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Tetracycline-regulated secretion of human (pro)insulin following plasmid-mediated transfection of human muscle

Lookup NU author(s): Dr Kathleen Scougall, Dr Elizabeth McIntyre, Professor James Shaw

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Abstract

Long-term secretion of insulin by host muscle following transduction with an insulin gene construct offers the potential of gene therapy for diabetes without immunosuppression. Clinical implementation will be dependent on proof of principle in human tissue and a system for safely regulating basal insulin levels. Liposomal co-transfection with a tetracycline-responsive wild type human preproinsulin (pTRE-hppl1) or mutant construct (pTRE-hppl4), in which PC2 and PC3 cleavage sites were altered to form tetrabasic consensus sites for furin, together with pTet-off (coding for a transactivating protein) was evaluated in the C2C12 mouse myoblast cell line and human myoblasts following establishment in primary culture. In the absence of tetracycline, (pro)insulin secretion in C2C12 and human myoblasts transfected with tetracycline-responsive hppl1 and hppl4 constructs was comparable to that following transfection with equivalent constructs under the control of a constitutively active cytomegaloviral promoter. Percentage processing to mature insulin was < 5% in C2C12 and human myoblasts transfected with pTet-off/pTRE-hppl1 but > 90% in C2C12 cells and 45-60% in human myoblasts on transfection with pTet-off/pTRE-hppl4. Incremental dose-responsive suppression of proinsulin secretion was demonstrated in C2C12 and human myoblasts expressing pTet-off/pTRE-hppl1 following incubation with tetracycline (0-100 μg/ml) for up to 72 h. Reversibility was confirmed following tetracycline withdrawal. Dose-responsive tetracycline-inducible repression of mature insulin secretion was confirmed in C2C12 cells following transfection with pTet-off/pTRE-hppl4. Regulation of human proinsulin biosynthesis and secretion has been attained in vivo following plasmid-mediated gene transfer to rat skeletal muscle and oral tetracycline administration. In conclusion, processing to mature insulin has been confirmed following plasmid-mediated gene transfer to human muscle in addition to in vitro- and in vivo-regulated human proinsulin secretion employing the safe and well-tolerated antibiotic, tetracycline. © 2005 Society for Endocrinology.


Publication metadata

Author(s): Wilson MO, Scougall KT, Ratanamart J, McIntyre EA, Shaw JAM

Publication type: Article

Publication status: Published

Journal: Journal of Molecular Endocrinology

Year: 2005

Volume: 34

Issue: 2

Pages: 391-403

Print publication date: 01/04/2005

ISSN (print): 0952-5041

ISSN (electronic): 1479-6813

Publisher: Society for Endocrinology

URL: http://dx.doi.org/10.1677/jme.1.01646

DOI: 10.1677/jme.1.01646

PubMed id: 15821105


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