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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

(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.
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f2: (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.

Mentions: Figure 2A shows the time course of fibrillation of insulin as seen by ThT fluorescence assay when incubated at pH 2.0 and 37 °C with stirring at 250 rpm. The formation of insulin fibrils was also confirmed by transmission electron microscopy and scanning electron microscopy which showed clear branched insulin fibrils when the images were taken after 600 min of incubation (Fig. 2C,D). It is seen from Fig. 2A that fibrillation follows a characteristic sigmoidal curve consisting of an initial lag phase, a subsequent elongation phase and a final saturation phase. The fitting of equation 1 (see Materials and methods) to the data thus obtained gives time periods of lag phase as (406 ± 5) min, elongation phase as 406–500 min, and saturation phase thereafter. The apparent rate constant (kapp) for the growth of fibrils is estimated as 0.04 min−1.


Inhibition of insulin fibrillation by osmolytes: Mechanistic insights.

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

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

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

f2: (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.
Mentions: Figure 2A shows the time course of fibrillation of insulin as seen by ThT fluorescence assay when incubated at pH 2.0 and 37 °C with stirring at 250 rpm. The formation of insulin fibrils was also confirmed by transmission electron microscopy and scanning electron microscopy which showed clear branched insulin fibrils when the images were taken after 600 min of incubation (Fig. 2C,D). It is seen from Fig. 2A that fibrillation follows a characteristic sigmoidal curve consisting of an initial lag phase, a subsequent elongation phase and a final saturation phase. The fitting of equation 1 (see Materials and methods) to the data thus obtained gives time periods of lag phase as (406 ± 5) min, elongation phase as 406–500 min, and saturation phase thereafter. The apparent rate constant (kapp) for the growth of fibrils is estimated as 0.04 min−1.

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