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Over-expression of Slc30a8/ZnT8 selectively in the mouse ? cell impairs glucagon release and responses to hypoglycemia.

ABSTRACT: BACKGROUND:The human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic ? cell (Solomou et al., J Biol Chem 290(35):21432-42) have also implicated ZnT8 in the control of glucagon release. Up to now, however, the possibility that increased levels of the transporter in these cells may impact glucagon secretion has not been explored. METHODS:Here, we use a recently-developed reverse tetracyline transactivator promoter-regulated ZnT8 transgene to drive the over-expression of human ZnT8 selectively in the ? cell in adult mice. Glucose homeostasis and glucagon secretion were subsequently assessed both in vivo during hypoglycemic clamps and from isolated islets in vitro. RESULTS:Doxyclin-dependent human ZnT8 mRNA expression was apparent in both isolated islets and in fluorescence-activated cell sorting- (FACS) purified ? cells. Examined at 12 weeks of age, intraperitoneal glucose (1 g/kg) tolerance was unchanged in transgenic mice versus wild-type littermates (n?=?8-10 mice/genotype, p?>?0.05) and sensitivity to intraperitoneal insulin (0.75U/kg) was similarly unaltered in transgenic animals. In contrast, under hyperinsulinemic-hypoglycemic clamp, a ~45 % (p??0.05). Over-expression of ZnT8 in glucagonoma-derived ?TC1-9 cells increased granule free Zn(2+) concentrations consistent with a role for Zn(2+) in this compartment in the action of ZnT8 on glucagon secretion. CONCLUSIONS:Increased ZnT8 expression, and a likely increase in intragranular free Zn(2+) concentration, is deleterious in pancreatic ? cells for stimulated glucagon release. These data provide further evidence that type 2 diabetes-associated polymorphisms in the SLC30A8/ZnT8 gene may act in part via alterations in glucagon release and suggest that ZnT8 activation may restrict glucagon release in some settings.

SUBMITTER: Solomou A

PROVIDER: S-EPMC4936320 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Over-expression of Slc30a8/ZnT8 selectively in the mouse α cell impairs glucagon release and responses to hypoglycemia.

Solomou Antonia A Philippe Erwann E Chabosseau Pauline P Migrenne-Li Stephanie S Gaitan Julien J Lang Jochen J Magnan Christophe C Rutter Guy A GA

Nutrition & metabolism 20160707

<h4>Background</h4>The human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic α cell (Solomou et al., J Biol Chem 290(35):21432-42) ...[more]

PMID: 27390586

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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

Over-expression of Slc30a8/ZnT8 selectively in the mouse ? cell impairs glucagon release and responses to hypoglycemia.

Publications

Over-expression of Slc30a8/ZnT8 selectively in the mouse α cell impairs glucagon release and responses to hypoglycemia.

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