I am having problems obtaining insulin stimulated glucose uptake in differentiated 3T3-L1 adipocytes. I have tried several differentiation protocols but I only get a 30% increase in glucose uptake after insulin stimulation. The protocol for [14C]-2-deoxy-D-glucose uptake I follow are from J.M Olefsky's group, in particular this paper: https://www.sciencedirect.com/science/article/pii/S0021925819482094
I do get differentiated 3T3-L1 adipocytes, using several different differentiation protocols. But for some reason the cells barely respond to insulin. I wonder about a few things that might cause my problem.
1. At what time point the cells are best suitable for glucose uptake experiments? Different papers write about 10-12 days post-differentiation, others talk about 10-12 days post differentiation initiation. since it takes a while before the cells are fully differentiated there is a difference between post differentiation and post differentiation initiation.
2. How long should the cells be serum starved and glucose starved?
3. Should DMEM with high or low glucose be used and with or without pyruvate?
4. I'm working with 12-well falcon plates. Does the type of plate/flask or perhaps with coating make a difference?
I hope someone can help, because we have no idea what causes the lack of insulin stimulated glucose uptake in the cells.
Hi,
We have usually conducted 2DG uptake assay. I will talk about my assay protocol.
1. 10-12days post differentiation initiation is suitable. I usually examined 2DG uptake assay at this timing. Perhaps 10-12 days post-differentiation were used interchangeably.
2and 3. Serum starvation is overnight (16h) in high glucose DMEM.
Glucose starvation is 2 hours in 1%BSA(FFA free) KRP-HEPES, PH7.4 (no pyruvate supplementation).
4. We usually used 6 well dish, but 12 well dish is also suitable for assay. Coating dish is not necessary.
I thing preparing good adipocytes is most important factor, since preadipocyte does not express sufficient insulin receptor, Akt2, and GLUT 4. Therefore, mixing well-differentiated adipocytes and poorly-differentiated adipocytes in the same well did not sufficiently increase glucose uptake by insulin stimulation.
Some of my publications including 2DG uptake in 3T3L1 adipocytes are listed below.
DOI: 10.1128/MCB.21.5.1633–1646.2001
doi:10.1152/ajpendo.90581.2008
doi: 10.1210/en.2008-1018
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