Abstract
Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.
Highlights
Insulin is one of the key hormones responsible for carbohydrate homeostasis and metabolic response
The cleavage of its signal sequence yields 81 amino acid proinsulin that is transported into the Golgi apparatus, in which the immature—proinsulin-containing—insulin secretory granules (ISGs) are formed that dominate the content of the β-cell [1]
The ISGs were isolated by a discontinuous Nycodenz and the subsequent Percoll gradients, where the ISGs were localized on dot-blots by the insulin (L6B10) mouse mAb
Summary
Insulin is one of the key hormones responsible for carbohydrate homeostasis and metabolic response. Insulin is synthesized in the endoplasmic reticulum (ER) as a 110 amino acid preproinsulin. The cleavage of its signal sequence yields 81 amino acid proinsulin that is transported into the Golgi apparatus, in which the immature—proinsulin-containing—insulin secretory granules (ISGs) are formed that dominate the content of the β-cell [1]. 10–20% of its total cell volume [2]), with each granule storing about 200 000 insulin molecules [3]. It is envisaged that proinsulin firstly forms there, soluble Zn2+-stabilized hexamers, which are processed into a fully functional, mature 51 amino acid two-chain insulin molecule that is still held in the Zn2+-maintained hexamer [4,5,6,7]. It is thought that the proinsulin → insulin hexamer transition lowers the solubility of the resulting insulin oligomers, which subsequently form some crystalline material [8]
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