Single-chain insulins as receptor agonists

Single-chain insulins (SCIs) are single polypeptide chains in which the insulin B-chain links contiguously with the insulin A-chain via an uncleaved connecting peptide. Although direct linkage of insulin B- and A-chains produces SCIs with little insulin receptor binding, biologists have been interested in bioengineering linker peptides that form a flexible reverse turn, allowing SCIs to activate insulin receptors. In this report, we have investigated a series of cDNAs intended to explore the significance of linker length, cleavability, and the impact of certain site-dependent residues for the bioactivity of recombinant SCIs on insulin receptors. SCI concentration is readily measured by RIA with a (proinsulin plus insulin)-specific polyclonal antibody. Although dibasic flanking residues may result in potential endoproteolytic susceptibility, a linker with -Gln-Arg- flanking sequences resisted cleavage even in secretory granules, ensuring single-chain behavior. Effective SCIs exhibit favorable and specific binding with insulin receptors. SCIs with linkers bearing an Arg residue immediately preceding the A-chain were most bioactive, although efficient receptor interaction was inhibited as SCI linker length increased, approaching that observed for proinsulin. SCIs activate downstream metabolic signaling, stimulating glucose uptake into adipocytes and suppressing gluconeogenic enzyme biosynthesis in hepatocytes, with only limited cross-reactivity on IGF-I receptors. SCIs might theoretically have utility either in immunotherapy or gene therapy in insulin-deficient diabetes.