Synthesis and Characterization of Molecular Hybrids of Insulin and Insulin - like Growth Factor I THE

Insulin and insulin-like growth factor (IGF) I have 50% homology in their primary amino acid sequences. IGF-I differs structurally from insulin because it has a connecting peptide between the Aand B-chains and an octapeptide sequence at the COOH-terminal of the IGF-I molecule termed the "D" region. To determine the role of the "D" region of IGF-I in its biological activity, we have synthesized a hybrid molecule in which this octapeptide is linked to the COOH-terminal of the A-chain of pork insulin and tested its properties in radioimmunoassay, radioreceptor assays, and bioassays. A control insulin analogue, A21 asparaginimid, which is altered at A21 but without the addition of the octapeptide sequence of IGF-I "D" region, was similarly tested. When pork insulin, insulin-IGF-I hybrid, A21-asparaginimid insulin, IGF-I, and IGF-II were tested for the stimulation of glucose oxidation in rat adipocytes, their potencies were 100%, 20%, 25%, 0.2%, and 2%, respectively. Similarly, in radioimmunoassay for pork insulin, the insulin-IGF-I hybrid, A21-asparaginimid insulin, IGF-I, and IGF-II were 11%, 10%, 0.2%, and 0.2% as potent as pork insulin. This was similar to their relative potency in competing with '2 5I-pork insulin for binding to the insulin receptors on rat adipocytes; insulin-IGFI hybrid and A21-asparaginimid insulin were only 28% and 25% as potent. IGF-I did not compete with 25'Iinsulin for binding even at 200 ,pg/ml. By contrast, in a bioassay measuring stimulation of thymidine incorporation into the DNA of human fibroblasts, the relative order of potency for these peptides was reversed: IGFI was 10-fold more potent than pork insulin, followed by IGF-II and insulin-IGF-I hybrid which were 5and 2.8-fold more potent than insulin, respectively, whereas A21-asparaginimid insulin was only 10% as potent. These data suggest that the terminal octapeptide of IGF-I plays a role in the increased growth-promoting activity of the IGFs, while it decreased IGF-I's potency on metabolic effects.