< Back to previous page

Publication

Defective insulin crystallisation in beta cells in the absence of ZnT8

Journal Contribution - Journal Article Conference Contribution

OBJECTIVE: In beta cells, zinc co-crystallises with insulin in dense-core secretory granules, but the exact role of zinc in insulin biosynthesis, storage and secretion is unknown. Polymorphism in the ZnT8 gene confers susceptibility to T2D, and autoantibodies to ZnT8 protein are observed in T1D. The aim of this study was to assess the role of ZnT8 in beta cells using mice that are deficient in ZnT8 expression (ZnT8-/- mice).MATERIALS AND METHODS: ZnT8-/- mice have a deletion of the promotor region and first exon. ZnT8 expression was assessed by RT-PCR, Western blots, and immunohistochemistry. Beta cell ultra-structure was investigated by transmission electron microscopy. Glucose tolerance was measured at age 6, 12, 25 and 52 weeks and after 10 weeks on high fat diet. Insulin processing was analysed via 35S pulse chase experiments and in situ mass spectral tissue profiling of pancreatic islets. Insulin release from isolated islets was measured in perifusion experiments, and zinc exocytosis from islet cell clusters was monitored by total internal fluorescence microscopy.RESULTS: ZnT8-/- mice were completely negative for the expression of islet ZnT8 mRNA and protein. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, indicating that ZnT8 is the only zinc transporter responsible for influx of zinc into the secretory granules. Also dense-core insulin granules were absent in ZnT8-/- beta cells (92.2 ± 6.6 vs 7.0 ± 2.7 %, p<0.0001); instead, immature, pale insulin “progranules” were detected, which were larger (433±83 vs 375±84 nm, p<0.001) than insulin granules in control cells. This phenotypic change was associated with loss of in situ and in vitro staining of islets with dithizone and disappearance of the bright light reflection of isolated islets. In contrast, rates of insulin biosynthesis, insulin content and insulin release after glucose stimulation were well preserved in ZnT8-/- mice. Moreover, when ZnT8-/- mice were fed a normal diet, glucose tolerance was preserved. However, when fed a high fat diet, the ZnT8-/- mice had the tendency to gain more weight and became glucose intolerant. CONCLUSIONS: This study shows the importance of beta cell secretory granule ZnT8 transporter in the formation of insulin crystals which -in turn- explains islet dithizone staining and the bright light reflection of isolated islets. Our in vivo data also suggest that zinc-insulin crystals allow a better insulin packaging efficiency in beta cell granule stores, which is relevant for resisting abnormal glucose homeostasis under the metabolic stress of a high fat diet.
Journal: DIABETOLOGIA
ISSN: 0012-186X
Volume: 52
Pages: S71 - S71
Publication year:2009