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Resveratrol as a nontoxic excipient stabilizes insulin in a bioactive hexameric form

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Abstract

Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as phenol and m-cresol, are routinely used to stabilize insulin in a hexameric form during its commercial preparation. However, long term usage of commercial insulin results in various adverse secondary responses, for which toxicity of the phenolic excipients is primarily responsible. In this study we aimed to find out a nontoxic insulin stabilizer. To that end, we have selected resveratrol, a natural polyphenol, as a prospective nontoxic insulin stabilizer because of its structural similarity with commercially used phenolic compounds. Atomic force microscopy visualization of resveratrol-treated human insulin revealed that resveratrol has a unique ability to arrest hINS in a soluble oligomeric form having discrete spherical morphology. Most importantly, resveratrol-treated insulin is nontoxic for HepG2 cells and it effectively maintains low blood glucose in a mouse model. Cryo-electron microscopy revealed 3D morphology of resveratrol-stabilized insulin that strikingly resembles crystal structures of insulin hexamer formulated with m-cresol. Significantly, we found that, in a condition inductive to amyloid fibrillation at physiological pH, resveratrol is capable of stabilizing insulin more efficiently than m-cresol. Thus, this study describes resveratrol as an effective nontoxic natural molecule that can be used for stabilizing insulin in a bioactive oligomeric form during its commercial formulation.

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Acknowledgements

This work was supported by SERB, DST (India) sponsored project, DBT contingency fund for Research Associate (DBT-RA, India), and CSIR-Indian Institute of Chemical Biology, Kolkata, India. BP sincerely thanks DBT for providing Research associate Fellowship. We acknowledge the Central Instrument Facility (CIF) (and all the technical staff associated with it) of CSIR-IICB for their help in all respect. We sincerely thank Dr. Ilic Zoran of Wadsworth Center, NY, USA, for critically checking the manuscript and Mr. Chiranjit Biswas for cryo-EM data collection. DD and SB acknowledge UGC and CSIR (India), respectively, for Senior Research Fellowship. Financial support from the DBT-RA Program in Biotechnology and Life Sciences is gratefully acknowledged.

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Author notes

  1. Bani Kumar Pathak and Debajyoti Das contributed equally.

Authors and Affiliations

  1. Structural Biology & Bio-Informatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India

    Bani Kumar Pathak, Sayan Bhakta & Jayati Sengupta

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Partha Chakrabarti & Jayati Sengupta

  3. Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India

    Debajyoti Das & Partha Chakrabarti

Authors
  1. Bani Kumar Pathak
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  2. Debajyoti Das
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  3. Sayan Bhakta
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  4. Partha Chakrabarti
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  5. Jayati Sengupta
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Contributions

BP and JS conceived the project and designed the experiments. BP carried out biochemical and biophysical experiments and 3D image processing of cryo-EM data. DD performed cell-based and animal experiments under the supervision of PC. SB assisted in cryo-EM data processing. BP, DD, PC and JS analyzed the data and wrote the manuscript.

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Correspondence to Partha Chakrabarti or Jayati Sengupta.

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The cryo-EM density map of reveratrol-stabilized oligomeric form of insulin was deposited in the Electron Microscopy Data Bank (EMDB) under the accession number EMD-9878, and the fitted model was deposited in the RCSB Protein Data Bank (PDB) under the accession code 6JR3.

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Pathak, B.K., Das, D., Bhakta, S. et al. Resveratrol as a nontoxic excipient stabilizes insulin in a bioactive hexameric form. J Comput Aided Mol Des 34, 915–927 (2020). https://doi.org/10.1007/s10822-020-00311-3

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  • DOI: https://doi.org/10.1007/s10822-020-00311-3

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