Hello everyone. I am curently working on a protocol to measure glucose uptake in HepG2 cells using 2-NDBG. The work involves the pre-treatment of cells with an active material and observe if it has an effect on increasing glucose uptake. Metformin is used as a positive control. I have come across several threads on ReaserchGate which gave me the impression that this experiment is kind of tricky.
I have read several references. In some references, the cells are pre-treated with the drug, next incubated with glucose-free medium, and lastly the fluorescent 2-NBDG (in glucose-free medium) is added. In others, after incubation with the drug and before the addition of 2-NBDG, there is an extra step in which cells are stimulated with insulin for only 15 min.
I have a couple of questions.
-Is the stimulation with insulin necessary? Or I can skip this step?
- What´s the mechanism that makes metformin or other drug pre-treated with cells and then completely washed away cause an increase in glucose uptake? Why the drug is not added simultanously with 2-NBDG?
-Why after treatment cells are incubated with glucose-free medium? How important is this step? What happens if we drop this step and incubate cells directly with 2-NBDG?
Hi Mazen,
Here are some of my thoughts:
1. It depends on if you were looking at insulin-dependent glucose uptake or insulin-independent glucose uptake. And that depends on what type of cells you were using, say skeletal muscle cells, which are most responsible for insulin-dependent glucose uptake (since they express GLUT4 which are insulin-dependent), you will need to add insulin. I am not familiar with the cells you were using, looks like they are liver cells. Liver cells express GLUT2 which are not insulin dependent. So you don't necessary have to add insulin, however, insulin can still indirectly stimulate glucose uptake in liver cells since it stimulates the liver to store glucose in the form of glycogen. By using insulin acute treatment, you might be able to see max effect on glucose uptake in the liver cells.
2. I don't know the answer and I am following.
3.The step using Glucose-free medium is very important and cannot be skipped.
Because you are using fluorescent 2-NBDG, which means you would determine the glucose uptake by the fluorescent. If you have glucose (non-fluorescent) in the medium then the cells might take up those glucose and affect your result.
Dear Liping. Thank you for your input.
1. I have based my questions on literature. I understand that insulin stimulates the uptake of glucose by HepG2 cells, but what is still not clear from is how previous treatment with metformin (and other drugs) followed by insulin stimulation can make a difference in the uptake. So we are tooking here about insulin-dependant and insulin-independant uptake happening simultanously. But it seems that insulin is also afecting the insulin-independant uptake. What would be the mechnism for this?
3. My qustion was why a pre-incubation with glucose-free medium is done, for 3-4 h, followed by the addition of 2-NBDG (also in glucose-free medium) for 20-30 min. Why not to incubate directly with 2-NBDG in glucose-free medium and skip the pre-2-NBDG step.
Here are some papers for clarification:
Article Calabash (Lagenaria siceraria) potency to ameliorate hypergl...
https://www.mdpi.com/1420-3049/23/12/3382
https://www.mdpi.com/2227-9717/8/1/33
Thank you again
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