Abstract
β-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-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 Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-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 Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of “slow” Zn2+-insulin into “fast” insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.
Highlights
The insulin concentration in blood is approximately 1 ng/ml, and the predominant form is monomeric [1]
To evaluate the potential of RIA technique to discern the different antigenic forms of insulin, Zn2+-insulin and Zn2+-free insulin were diluted in KrebsRinger bicarbonate (KRB) buffer without bovine serum albumin (BSA) at the same known concentration and incubated with I125 insulin in antibody-coated tubes
No significant differences were found between Zn2+ free insulin and Zn2+-insulin plus 3% BSA (262.9 ± 45.5 and 279.9 ± 60.8 pmol/l respectively), most likely due to chelation of the Zn2+ by serum albumin and the subsequent dissociation of Zn2+-insulin into Zn2+-free insulin
Summary
The insulin concentration in blood is approximately 1 ng/ml, and the predominant form is monomeric [1]. Albumin and Zn2+-insulin decision to publish, or preparation of the manuscript
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