Dataset Information

ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and ?-Cell Phenotype via ZIP-ZnT8 Coregulation.

ABSTRACT: The zinc transporter ZnT8 (SLC30A8) localises to insulin secretory granules of ?-cells where it facilitates zinc uptake for insulin crystallisation. ZnT8 abundance has been linked to ?-cell survival and functional phenotype. However, the consequences of ZnT8 haploinsufficiency for ?-cell zinc trafficking and function remain unclear. Since investigations in human populations have shown SLC30A8 truncating polymorphisms to decrease the risk of developing Type 2 Diabetes, we hypothesised that ZnT8 haploinsufficiency would improve ?-cell function and maintain the endocrine phenotype. We used CRISPR/Cas9 technology to generate ZnT8 haploinsufficient mouse MIN6 ?-cells and showed that ZnT8 haploinsufficiency is associated with downregulation of mRNAs for Slc39a8 and Slc39a14, which encode for the zinc importers, Znt- and Irt-related proteins 8 (ZIP8) and 14 (ZIP14), and with lowered total cellular zinc content. ZnT8 haploinsufficiency disrupts expression of a distinct array of important ?-cell markers, decreases cellular proliferation via mitogen-activated protein (MAP) kinase cascades and downregulates insulin gene expression. Thus, ZnT8 cooperates with zinc importers of the ZIP family to maintain ?-cell zinc homeostasis. In contrast to the hypothesis, lowered ZnT8 expression reduces MIN6 cell survival by affecting zinc-dependent transcription factors that control the ?-cell phenotype.

SUBMITTER: Lawson R

PROVIDER: S-EPMC6861948 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and β-Cell Phenotype via ZIP-ZnT8 Coregulation.

Lawson Rebecca R Maret Wolfgang W Hogstrand Christer C

International journal of molecular sciences 20191104 21

The zinc transporter ZnT8 (<i>SLC30A8</i>) localises to insulin secretory granules of β-cells where it facilitates zinc uptake for insulin crystallisation. ZnT8 abundance has been linked to β-cell survival and functional phenotype. However, the consequences of ZnT8 haploinsufficiency for β-cell zinc trafficking and function remain unclear. Since investigations in human populations have shown <i>SLC30A8</i> truncating polymorphisms to decrease the risk of developing Type 2 Diabetes, we hypothesis ...[more]

PMID: 31690008

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Project description:The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn2+/H+ antiporter. We also purified E. coli-expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Dataset Information

ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and ?-Cell Phenotype via ZIP-ZnT8 Coregulation.

Publications

ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and β-Cell Phenotype via ZIP-ZnT8 Coregulation.

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