Inhibition of insulin fibrillation by osmolytes: Mechanistic insights

Abstract

We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50-250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies.

Figure 1. Chemical structures of osmolytes (A) betaine, (B) citrulline, (c) proline and (d) sorbitol.

Figure 2

(A) Kinetics of the insulin amyloid formation monitored by the binding of ThT with insulin amyloid fibrils, (B) far-UV CD spectra of insulin at different time intervals, (C) transmission electron microscopic and (D) scanning electron microscopic images of insulin fibrils after 600 min of incubation.

Figure 3. Kinetics of insulin fibril extension in absence and in presence of different concentration of osmolytes (A) betaine, (B) citrulline, (C) proline and (D) sorbitol studied by monitoring the changes in fluorescence emission intensity as a function of time.

Figure 4. Maximum ThT intensity observed during insulin fibril formation in presence of 250 mM osmolytes.

Figure 5. Transmission electron microscopic images of insulin after 600 min of incubation in presence of 250 mM (A) betaine, (B) citruine, (C) proline and (D) sorbitol.

Figure 6. Limiting enthalpies of interaction of insulin with (A) betaine, (B) citrulline, (C) proline, and (D) sorbitol at different stages of fibrillation.

Figure 7. Schematic representation of mechanism of inhibition of insulin fibrillation by osmolytes.