Thermodynamic Contributions to the Stability of the Insulin Hexamer

Lisi, George P.; Png, C.Y. Maximilian; Wilcox, Dean E.

doi:10.1021/bi401678n

Cited by 26 publications

(38 citation statements)

References 49 publications

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“…These structural and spectroscopic data provide evidence for the T 3 -R 3 trimers cross-talk and, as postulated previously, for a heterotropic allostery within the insulin hexamer (22, 30, 44–47). However, the exact molecular detail behind this phenomenon has been elusive, likely involving a network of propagating longer range interactions, which fit the Seydoux, Malhotra, and Bernhard (SMB) cooperativity model (48).…”

Section: Discussionsupporting

confidence: 85%

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules

Palivec

Viola

Kozak

et al. 2017

Journal of Biological Chemistry

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Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3Rf3, and R6. As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic β-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.

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Section: Discussionsupporting

confidence: 85%

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules

Palivec

Viola

Kozak

et al. 2017

Journal of Biological Chemistry

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“…In solution, insulin can correspond to four conformations: monomers, dimers, tetramers or hexamers [ 21 ]. In pharmaceutical formulations, insulin is presented as a hexamer, because this is the most resistant to fibrillation and degradation [ 22 ]. In the bloodstream however, hexamers dissociate to become monomers binding to insulin receptors [ 23 ].…”

Section: Discussion/ Conclusionmentioning

confidence: 99%

In vitro assessment of the influence of intravenous extension set materials on insulin aspart drug delivery

Masse¹,

Maton

Genay³

et al. 2018

PLoS ONE

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Insulin is a frequently prescribed drug in hospitals and is usually administered by syringe pumps with an extension line which can be made of various materials. Two insulin solutions were studied: an insulin analogue, Novorapid® which contains insulin aspart and two phenolic preservatives (e.g. phenol and metacresol) and Umuline rapide® with human insulin and metacresol as preservative. Some studies have indicated interactions between insulin, polyvinyl chloride (PVC) and polyethylene (PE). The aim of this work was to study such interactions between Novorapid® or Umuline rapide® and infusion extension line materials (PVC, PE and coextruded (PE/PVC)). Insulin solution at 1 IU/mL was infused at 2 mL/h over 24 hours with 16 different extension lines (8 in PVC, 3 in PE and 5 in PE/PVC). Ultra-Fast Liquid Chromatography with diode array detection (UFLC-DAD) was performed to quantify insulin (human and aspart) and preservatives (metacresol and phenol). Limited human insulin sorption was observed thirty minutes after the onset of infusion: 24.3 ± 12.9%, 3.1 ± 1.6% and 18.6 ± 10.0% for PVC, PE and PE/PVC respectively. With insulin aspart, sorption of about 5% was observed at the onset of infusion for all materials. However, there were interactions between phenol and especially metacresol with PVC, but no interactions with PE and PE/PVC. This study shows that insulin interacts with PVC, PE and PE/PVC at the onset of infusion. It also demonstrates that insulin preservatives interact with PVC, which may result in problems of insulin conservation and conformation. Some more studies are required to understand the clinical impact of the latter during infusion.

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“…In vivo , such as Zn 2+ ions, and in pharmaceutical formulations Zn 2+ ions and phenol. Zn 2+ is expected to stabilize the hexamer by coordinating His B10 43–46 in the insulin interface and expected to enhance the dimer to hexamer transition 13,14 . We quantified the effect of Zn 2+ by addition of 100 μM Zn 2+ , an excess amount of Zn 2+ in solution compared to HI.…”

Section: Resultsmentioning

confidence: 99%

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Bohr

Mishra

et al. 2022

Preprint

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Insulin formulations are the hallmark of interventions for treatment of diabetes. Understanding the mechanism that governs insulin self assembly or disassembly, and the role of stabilizing additives, are essential for improving insulin formulations. We report here the real-time direct observation of single insulin self-assembly and disassembly events using single molecule fluorescence microscopy. Our direct observations revealed previously unaccounted monomeric additions to occur to all types of assemblies and allowed us to quantify the existence, abundance and kinetic characterization of diverse assembly pathways involving monomeric dimers or tetrameric insulin species. We proposed and experimentally validated a model where the insulin self-assembly pathway is rerouted favoring monomeric or oligomeric assembly events by solution concentration, additives and formulations. Our rate simulation predicted the abundance of each oligomeric species across a concentration range of 6 orders of magnitude. Besides providing fundamental new insights, the results and toolbox here can be universally applied contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for other proteins.

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Thermodynamic Contributions to the Stability of the Insulin Hexamer

Cited by 26 publications

References 49 publications

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules

In vitro assessment of the influence of intravenous extension set materials on insulin aspart drug delivery

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

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