Abstract
Zinc has an important role in normal pancreatic beta cell physiology as it regulates gene transcription, insulin crystallization and secretion, and cell survival. Nevertheless, little is known about how zinc is transported through the plasma membrane of beta cells and which of the class of zinc influx transporters (Zip) is involved. Zip4 was previously shown to be expressed in human and mouse beta cells; however, its function there is still unknown. Therefore, the aim of this study was to define the zinc transport role of Zip4 in beta cells. To investigate this, Zip4 was over-expressed in MIN6 beta cells using a pCMV6-Zip4GFP plasmid. Organelle staining combined with confocal microscopy showed that Zip4 exhibits a widespread localization in MIN6 cells. Time-lapse zinc imaging experiments showed that Zip4 increases cytoplasmic zinc levels. This resulted in increased granular zinc content and glucose-stimulated insulin secretion. Interestingly, it is unlikely that the increased glucose stimulated insulin secretion was triggered by a modulation of mitochondrial function, as mitochondrial membrane potential remained unchanged. To define the role of Zip4 in-vivo, we generated a beta cell-specific knockout mouse model (Zip4BKO). Deletion of the Zip4 gene was confirmed in Zip4BKO islets by PCR, RT-PCR, and immuno-histochemistry. Zip4BKO mice showed slightly improved glucose homeostasis but no change in insulin secretion during an oral glucose tolerance test. While Zip4 was not found to be essential for proper glucose homeostasis and insulin secretion in vivo in mice, this study also found that Zip4 mediates increases in cytoplasmic and granular zinc pools and stimulates glucose dependant insulin secretion in-vitro.
Highlights
The highest cellular zinc concentration in the body is within pancreatic beta cells where it plays an essential role in insulin processing, including biosynthesis, secretory granule maturation, and exocytosis [1, 2]
As GFP is fused to the c-terminal end of Zip4, it can be localized with a fluorescent capable microscope (Fig. 2A). quantitative real-time polymerase chain reaction (qPCR) was performed on control and Zip4 plasmid transfected MIN6 cells. qPCR demonstrated that transfection significantly increased expression of Zip4 (Fig. 2B; 3 independent experiments)
Because we observed that up-regulation of Zip4 in MIN6 cells produced an increase in cytosolic zinc accumulation along with a stimulation of insulin secretion in the presence of high glucose, we studied the function of Zip4 in vivo
Summary
The highest cellular zinc concentration in the body is within pancreatic beta cells where it plays an essential role in insulin processing, including biosynthesis, secretory granule maturation, and exocytosis [1, 2]. The essential role of zinc in regulating human beta cell function was first identified based on genome-wide association studies that identified zinc efflux transporter 8 (ZnT8) as a significant risk factor for type 2 diabetes [3,4,5,6,7]. While ZnT8, and zinc entry into insulin secretory granules, has been extensively characterized, our knowledge on how zinc enters beta cells through the plasma membrane, and the zinc influx transporters (Zip) involved is still limited. All that is known is that some zinc enters beta cells through voltage gated calcium channels [13]
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