Insulin receptor activation and down-regulation by cationic lipid transfection reagents

<p>Abstract</p> <p>Background</p> <p>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 stimula...

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Main Authors: Renström Ing-Marie, Kaiser Christina, Danielsson Eva, Andersson Monica, Lanner Johanna, Pramfalk Camilla, Warolén Malin, James Stephen R
Format: Article
Language:English
Published: BMC 2004-01-01
Series:BMC Cell Biology
Online Access:http://www.biomedcentral.com/1471-2121/5/7
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spelling doaj-c7f4fef8960a4894a9bb48c692c5f1022020-11-25T01:31:31ZengBMCBMC Cell Biology1471-21212004-01-0151710.1186/1471-2121-5-7Insulin receptor activation and down-regulation by cationic lipid transfection reagentsRenström Ing-MarieKaiser ChristinaDanielsson EvaAndersson MonicaLanner JohannaPramfalk CamillaWarolén MalinJames Stephen R<p>Abstract</p> <p>Background</p> <p>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</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p> http://www.biomedcentral.com/1471-2121/5/7
collection DOAJ
language English
format Article
sources DOAJ
author Renström Ing-Marie
Kaiser Christina
Danielsson Eva
Andersson Monica
Lanner Johanna
Pramfalk Camilla
Warolén Malin
James Stephen R
spellingShingle Renström Ing-Marie
Kaiser Christina
Danielsson Eva
Andersson Monica
Lanner Johanna
Pramfalk Camilla
Warolén Malin
James Stephen R
Insulin receptor activation and down-regulation by cationic lipid transfection reagents
BMC Cell Biology
author_facet Renström Ing-Marie
Kaiser Christina
Danielsson Eva
Andersson Monica
Lanner Johanna
Pramfalk Camilla
Warolén Malin
James Stephen R
author_sort Renström Ing-Marie
title Insulin receptor activation and down-regulation by cationic lipid transfection reagents
title_short Insulin receptor activation and down-regulation by cationic lipid transfection reagents
title_full Insulin receptor activation and down-regulation by cationic lipid transfection reagents
title_fullStr Insulin receptor activation and down-regulation by cationic lipid transfection reagents
title_full_unstemmed Insulin receptor activation and down-regulation by cationic lipid transfection reagents
title_sort insulin receptor activation and down-regulation by cationic lipid transfection reagents
publisher BMC
series BMC Cell Biology
issn 1471-2121
publishDate 2004-01-01
description <p>Abstract</p> <p>Background</p> <p>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</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>
url http://www.biomedcentral.com/1471-2121/5/7
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