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.

Graphic Abstract

中文翻译：

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白藜芦醇作为一种无毒赋形剂以生物活性六聚体形式稳定胰岛素。

摘要

胰岛素聚集是导致用于糖尿病管理的液体制剂中活性药物分子数量显着减少的主要原因。酚类化合物，如苯酚和间甲酚，通常用于在其商业制备过程中稳定六聚体形式的胰岛素。然而，长期使用市售胰岛素会导致各种不良的继发反应，而酚类赋形剂的毒性是造成这种反应的主要原因。在这项研究中，我们旨在找出一种无毒的胰岛素稳定剂。为此，我们选择了白藜芦醇，一种天然多酚，作为一种预期的无毒胰岛素稳定剂，因为它与商业使用的酚类化合物的结构相似。白藜芦醇处理的人胰岛素的原子力显微镜可视化显示，白藜芦醇具有独特的能力，可以将 hINS 以具有离散球形形态的可溶性寡聚形式捕获。最重要的是，经白藜芦醇处理的胰岛素对 HepG2 细胞无毒，可在小鼠模型中有效维持低血糖。冷冻电子显微镜显示白藜芦醇稳定胰岛素的 3D 形态与间甲酚配制的胰岛素六聚体的晶体结构极为相似。值得注意的是，我们发现，在生理 pH 值诱导淀粉样蛋白纤维化的条件下，白藜芦醇能够比间甲酚更有效地稳定胰岛素。因此，

图形摘要