Combined Deletion of Slc30a7 and Slc30a8 Unmasks a Critical Role for ZnT8 in Glucose-Stimulated Insulin Secretion

Abstract

Polymorphisms in the SLC30A8 gene, which encodes the ZnT8 zinc transporter, are associated with altered susceptibility to type 2 diabetes (T2D), and SLC30A8 haploinsufficiency is protective against the development of T2D in obese humans. SLC30A8 is predominantly expressed in pancreatic islet β-cells, but surprisingly, multiple knockout mouse studies have shown little effect of Slc30a8 deletion on glucose tolerance or glucose-stimulated insulin secretion (GSIS). Multiple other Slc30a isoforms are expressed at low levels in pancreatic islets. We hypothesized that functional compensation by the Slc30a7 isoform, which encodes ZnT7, limits the impact of Slc30a8 deletion on islet function. We therefore analyzed the effect of Slc30a7 deletion alone or in combination with Slc30a8 on in vivo glucose metabolism and GSIS in isolated islets. Deletion of Slc30a7 alone had complex effects in vivo, impairing glucose tolerance and reducing the glucose-stimulated increase in plasma insulin levels, hepatic glycogen levels, and pancreatic insulin content. Slc30a7 deletion also affected islet morphology and increased the ratio of islet α- to β-cells. However, deletion of Slc30a7 alone had no effect on GSIS in isolated islets, whereas combined deletion of Slc30a7 and Slc30a8 abolished GSIS. These data demonstrate that the function of ZnT8 in islets can be unmasked by removal of ZnT7 and imply that ZnT8 may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.

Figure 1.

Generation and analysis of islet zinc content and fasting metabolic parameters in ZnT7 KO and DKO mice. A, Schematic representation of the WT murine Slc30a7 locus and the targeting construct used to generate ZnT7 KO mice by homologous recombination in ES cells. Exons 1 and 2 were replaced with a cassette containing the LacZ gene and a TK-neomycin selectable marker. B, Southern blot analysis of the Slc30a7 locus using genomic DNA extracted from the indicated targeted ES cell lines, or WT ES cell gDNA, designated Lex-2, as a control, using 5′ and 3′ diagnostic probes (A). The sizes of the WT locus, targeted allele, and DNA markers are indicated. Clone 1E9 was used to achieve germline transmission. C, Zinc content in isolated approximately 17-week-old ZnT7 KO and DKO male mouse islets. Results represent the mean ± SEM (n = 3–14); P < .0001, one-way ANOVA; *, differences with WT. D–I, Phenotypic parameters in 6-hour fasted 16-week-old ZnT7 KO and DKO mice. Results are the mean ± SEM of data with the genotype, gender, and number of animals indicated. W, WT; H, HET; K, KO; DKO; F, female; M, male. F, P < .0001 and M, P = .001 (D); F, P < .0001 and M, P = .0002 (E); M, P = .0010 (F); M, P = .0007 (I); one-way ANOVA; *, differences with matching WT are indicated.

Figure 2.

Analysis of insulin sensitivity, glucose tolerance, and plasma insulin in ZnT7 KO and DKO mice. A and B, ITTs were performed on 21- to 24-week-old 5-hour fasted conscious male mice. Results show the mean glucose concentrations ± SEM expressed as mg/dL (A) or as a percentage of blood glucose at t = 0 (B). A, P = .0141, two-way ANOVA. C and D, OGTTs were performed on 6-hour fasted 20-week-old female (C) and male (D) conscious mice. Results show the mean glucose concentrations in tail blood ± SEM. C and D, P < .0001, two-way ANOVA. E, OGTTs were performed on 6-hour fasted 29- to 30-week-old male conscious mice. Results show the mean glucose and insulin concentrations in blood isolated from the retroorbital plexus ± SEM. Glucose and insulin data: P < .0001, two-way ANOVA; *, differences with WT; **, differences between ZnT7 KO and DKO; #, differences between t = 0 and t = 15. F and G, IPGTTs were performed on 6-hour fasted 10-week-old female (F) and male (G) conscious mice. Results show the mean glucose concentrations in tail blood ± SEM. F and G, P < .0001, two-way ANOVA; *, differences with WT; **, differences between ZnT7 KO and DKO. H, IPGTTs were performed on 6-hour fasted 22- to 25-week-old male conscious mice. Results show the mean glucose and insulin concentrations in blood isolated from the retroorbital plexus ± SEM. Glucose data, P < .0099, two-way ANOVA. Insulin data: P < .0017, two-way ANOVA; *, differences with WT; #, differences between t = 0 and t = 15.

Figure 3.

Analysis of pancreatic insulin content and islet structure in ZnT7 KO and DKO mice. A, Insulin content in whole pancreas from 20- to 24-week-old 6-hour fasted male mice. Results represent the mean ± SEM (n = 3); P = .0148, one-way ANOVA; *, differences with WT. B–D, β-Cell mass (B), isolated islet insulin content (C), isolated islet DNA content (D), β-cell size (E), and α- to β-cell ratio (F) in male mice. Pancreas and islet results represent the mean ± SEM; P = .0174, one-way ANOVA (B); P = .0029, one-way ANOVA (F); *, differences with WT. G, Immunofluorescent staining of 18-month-old male mouse pancreata with antisera raised to insulin and glucagon. Representative pictures (×15 magnifications) are shown. H, Analysis of insulin secretory granule structure in 12-week-old male mice using EM. Representative micrographs are shown.

Figure 4.

Analysis of insulin secretion in vitro in islets isolated from ZnT7 KO and DKO mice and the molecular basis for altered plasma insulin levels in vivo. A, GSIS in islets isolated from approximately 34-week-old male ZnT7 KO and DKO mice. Results show the mean data ± SEM from 5–10 islet preparations. P = .0046, two-way ANOVA; *, differences between WT and DKO; **, differences between ZnT7 KO and DKO; #, differences between 5mM and 16.7mM glucose. B, Ratio of plasma glucose-stimulated insulin at t = 15 during IPGTTs in 6-hour fasted 20- to 25-week-old male conscious mice relative to pancreatic insulin content. Results represent the mean ± SEM; P = .0125, one-way ANOVA; *, difference between WT and DKO; **, difference between ZnT7 KO and DKO. C and D, Hepatic glycogen content (C) and Gsy2 expression (D) in 6-hour fasted 28- to 36-week-old male mice. Results represent the mean ± SEM; P < .0001, one-way ANOVA (C); P = .0140, one-way ANOVA (D); *, differences with WT; **, differences between ZnT7 KO and DKO. E and F, IPGTTs (E) or OGTTs (F) were performed on 6-hour fasted 22- to 30-week-old conscious male mice. Results show the mean C peptide concentration in blood isolated from the retroorbital plexus ± SEM. Matching insulin data are from Figure 2, E and H. C peptide data: P = .0362 (E); C peptide data: P < .0001 (F); C peptide:insulin data: P = .0105 (F), two-way ANOVA; *, differences with WT; **, differences between ZnT7 KO and DKO; #, differences between t = 0 and t = 15. G, Plasma proinsulin levels in 6-hour fasted 29- to 30-week-old conscious male mice. Results represent the mean ± SEM. H, Absence of ZnT7:ZnT8 dimer formation as assessed using pulldown assays. Representative blots are shown. I, Arginine tolerance tests were performed on 6-hour fasted 28- to 34-week-old conscious male mice. Results show the mean glucose and insulin concentrations in blood isolated from the retroorbital plexus ± SEM. Glucose data: P = .0005; insulin data: P = .0161, two-way ANOVA; *, differences with WT; #, differences between t = 0 and t = 2.