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Inhibition of insulin fibrillation by osmolytes: Mechanistic insights.

Choudhary S, Kishore N, Hosur RV - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Mumbai 400098, India.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: Figure 3A represents the kinetic studies of insulin fibrillation in the absence and presence of different concentrations of betaine. The ThT fluorescence kinetics plot indicates that the lag period of insulin fibrillation has slightly decreased at 50, 100 and 250 mM concentrations to (322 ± 3) min, (319 ± 8) min and (360 ± 5) min, respectively, under the studied conditions. However, there is a significant reduction in the ThT fluorescence intensity. It is clear from the figure that betaine has successfully suppressed fibril formation although it has not delayed the onset of fibrillation, wherever it is seen. In the case of citrulline, the lag periods of insulin fibrillation at concentrations of 50 and 100 mM, are found to be (335 ± 4) and (351 ± 3) minutes which becomes (547 ± 6) minutes when the concentration of the osmolyte is raised to 250 mM (Fig. 3B). There is significant decrease in the ThT fluorescence intensity which suggests that the amount of fibrils formed is substantially decreased in the presence of citrulline. The lag periods are (413 ± 3), (472 ± 5) and (569 ± 5) min in the presence of 50, 100 and 250 mM proline, respectively. Here, the fluorescence emission intensity has also drastically decreased as compared to the other two osmolytes (Fig. 4). These results suggest that proline is most effective in both delaying and suppression of the fibrillation. When the studies were done in the presence of different concentrations of sorbitol (Fig. 3D) it was found that there is only a slight delay in the lag period of insulin fibrillation. In the presence of 50, 100 and 250 mM sorbitol, the lag periods were found to be (447 ± 2), (466 ± 3) and (478 ± 5) min, respectively, with slight decrease in the ThT fluorescence intensity. Figure 4 shows the extent of fibrillation in the presence of the above four osmolytes at 250 mM concentration. Clearly, betaine, citrulline and proline are very effective in preventing the fibrillation of insulin.


Inhibition of insulin fibrillation by osmolytes: Mechanistic insights.

Choudhary S, Kishore N, Hosur RV - Sci Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663473&req=5

f3: 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.
Mentions: Figure 3A represents the kinetic studies of insulin fibrillation in the absence and presence of different concentrations of betaine. The ThT fluorescence kinetics plot indicates that the lag period of insulin fibrillation has slightly decreased at 50, 100 and 250 mM concentrations to (322 ± 3) min, (319 ± 8) min and (360 ± 5) min, respectively, under the studied conditions. However, there is a significant reduction in the ThT fluorescence intensity. It is clear from the figure that betaine has successfully suppressed fibril formation although it has not delayed the onset of fibrillation, wherever it is seen. In the case of citrulline, the lag periods of insulin fibrillation at concentrations of 50 and 100 mM, are found to be (335 ± 4) and (351 ± 3) minutes which becomes (547 ± 6) minutes when the concentration of the osmolyte is raised to 250 mM (Fig. 3B). There is significant decrease in the ThT fluorescence intensity which suggests that the amount of fibrils formed is substantially decreased in the presence of citrulline. The lag periods are (413 ± 3), (472 ± 5) and (569 ± 5) min in the presence of 50, 100 and 250 mM proline, respectively. Here, the fluorescence emission intensity has also drastically decreased as compared to the other two osmolytes (Fig. 4). These results suggest that proline is most effective in both delaying and suppression of the fibrillation. When the studies were done in the presence of different concentrations of sorbitol (Fig. 3D) it was found that there is only a slight delay in the lag period of insulin fibrillation. In the presence of 50, 100 and 250 mM sorbitol, the lag periods were found to be (447 ± 2), (466 ± 3) and (478 ± 5) min, respectively, with slight decrease in the ThT fluorescence intensity. Figure 4 shows the extent of fibrillation in the presence of the above four osmolytes at 250 mM concentration. Clearly, betaine, citrulline and proline are very effective in preventing the fibrillation of insulin.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Mumbai 400098, India.

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.

No MeSH data available.


Related in: MedlinePlus