A colorimetric bioassay for quantitation of both basal and insulin-induced glucose consumption in 3T3-L1 adipose cells.

Introduction : The quantitation of glucose consumption in animal cell cultures is mainly based on the use of radiolabelled or fluorescent analogues, resulting in expensive and tedious procedures, requiring special equipment and, sometimes, with potential healthand environmental risks.
Objectives : The objective of this work was to adapt and evaluate the application of a blood plasma colorimetric assay to quantify glucose consumption in in vitro cultures of adipose cells.
Methods : We worked with 3T3-L1 adipose cells differentiated by 7-8 days, which were exposed to different glucose concentrations (5.5, 2.8 and 1.4 mM) for variable times, either in the absence or the presence of 100 nM insulin. Using a commercial colorimetric glucose assay, extracellular glucose was determined, and glucose uptake was calculated as the difference between the initial and final glucose concentration.
Results : The applied bioassay allowed us to quantify glucose uptake in our cell model and showed a linear response over time (r 2 =0.9303) to the different glucose concentrations, either in the absence (basal) or presence of insulin (stimulated). The kinetic parameters calculated for uptake were V ma x 4.1 and 5.9 nmol/ml/min (for basal and insulin-stimulated, respectively) and a K app of 1.1 mM under both conditions. In addition, insulin-stimulated uptake was significantly higher than basal uptake at ≥120 min in glucose 5.5 mM (p≤0.01, n=5), and 240 min in glucose 1.4 mM (p≤0.01, n=5). Finally, at 1.4 mM of initial glucose, the insulin response showed a concentration-dependent effect, with an EC50 of 18.4 ± 1.1 nM.
Conclusions : A simple and low-cost bioassay is proposed to quantify glucose
consumption in 3T3-L1 adipose cells.