The efficiency of insulin production and its content in insulin-expressing model β-cells correlate with their Zn ²⁺ levels

Abstract

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn 2+ -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 Zn 2+ 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 Zn 2+ 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.

Characterization of insulin crystalline form in isolated β-cell secretory granules

“…This revealed that MIN6 cells are more glucose-responsive than INS-1E cells, both in terms of the quantity of secreted insulin and the reproducibility of results, despite a lower total insulin content in these cells. It is possible that the ability of INS-1E cells to secrete insulin in response to glucose is altered, possibly by an impaired proinsulin processing [43], and that higher intracellular concentration of ( pro)insulin in INS-1E cells is caused by their reduced ability to secrete insulin. Therefore, considering all these results, and the initial analysis of the β-cells by FIB/ SEM microscopy performed here, we selected the MIN6 cells for further isolation and analysis of insulin secretory granules, as they appear as a more native-like model of the β-cells than INS-1E or BRIN-BD11 cells.…”

“…The insulin secretory granules from MIN-6 cells were isolated according to our protocol, described earlier by Dzianova et al [43], and TEM images of isolated MIN6 cell secretory granules were collected (figure 3). Smaller particles representing insulin secretory granules were visible there (figure 3a-c), together with bigger mitochondria that were apparently copurified with insulin granules, a phenomenon already described by Brunner et al [46].…”

“…The isolation of granules was performed according to the protocol published by Brunner et al [46] and described in detail in Dzianova et al [43]. The final pellet of isolated insulin secretory granules was re-suspended in buffer composed of 0.27 M sucrose, 10 mM MOPS/1 M Tris, pH 6.8 (SMT buffer), kept at 0-4°C without freezing, and used for further analyses within the next 24 h. The protein content in the granule fraction was determined by the Bradford assay.…”

“…However, we were not able to register any X-ray diffraction from the isolated secretory granules from rat pancreatic Islets of Langerhans and from rat INS-1E cells. Nevertheless, X-ray fluorescence (XRF) scans revealed a striking difference in Zn 2+ content between INS-1E secretory granules and native rat pancreatic Islet granules [43].…”

On the Electroanalytical Detection of Zn Ions by a Novel Schiff Base Ligand-SPCE Sensor

“…Among the various heavy metal ions, Zn 2+ plays an important role in the regulation of many reactions catalyzed by enzymes in the human body and therefore maintains physiological processes at the optimal level. Zinc is considered as an essential trace element for human beings due to its relationship with insulin production [ 8 , 9 ], and for the regulation of biological processes, being involved as catalysts for more than 200 enzymes [ 10 ]. In addition, zinc is also present in food, e.g., proteins that are high in zinc content.…”