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Endocrine Abstracts (2004) 8 OC2

SFE2004 Oral Communications Growth and Development (8 abstracts)

Insulin transgene overexpression: a tool for characterisation of beta-cell phenotype in transdifferentiation studies

A Aldibbiat , KT Scougall , SC Campbell , WM Macfarlane & JAM Shaw


Diabetes Research Group, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.


Transdifferentiation of non-endocrine pancreatic cells towards a beta-cell phenotype may enable insulin independence in many individuals from a single donor. This will be dependent on near-physiological proinsulin biosynthesis, processing, storage and secretion. The aim of this study was to fully characterise endocrine potential of pancreatic acinar cells before and after transdifferentiation.

Expression of exocrine/endocrine markers in the AR42J rat acinar cell line was determined by RT-PCR. Transdifferentiation regimes comprised culture on Matrigel basement membrane and supplementation with a range of growth factors (GLP1(7-36); activin A; betacellulin; HGF). Potential for physiological insulin processing/storage was determined by differential ELISA following human preproinsulin transgene (pIRES-hppI1) transfection.

Exocrine acinar phenotype and epithelial origin of AR42J cells was confirmed (amylase/cytokeratin immuno-staining). Expression of PDX1; glucokinase; pancreatic polypeptide in addition to insulin mRNA was demonstrated (RT-PCR). A morphological shift, induction of GLUT2 and upregulation of insulin gene expression was attained following culture on Matrigel. Maximal PDX1, GLUT2 and insulin mRNA expression was confirmed following 10nM GLP1 supplementation. Pro-hormone convertase (PC3) expression was undetectable in all studies. Western blot for PDX1 protein was negative. Endogenous insulin was undetectable by ultrasensitive rat insulin ELISA in wild type or transdifferentiated cells. Secretion of human (pro)insulin was confirmed following transient transfection of cells grown on Matrigel with pIRES-hppI1 plasmid. Less than 5% processing to mature insulin and less than 5% intracellular (pro)insulin storage was demonstrated in the absence or presence of 10nM GLP1 supplementation.

Expression of beta-cell markers including insulin mRNA has been demonstrated in the rat AR42J acinar cell line in addition to induction of GLUT2 following culture on Matrigel and GLP1 treatment. Absence of true beta-cell phenotype was confirmed by undetectable PDX1 and rat insulin at the protein level and by minimal proinsulin processing and storage following transfection with a human preproinsulin construct.

Volume 8

195th Meeting of the Society for Endocrinology joint with Diabetes UK and the Growth Factor Group

Society for Endocrinology 

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