Dissociation of insulin oligomers by bile salt micelles and its effect on alpha-chymotrypsin-mediated proteolytic degradation.

Bile salts have been found to be effective absorption promoters of insulin across mucosal barriers, i.e., nasal and gastrointestinal. One of the mechanisms proposed for absorption enhancement is the dissociation of insulin oligomers to monomers, rendering a higher insulin diffusivity. alpha-Chymotryptic degradation and circular dichroism studies were used to characterize such a transition. When zinc insulin (hexamers) and sodium insulin (dimers) were subjected to alpha-chymotryptic degradation, a 3.2-fold difference in the apparent first-order rate constants was observed (zinc insulin being slower than sodium insulin), representing the intrinsic difference in the concentration of total associated species in solution (three times). In the presence of a bile salt, sodium glycocholate (NaGC), the rate of degradation of both zinc and sodium insulin increased in an asymptotic manner. A maximum increase of 5.4-fold was observed for zinc insulin at a 30 mM NaGC concentration and a 2.1-fold increase was noted for sodium insulin at 10 mM NaGC, both values being close to the theoretical numbers of 6- and 2-fold as predicted by the complete dissociation of hexamers and dimers to monomers. The result indicates dissociation of insulin oligomers to monomers by bile salt micelles, probably by hydrophobic micellar incorporation of monomeric units. Circular dichroism studies also revealed progressive attenuation of molecular ellipticities at negative maxima of 276, 222, and 212 nm for zinc insulin solution in the presence of NaGC. Therefore, both alpha-chymotryptic degradation and circular dichroism studies have consistently demonstrated that the bile salts may be capable of dissociating insulin oligomers to monomers, a fact which may play an important role in enhancing insulin bioavailability.