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.
Highlights
Diabetes mellitus in dogs is characterised by the presence of hyperglycaemia caused by an absolute or relative deficiency in the pancreatic β cell hormone, insulin (Catchpole et al., 2008)
Whereas recombinant insulin is used to treat human diabetic patients, in the UK there is currently only one licensed insulin product available for treatment of diabetes in companion animals, which consists of purified porcine insulin
RcPROINS showed a degree of binding to the insulin receptor (INSR) and IGF-1R, binding to the INSR was greater and more selective, compared to binding to IGF-1R for the three single-chain insulin (SCI) analogues assessed (Fig. 4A)
Summary
Diabetes mellitus in dogs is characterised by the presence of hyperglycaemia caused by an absolute or relative deficiency in the pancreatic β cell hormone, insulin (Catchpole et al., 2008). All diabetic dogs require administration of exogenous insulin to control their blood glucose concentration. Whereas recombinant insulin is used to treat human diabetic patients, in the UK there is currently only one licensed insulin product available for treatment of diabetes in companion animals, which consists of purified porcine insulin Recombinant human insulin has been used for many years in North America for treatment of canine diabetes, where until recently there was no FDA-approved insulin for companion animals (Rucinsky et al, 2010). Since the supply of bovine and porcine insulin for veterinary use generally relies on human market availability, insulin for diabetic dogs is likely to become increasingly limited. In recent years, bovine insulin products (formerly Insuvet soluble, lente and protamine zinc insulin, Zoetis) have been withdrawn from the veterinary market. There is an anticipated need for development of recombinant canine insulin preparations
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