Molecular Genetic Regulation of Slc30a8/ZnT8 Reveals a Positive Association With Glucose Tolerance.

Zinc transporter 8 (ZnT8), encoded by SLC30A8, is chiefly expressed within pancreatic islet cells, where it mediates zinc (Zn(2+)) uptake into secretory granules. Although a common nonsynonymous polymorphism (R325W), which lowers activity, is associated with increased type 2 diabetes (T2D) risk, rare inactivating mutations in SLC30A8 have been reported to protect against T2D. Here, we generate and characterize new mouse models to explore the impact on glucose homeostasis of graded changes in ZnT8 activity in the β-cell. Firstly, Slc30a8 was deleted highly selectively in these cells using the novel deleter strain, Ins1Cre. The resultant Ins1CreZnT8KO mice displayed significant (P < .05) impairments in glucose tolerance at 10 weeks of age vs littermate controls, and glucose-induced increases in circulating insulin were inhibited in vivo. Although insulin release from Ins1CreZnT8KO islets was normal, Zn(2+) release was severely impaired. Conversely, transgenic ZnT8Tg mice, overexpressing the transporter inducibly in the adult β-cell using an insulin promoter-dependent Tet-On system, showed significant (P < .01) improvements in glucose tolerance compared with control animals. Glucose-induced insulin secretion from ZnT8Tg islets was severely impaired, whereas Zn(2+) release was significantly enhanced. Our findings demonstrate that glucose homeostasis in the mouse improves as β-cell ZnT8 activity increases, and remarkably, these changes track Zn(2+) rather than insulin release in vitro. Activation of ZnT8 in β-cells might therefore provide the basis of a novel approach to treating T2D.

The regulation of insulin secretion by glucose involves the uptake and metabolism of the sugar by pancreatic β-cells (1), stimulation of mitochondrial oxidative metabolism (2), Ca2+ influx (3), and the exocytosis of the hormone from dense core secretory granules (4, 5) where it is stored in a near-crystalline form alongside Zn2+ and Ca2+ ions (6). Although it is increasingly accepted that impaired insulin secretion underlies the development of type 2 diabetes (T2D) (7), a disease affecting more than 8% of the adult population worldwide (8), the mechanisms involved remain poorly understood (9). Nonetheless, disease risk is strongly influenced by both genetic (10) and environmental (11) factors.

A nonsynonymous variant in the SLC30A8 gene associated with elevated T2D risk was identified by genome-wide association studies (GWAS) in 2007 (12). Expressed almost exclusively in pancreatic β- and α-cells (13–15), SLC30A8 encodes a secretory granule-resident zinc transporter, zinc transporter 8 (ZnT8), implicated in the accumulation of zinc within these organelles and thus in insulin storage (16). Given these likely roles, SLC30A8/ZnT8 has been mooted as a potentially tractable new target for personalized disease therapy.

Subsequent functional studies on the expressed ZnT8 protein (14, 17) demonstrated that the risk (R325) variant is a less active zinc transporter than the protective (W) form. Consequently, possession of risk alleles seems likely to impair insulin crystallization and storage. Supporting this view, mice inactivated globally (14, 18) or selectively in the β-cell (15, 19) for Slc30a8 revealed striking abnormalities in the formation of dense cores within insulin granules. Surprisingly, however, measurements of insulin release from isolated islets from Slc30a8 null mice revealed either no change (18) or improved (14, 19) glucose-stimulated insulin secretion from isolated islets or the perfused pancreas, and unchanged insulin content. Despite this, glucose homeostasis and circulating insulin levels were both lowered in ZnT8 null animals. Providing a possible explanation for this conundrum, Tamaki et al (19) demonstrated that the enhanced release of Zn2+ ions alongside insulin in W-variant carriers suppresses insulin clearance (and presumably nonproductive insulin signaling) by the liver, favoring insulin action on this, as well as other tissues (notably, adipocytes and skeletal muscle). An observed increase in C-peptide to insulin ratio in human R-carriers supported this model, because the mature hormone, but not proinsulin, is expected to be cleared by the liver. Moreover, Slc30a8 elimination from the mouse has no effect on insulin processing (14, 18), arguing against a β-cell-autonomous action of the variant on the release of mature vs partially processed forms. Together, the above findings have stimulated the search for activators of the transporter which, by favoring Zn2+ accumulation by β-cell secretory granules, may eventually prove useful in the clinic.

