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Establishment and phenotyping of disease model cells created by cell-resealing technique

Cell-based assays are growing in importance for screening drugs and investigating their mechanisms of action. Most of the assays use so-called “normal” cell strain because it is difficult to produce cell lines in which the disease conditions are reproduced. In this study, we used a cell-resealing te...

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Autores principales: Kano, Fumi, Noguchi, Yoshiyuki, Murata, Masayuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680332/
https://www.ncbi.nlm.nih.gov/pubmed/29123170
http://dx.doi.org/10.1038/s41598-017-15443-0
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author Kano, Fumi
Noguchi, Yoshiyuki
Murata, Masayuki
author_facet Kano, Fumi
Noguchi, Yoshiyuki
Murata, Masayuki
author_sort Kano, Fumi
collection PubMed
description Cell-based assays are growing in importance for screening drugs and investigating their mechanisms of action. Most of the assays use so-called “normal” cell strain because it is difficult to produce cell lines in which the disease conditions are reproduced. In this study, we used a cell-resealing technique, which reversibly permeabilizes the plasma membrane, to develop diabetic (Db) model hepatocytes into which cytosol from diabetic mouse liver had been introduced. Db model hepatocytes showed several disease-specific phenotypes, namely disturbance of insulin-induced repression of gluconeogenic gene expression and glucose secretion. Quantitative image analysis and principal component analysis revealed that the ratio of phosphorylated Akt (pAkt) to Akt was the best index to describe the difference between wild-type and Db model hepatocytes. By performing image-based drug screening, we found pioglitazone, a PPARγ agonist, increased the pAkt/Akt ratio, which in turn ameliorated the insulin-induced transcriptional repression of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1. The disease-specific model cells coupled with image-based quantitative analysis should be useful for drug development, enabling the reconstitution of disease conditions at the cellular level and the discovery of disease-specific markers.
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spelling pubmed-56803322017-11-17 Establishment and phenotyping of disease model cells created by cell-resealing technique Kano, Fumi Noguchi, Yoshiyuki Murata, Masayuki Sci Rep Article Cell-based assays are growing in importance for screening drugs and investigating their mechanisms of action. Most of the assays use so-called “normal” cell strain because it is difficult to produce cell lines in which the disease conditions are reproduced. In this study, we used a cell-resealing technique, which reversibly permeabilizes the plasma membrane, to develop diabetic (Db) model hepatocytes into which cytosol from diabetic mouse liver had been introduced. Db model hepatocytes showed several disease-specific phenotypes, namely disturbance of insulin-induced repression of gluconeogenic gene expression and glucose secretion. Quantitative image analysis and principal component analysis revealed that the ratio of phosphorylated Akt (pAkt) to Akt was the best index to describe the difference between wild-type and Db model hepatocytes. By performing image-based drug screening, we found pioglitazone, a PPARγ agonist, increased the pAkt/Akt ratio, which in turn ameliorated the insulin-induced transcriptional repression of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1. The disease-specific model cells coupled with image-based quantitative analysis should be useful for drug development, enabling the reconstitution of disease conditions at the cellular level and the discovery of disease-specific markers. Nature Publishing Group UK 2017-11-09 /pmc/articles/PMC5680332/ /pubmed/29123170 http://dx.doi.org/10.1038/s41598-017-15443-0 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Kano, Fumi
Noguchi, Yoshiyuki
Murata, Masayuki
Establishment and phenotyping of disease model cells created by cell-resealing technique
title Establishment and phenotyping of disease model cells created by cell-resealing technique
title_full Establishment and phenotyping of disease model cells created by cell-resealing technique
title_fullStr Establishment and phenotyping of disease model cells created by cell-resealing technique
title_full_unstemmed Establishment and phenotyping of disease model cells created by cell-resealing technique
title_short Establishment and phenotyping of disease model cells created by cell-resealing technique
title_sort establishment and phenotyping of disease model cells created by cell-resealing technique
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680332/
https://www.ncbi.nlm.nih.gov/pubmed/29123170
http://dx.doi.org/10.1038/s41598-017-15443-0
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