Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes

Abstract

Edited by F. Peter GuengerichZinc is a critical element for insulin storage in the secretory granules of pancreatic beta cells. The islet-specific zinc transporter ZnT8 mediates granular sequestration of zinc ions. A genetic variant of human ZnT8 arising from a single nonsynonymous nucleotide change contributes to increased susceptibility to type-2 diabetes (T2D), but it remains unclear how the high risk variant (Arg-325), which is also a higher frequency (>50%) allele, is correlated with zinc transport activity. Here, we compared the activity of Arg-325 with that of a low risk ZnT8 variant (Trp-325). The Arg-325 variant was found to be more active than the Trp-325 form following induced expression in HEK293 cells. We further examined the functional consequences of changing lipid conditions to mimic the impact of lipid remodeling on ZnT8 activity during insulin granule biogenesis. Purified ZnT8 variants in proteoliposomes exhibited more than 4-fold functional tunability by the anionic phospholipids, lysophosphatidylcholine and cholesterol. Over a broad range of permissive lipid compositions, the Arg-325 variant consistently exhibited accelerated zinc transport kinetics versus the Trp-form. In agreement with the human genetic finding that rare loss-offunction mutations in ZnT8 are associated with reduced T2D risk, our results suggested that the common high risk Arg-325 variant is hyperactive, and thus may be targeted for inhibition to reduce T2D risk in the general populations.Zinc forms stable complexes with insulin hexamers, enabling crystalline insulin packing in secretory granules of pancreatic beta cells. Defective insulin secretion in the face of insulin resistance is a characteristic feature of T2D, 4 a complex multifactorial polygenetic disease with more than 80 T2D susceptibility loci/genes identified so far by genome-wide association studies (GWASs). A nonsynonymous single nucleotide polymorphism in SLC30A8 (rs13266634 C3 T), which causes an arginine to tryptophan change at position 325, is associated with increased risk of developing T2D (1). The risk allele is widespread in more than 50% of the population according to HapMap data (build 35). SLC30A8 encodes a granular zinc transporter known as ZnT8. In pancreatic beta cells, ZnT8 is highly expressed and responsible for transporting cytosolic zinc into insulin granules (2). However, the molecular mechanism underlying the genetic susceptibility of ZnT8 polymorphisms remains controversial. ZnT8 inactivation in various mouse models revealed large phenotypic variations ranging from decreased, unchanged to even enhanced insulin secretion (3). Functional characterization of overexpressed polymorphic alleles in pancreatic beta cells suggested an attenuated zinc transport activity associated with an increased T2D susceptibility (4, 5), whereas genotyping rare nonsense and frameshift mutations of ZnT8 in humans indicated an opposite causal relationship, suggesting that a reduction of ZnT8 expression actually decreased T2D risk (6). The conflicting results conce...

Exploring the Association Between Demographics, SLC30A8 Genotype, and Human Islet Content of Zinc, Cadmium, Copper, Iron, Manganese and Nickel

“…The results reported by Merriman et al support this notion 27 . However, a potential additional role of ZnT8 at the level of the cell membrane cannot be ruled out.…”

Coupling of Insulin Secretion and Display of a Granule-resident Zinc Transporter ZnT8 on the Surface of Pancreatic Beta Cells

“…The folding of the tagged-ZnT8 in the detergent extract of stably transfected INS-1E cells was examined by fluorescence size exclusion HPLC (FSEC). ZnT8 with a C-terminal GFP fusion tag (termed ZnT8-GFP) revealed a major monodispersed GFP fluorescence peak as reported previously (34). Inserting a FLAG epitope at one of the nine sites on the ZnT8 extracellular surface yielded only one tagged construct that exhibited a monodispersed FSEC profile.…”

The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant

“…A recent report, in which the activity of ZnT8 reconstituted in liposomes was investigated, concluded that the transport activity is dependent on the lipid environment, and inferred that the lipid environment affects zinc loading during insulin granule biogenesis . This report also noted that the T2D‐risk R325 ZnT8 variant consistently showed a small increase in zinc transport activity compared to the T2D‐protective W325 variant, which was revealed only with specific lipid compositions of the liposomes.…”

“…Second, this is the first report detailing that the W325R mutation causes significant differences in ZnT8 CTD dimer formation and stability. How or even if these effects in the CTDs lead to the altered transport function of the full-length proteins reported elsewhere [9], and ultimately to the relatively large increased risk of developing T2D for carriers of the R325 variant, will require further investigation. That the T2D-risk ZnT8 R325 variant is the more active form of the transporter suggests that people with the R325 variant may have an increased zinc content in their insulin granules as indicated by the data on human islets [41].…”