However, and challenging the above view, a recent study based largely on Swedish, Finnish, and other Northern European populations, but also including individuals from elsewhere, identified rare (<0.1% of the population) nonsense (truncating) or missense mutations in the SLC30A8 gene. Unexpectedly, the carrier population showed an approximately 3-fold enrichment for healthy individuals vs those with T2D, implying a protective role for the mutant transporter. Although only a small number of carriers was involved (345 in total of ∼150 000 subjects sequenced) a range of structurally distinct variants was found in cohorts with differing ancestry, providing evidence that the SLC30A8 mutations, rather than other polymorphisms in the same linkage disequilibrium block, were likely to explain the changes in disease risk.

The above findings are nonetheless difficult to reconcile with the observed increase in T2D risk in carriers of the common risk alleles. Although an activating effect of the identified mutants on the remaining allele cannot be excluded absolutely, an alternative explanation (20) is that a complex interplay between insulin storage and Zn2+ release by β-cells, and downstream effects on target tissues including the liver, results in a bimodal (bell-shaped) dependence of T2D risk on ZnT8 activity. Thus, modest decreases in β-cell ZnT8 activity, as observed in carriers of the common risk (R) variants, may act chiefly by lowering β-cell Zn2+ secretion, thus enhancing insulin clearance by the liver. On the other hand, a more substantial lowering of ZnT8 activity, engendered by rare loss-of-function alleles, may lead to a more dramatic increase in insulin release from the pancreas, an effect outweighing impaired Zn2+ release and altered insulin clearance.

The impact of deleting ZnT8 from the β-cell in mice has also been the subject of some debate. Thus, one recent study (21) reported that global knockout on a pure C57BL6 background exerted no effects on glucose tolerance, in contrast to findings on more mixed backgrounds (14, 18). Moreover, several previously reported β-cell-selective deletion models are complicated by deletion in other tissues, including the brain, when Cre deleter strains (notably, RIP2Cre and Pdx1), with activity in these tissues (22), are used. Correspondingly, RIP2Cre:ZnT8 mice gain more weight vs controls on a high fat diet than observed with globally deleted animals (23). The latter findings argue for a role for ZnT8 in a small number of neuronal cells in which the Pdx1 or Ins2 promoter may be at least transiently active during development or at later stages. On the other hand, the mouse insulin promoter 1 Cre (MIPCre) used in Ref. 19 may also be affected by the coexpression of GH encoded by the cDNA included in this transgene (24).

Our first goal here was therefore to explore the impact of deleting ZnT8 more specifically in the β-cell, and on a pure C57BL6 background, using a new deleter strain in which the Ins1 promoter, which is inactive in brain and other tissues (22), drives expression of Cre after introduction into the endogenous locus (“knock-in”) (25, 26). Importantly, Ins1Cre mice do not express the GH minigene, unlike both RIP2Cre (24) and MIPCre mice (27), and mice bearing the transgene alone display no abnormalities in glucose tolerance (22) (Rutter, G.A., unpublished results).

Up to now, there have been no attempts to examine the effect of overexpressing ZnT8 selectively in the β-cell, thus mimicking one of the likely actions of agents capable of stimulating the activity of the transporter. Our second goal here was therefore to generate a series of transgenic mouse lines in which ZnT8 expression is under the control of rat insulin promoter Tet-On system (28).

We demonstrate that highly selective deletion of ZnT8 in the β-cell leads to dense core granule misformation and glucose intolerance. By contrast, overexpression of the transporter in the β-cell in adults leads to improved glucose tolerance but reduced insulin secretion, whereas Zn2+ release is markedly enhanced. A positive relationship thus pertains between β-cell ZnT8 expression (and Zn2+ secretion), and glucose tolerance. If reflective of human physiology, these results lend weight to the view that ZnT8 activation might prove beneficial in the context of T2D.

