I need to establish a diabetic condition In HepG2 cells. Kindly tell me the method.
There are several methods of inducing insulin resistance in cells. I've never worked with Hep G2 cells though, only fat and muscle cells. Different labs have published protocols using various compounds, with differing concentrations and length of incubation. The key things to look for are conditions seen in obesity and type 2 diabetes.
I have previously used insulin (5nM; 24 hours), TNFalpha (40nM; 24 hours), Angiotensin II (10nM; 24 hours), Endothelin-1 (100nM; 24 hours) and hydrogen peroxide (100uM; 3 hours).
If you are looking for a "diabetic condition" I would recommend using high insulin, high glucose and high fatty acids as this is metabolically relevant. However, many of the inflammatory protein I've used are also increased in diabetics.
Insulin resistance in fat and muscle is typically measured in terms of reduced Glut4 translocation to membrane despite presence of insulin, which implies reduced glucose uptake. However, in hepatocytes, insulin resistance is measured in terms of hampered gluconeogenesis (one of the ways) and you have to look for those markers after giving appropriate treatments as mentioned in the above post. That would be your insulin resistance model of hepatocytes. You can also look for other signalling intermediates like IRS1/2 states, MAPK etc. to determine it is showing such symptoms.
Hi,
In my previous work on diabetes, I used to use high glucose and high insulin condition to mimic diabetic conditions: Insulin 100 microU/ml and glucose 25 mM.
https://www.jstage.jst.go.jp/article/jat/19/12/19_13482/_pdf
To evaluate the best conditions for your diabetic status you can see by westernblot the Akt phosphorylation level after 10 min of cell culture.
Good luck.
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