title: Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose uptake.
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.
authors.affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, University of London, Hawkshead Lane, Hatfield, Hertfordshire AL9 7TA, United Kingdom.
authors.forename: Jamie P
authors.initials: JP
authors.lastname: Adams
authors.affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, University of London, Hawkshead Lane, Hatfield, Hertfordshire AL9 7TA, United Kingdom.
authors.forename: Angela L
authors.initials: AL
authors.lastname: Holder
authors.affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, University of London, Hawkshead Lane, Hatfield, Hertfordshire AL9 7TA, United Kingdom. Electronic address: bcatchpole@rvc.ac.uk.
authors.forename: Brian
authors.initials: B
authors.lastname: Catchpole
mesh_terms: D041721:3T3-L1 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002096:C-Peptide / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002096:C-Peptide; D007328:Insulin; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D005947:Glucose
keywords: Canine diabetes mellitus; Glucose uptake; Insulin; Insulin receptor