Results

Impaired glucose tolerance and insulin secretion in Ins1Cre:ZnT8fl/fl mice

β-Cell-selective deletion of ZnT8 with a variety of Cre deleter strains (eg, RIP2 [15] and MIP [19]) display varying degrees of recombination at extrapancreatic sites, due to ectopic expression of Cre. By contrast, Ins1Cre knockin mice display no detectable expression of the recombinase in the brain, only very minor recombination in other islet cells (<3% of α-cells in utero), but more than 94% recombination in β-cells (25, 29). We therefore used this model to inactivate ZnT8 selectively in β-cells (Figure 1A). Confirming efficient deletion of the endogenous ZnT8 alleles in the β-cell with Ins1Cre, islets from Ins1Cre+/−:ZnT8fl/fl (Cre+) mice showed more than 80% reduction in ZnT8 mRNA levels (**, P < .01; two-way ANOVA; Cre− vs Cre+; n = 3 and 4, respectively) compared with litter mate controls (Ins1 Cre−/−:ZnT8fl/fl; Cre−), with no changes in the expression of other ZnT family members (Figure 1B). Loss of ZnT8 immunoreactivity was seen specifically in the β-cell, and not the α-cell, compartment, as demonstrated using immunohistochemical analysis of isolated islets, staining for insulin and glucagon, respectively (Figure 1C), and counting the number of ZnT8 positive cells colocalized with insulin and glucagon (Figure 1D). A decrease in overall immunoreactivity of more than 90% for monomeric ZnT8 was shown using Western (immuno)blotting analysis (Figure 1E) compatible with an islet composition of approximately 70%–80% β-cells (30) and levels of ZnT8 expression in α-cells about 50% of those in β-cells (Figure 1C and Ref. 14) .

Figure 1.

Ins1Cre-mediated deletion of ZnT8 in pancreatic β-cells. Mice carrying a LoxP site together with a flippase recognition target-flanked neomycin selection cassette within intron 1, and a single distal LoxP site within the upstream exon 1 containing the translational start codon, were bred with the Ins1Cre deleter strain, leading to the removal of exon 1 of Slc30A8/ZnT8 (A). Ins1Cre-mediated deletion of ZnT8 resulted in an approximate 80%–90% reduction in Slc30a8 expression (B) (**, P < .001 vs 3 Cre−, 4 Cre+), with no significant changes in the expression of other ZnT family members (P > .05 by two-way ANOVA). Gene expression was normalized to β-actin, and fold change gene expression was determined using 2−ΔΔCT. B, ZnT8 protein expression is reduced in islets from Cre+ animals, as shown by immunofluorescence staining, which demonstrates deletion specifically in β-cells (n = 90) but not α-cells (n = 24) (C and D). Deletion of ZnT8 revealed by Western (immuno)blotting of isolated islets from Ins1Cre+/−:ZnT8fl/fl mice and controls (E). Values represent mean ± SEM. Scale bar in C, 12.5 μm.

Maintained on a regular chow diet, male Ins1Cre+/−:ZnT8fl/fl mice displayed normal glucose tolerance at 6 weeks of age (not significant [ns]; repeated measures two-way ANOVA; n = 8 Cre− and n = 14 Cre+, respectively) (Figure 2A) but impaired responses to the sugar by 10 weeks (11.5 ± 0.59 vs 13.6 ± 0.74 mmol/L; Cre− vs Cre+; P < .05; 30-min time point; repeated measures two-way ANOVA; n = 8 and 11, respectively) (Figure 2B). These changes gradually resolved with age (ns; repeated measures two-way ANOVA; n = 7 Cre− and n = 10 Cre+) (Figure 2C). Female knockout (KO) mice showed no evident abnormalities at either age (Supplemental Figure 1, A–F). Ins1Cre+/−:ZnT8fl/fl (aged 10 wk) showed significantly higher glucose responses (P < .001 15- and 30-min time point, repeated measures two-way ANOVA, n = 9 Cre− and n = 12 Cre+) (Figure 2D) but lower insulin responses (0.70 ± 0.073 vs 0.49 ± 0.072; Cre− vs Cre+; P < .05; 30-min time point; repeated measures two-way ANOVA; n = 14 Cre− and n = 13 Cre+) (Figure 2E) in response to a 3-g/kg bodyweight glucose injection, consistent with impaired insulin secretion or enhanced clearance of the hormone. Insulin sensitivity measured using an insulin tolerance test was unchanged in both male (Figure 2F) and female (Supplemental Figure 2) Ins1Cre+/−:ZnT8fl/fl mice, as assessed at 10 or 8 weeks, respectively. These findings are in line with those in global (14) or mouse insulin 1 promoter-deleted animals (19).

