Insulin receptor activation and down-regulation by cationic lipid transfection reagents.
BACKGROUND
Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000 and FuGENE-6. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types


RESULTS
We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000 on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000 can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells.


CONCLUSIONS
Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.
Pramfalk
Camilla
C
Department of Biosciences, Novum, Karolinska Institute, Huddinge, Stockholm, Sweden. Camilla.Pramfalk@biosci.ki.se
Lanner
Johanna
J
Andersson
Monica
M
Danielsson
Eva
E
Kaiser
Christina
C
Renström
Ing-Marie
IM
Warolén
Malin
M
James
Stephen R
SR
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005947:Glucose / Q000493:pharmacokinetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008055:Lipids / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
C411955:FuGene; D007328:Insulin; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D011972:Receptor, Insulin; D005947:Glucose