Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake

Adams, J P; Holder, A L; Catchpole, B

Abstract

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes.

All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs.

Citation

Adams, J. P., Holder, A. L., & Catchpole, B. (2014). Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake. https://doi.org/10.1016/j.tvjl.2014.09.027

• Journal Article Type: Article
• Acceptance Date: Sep 29, 2014
• Publication Date: Dec 4, 2014
• Deposit Date: Jul 8, 2015
• Publicly Available Date: Jul 8, 2015
• Journal: VETERINARY JOURNAL
• Peer Reviewed: Peer Reviewed
• Volume: 202
• Issue: 3
• Pages: 436-42
• DOI: https://doi.org/10.1016/j.tvjl.2014.09.027
• Public URL: https://rvc-repository.worktribe.com/output/1404629