Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

Abstract

OBJECTIVEZinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.RESEARCH DESIGN AND METHODSSlc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols.RESULTSZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay.CONCLUSIONSZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

A low-risk ZnT-8 allele (W325) for post-transplantation diabetes mellitus is protective against cyclosporin A-induced impairment of insulin secretion

“…More efficient accumulation of zinc in secretory granules in cells expressing ZnT-8 W325, as demonstrated by previous studies, 13,25 appears to account for this effect. Importantly, the different effects of the two variants on insulin secretion became more pronounced when b-cell insulin secretion was suppressed by CsA.…”

“…In addition, measurement of mRNA expression using real-time PCR showed that neither CsA nor high glucose had a measurable effect on insulin mRNA expression, at least in the experimental time frame used for GSIS study (Figure 3b). As the zinc transport activity of ZnT-8 has been positively correlated with insulin-secreting capacity, 11,14,25 we performed a vesicle transport assay to determine whether the polymorphism confers differential zinc transport activity. Vesicles expressing ZnT-8 W325 showed increased transport of 65 Zn into vesicles when compared with those expressing ZnT-8 R325 (Figure 4).…”

“…This is consistent with a previous study showing that ZnT-8 W325 increased the uptake of zinc into secretory vesicles compared with R325, as identified by zinquin fluorescent dye. 25 To further characterize the mechanism by which the ZnT-8 polymorphism affects insulin secretory capacity, we imaged insulin vesicle fusion events using total internal reflection fluorescence microscopy. Secretory vesicles containing EGFP-tagged insulin underwent fusion exocytosis upon glucose stimulation.…”

“…12 A previous study showed that uptake of zinc into secretory vesicles was increased in cells expressing ZnT-8 W325 compared with those expressing R325, as demonstrated using zinquin fluorescent dye. 25 Although we adopted a different zinc transport assay, the in vitro vesicle 65 Zn uptake assay, we obtained the same results as those from the zinc fluorescence-based cell assay. This finding strongly supports the conclusion that ZnT-8 W325 is more efficient in promoting zinc ion uptake into insulin vesicles, which might contribute to enhanced insulin secretion.…”

Zinc and diabetes

“…A crystal structure of mammalian ZnT8 is not yet available; consequently, the data on protein structure-function relationships and effect of the SNPs on them are based on the bacterial homolog YiiP, which has 51.8% sequence homology with the mammalian protein [74,75]. Arginine/Tryptophan-325 is located in an area involved in transporter homodimerization but as the side chains point away from the dimerization interface, it is unlikely that the R325W substitution prevents dimerization or Zn 2+ binding (Figure 2) [76,77].However, it is conceivable that the positive charge of the R-side chain may hamper molecular interactions, for instance with unidentified Zn 2+ -binding proteins which might interact with ZnT8 to facilitate zinc transport. Supporting this view, we found by overexpressing either isoform in a β cell line and measuring zinc in vesicles with Zinquin that in presence of supraphysiological zinc concentration, zinc accumulation into granules was significantly higher for W-versus R-form.…”