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Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice

Gene Therapy volume 12, pages 655–667 (2005)Cite this article

Abstract

We describe the durable correction of streptozotocin-induced murine diabetes by in vivo implantation of primary mouse hepatocytes electroporated ex vivo with a human proinsulin cDNA plasmid construct controlled by glucose and zinc regulatory elements. Transfected hepatocytes increased insulin transgene transcription and secretion within 10–20 min of exposure to 25 mM glucose or 60 μM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes (∼8 × 105 viable cells) were implanted in C57BL/6J diabetic mice in one of three sites: unresected liver, regenerating liver or mesentery. Control diabetic mice were implanted with untransfected hepatocytes. At 30 days after implantation, 8/15 control mice were alive, while 19/19 treated mice were alive. The ratio of body weight on day 30/nadir body weight was significantly higher for all treated groups compared with controls. All eight surviving control mice were hyperglycemic 30 days post-implantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (P⩽0.001) without fasting hypoglycemia and better glucose tolerance (P⩽0.0003) than untreated controls. All (6/6) diabetic mice implanted in regenerating liver and 71% (5/7) implanted in unresected liver were alive 77 days after implantation. Engrafted hepatocytes were identified, mainly around central veins, by staining for β-galactosidase activity and with anti-human insulin antibody.

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Acknowledgements

This work was supported by grants from the Biomedical Research Council, National Medical Research Council and National Cancer Centre, all in Singapore. We thank Adrian Khoo for useful discussions on hepatocyte preparation, Serene Lok for technical assistance with hepatocyte implantations, Elsie Kok, Magdalene Koh and Leong Siew Hong for assistance with immunohistochemistry and image capture, Patricia Netto for help with electron microscopy and Dr Li Huihua for statistical guidance.

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Authors and Affiliations

  1. Division of Medical Sciences, National Cancer Centre, Singapore, Republic of Singapore

    N K F Chen, J Sivalingam & O L Kon

  2. Department of Pathology and Laboratory Medicine, Tan Tock Seng Hospital, Singapore, Republic of Singapore

    S Y Tan

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  1. N K F Chen
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Chen, N., Sivalingam, J., Tan, S. et al. Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice. Gene Ther 12, 655–667 (2005). https://doi.org/10.1038/sj.gt.3302446

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