Fig. 2.

Acute insulin-induced leptin secretion from adipocytes differentiated with Ibmx/Dex/Ins plus PPARγ agonist. (A) Scheme of a designed secretion assay with short-term insulin stimulation (170 nM). 3T3-L1 fibroblasts were differentiated with Ibmx/Dex/Ins plus Tzd cocktail and cultured until fully differentiated (day 14). On the night before the experiment medium was changed to minimal amounts (0.5 ml) in order to allow leptin to accumulate for 15 h overnight. In the morning, an overnight point was taken as a starting concentration for leptin secretion and cells were stimulated with 170 nM insulin for 2 h. Medium was collected from 3 wells of a 6-well plate, concentrated using Millipore filter units and leptin content was assayed using a Quantikine Elisa kit. Accumulated leptin was normalized to total amount of protein from each well. (B) Representative experiment of insulin-induced leptin secretion over 15 min and 2 h from adipocytes. The experiment was repeated over 10 times all with equivalent data.