Abstract
An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic β cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose–response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for β cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic β cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic β cells and become a therapy for the treatment of type I diabetes.
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Acknowledgements
This work was supported by a Project Grant from the National Health and Medical Research Council of Australia. Three of the authors were recipients of Postgraduate Scholarships, one from the University of Technology, Sydney (BZ); one from The University of New South Wales (MTT); and one an Australian Postgraduate Award from the Department of Education, Training and Youth Affairs, Australia (RKBH). We wish to thank Dr Murray Smith for his constructive comments on the manuscript, Dr Anil Amaratunga, Mr Appavoo Mathiyalagan and Dr Chang Tao for technical assistance with Western blots, Mrs Pauline Khoury for assistance with the transplants, Ms Robyn Baum and Ms Lillian Tan for technical assistance with the proinsulin and C-peptide assay, and Mr Roland Smith, Anatomy, Sydney University for printing the electron micrographs in Figures 6 and 7a–c.
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Tuch, B., Szymanska, B., Yao, M. et al. Function of a genetically modified human liver cell line that stores, processes and secretes insulin. Gene Ther 10, 490–503 (2003). https://doi.org/10.1038/sj.gt.3301911
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DOI: https://doi.org/10.1038/sj.gt.3301911
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