Zn2+ chelation by serum albumin improves hexameric Zn2+ -insulin dissociation into monomers after exocytosis

β-cells release hexameric Zn-insulin into the extracellular space, but monomeric Zn-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn concentration-dependent. In this study, we investigate the influence of albumin binding to Zn on Zn-insulin dissociation into Zn-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn-free insulin but strongly affects RIA measurements of Zn-insulin. In contrast, accurate determination of Zn-insulin was obtained only when bovine serum albumin or Zn chelators were present in the assay buffer solution. Albumin and Zn chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn chelators promotes the conversion of “slow” Zn-insulin into “fast” insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn chelators. These observations support the notion that the Zn-binding properties of albumin improve the dissociation of Zn-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.Our research is supported by Fondos FEDER and the Instituto de Salud Carlos III (CIBERDEM, CB07/08/0006; FIS PI14/010015 and Red TerCel RD/0012/0028 to BS and FM) and Junta de Andalucía (PAI-BIO311 (to FM), CTS-576 (to FB) and CTS 11-727).Peer Reviewe