubator at 37°C for the appropriate time for the assay. 
8. Harvest the cells. 

Note: For the original publication step 7 was 10 minutes for AKT2 phosphorylation and 40 minutes for glucose uptake assays. 

Insulin concentrations can be varied as desired.