Figure 2.

In vivo assessment of glucose homeostasis in Ins1CreZnT8KO mice. Intraperitoneal glucose tolerance test (IPGTT) and AUC of 6-week-old (A), 10-week-old (*, P < .05, 30-min time point) (B), and 14-week-old (C) male Ins1Cre+/−ZnT8fl/fl (Cre+) and littermate control (Ins1CreZnT8−/−ZnT8fl/fl, Cre−) mice. Animals were injected with 1-g/kg bodyweight glucose and blood glucose measured at time point 0, 15, 30, 60, 90, and 120 minutes after glucose injection (n = 7–14 animals). Glucose (*** P < .01, 15-min time point; ****, P < .001, 30-min time point) (D) and insulin (*, P < .05, 30-min time point) (E) responses of 10-week-old male Ins1CreZnT8 mice after 3-g/kg bodyweight glucose injection (n = 13–14 animals per genotype). F, Insulin tolerance test of 10-week-old Ins1CreZnT8 and littermate control male mice. Animals were injected with 0.75-U/kg bodyweight insulin and blood glucose measured as per IPGTT. Numbers in solid bars in the histograms indicate the number of animals studied. Values are mean ± SEM.

Unchanged glucose-, incretin-, and KCl-stimulated insulin secretion but altered Zn2+ dynamics in isolated Ins1Cre+/−:ZnT8fl/fl islets

Islets isolated from 10-week-old male Ins1Cre+/−:ZnT8fl/fl mice showed no change with respect to control islets in in vitro insulin secretion in response to 16.7 mmol/L glucose, incretin or depolarization induced with KCl (ns; two-way ANOVA; n = 12–16 replicates per genotype) (Figure 3A). Secretion in response to lower (8 mmol/L) glucose concentrations was also unchanged (Supplemental Figure 3A) and, similarly, glucose-stimulated insulin release was not different between null and wild-type islets assayed during perifusion at 16.7 mmol/L glucose (Supplemental Figure 3B). Likewise, deletion of ZnT8 did not affect the amplitude (0.50 ± 0.05 vs 0.47 ± 0.06; Cre− vs Cre+; ns; Student's t test; n = 17 and 9 islets, respectively) or area under the curve (AUC) (1132 ± 19.7 vs 1107 ± 24.2; Cre− vs Cre+; ns; Student's t test; n = 17 and 9 islets, respectively) of glucose (Figure 3B) or KCl-stimulated intracellular free Ca2+ ([Ca2+]i) increases (amplitude, 1.38 ± 0.11 vs 1.23 ± 0.15; AUC, 443 ± 9.71 vs 450 ± 17.;3 Cre− vs Cre+; ns; Student's t test; n = 5 and n = 13 islets, respectively) (Figure 3C). Finally, β-cell-β-cell connectivity (31), known to contribute to the regulation of insulin release from intact islets, was unaltered in ZnT8 null islets (Figure 3, D and E).

Figure 3.

In vitro assessment of islets isolated from Ins1CreZnT8KO mice. Insulin secretion from 5 size-matched islets was assessed using a Homogenous Time-Resolved Fluorescence-based assay (Materials and Methods). Briefly, islets were pretreated at 3mM glucose for 1 hour at 37°C before being exposed to either 3mM glucose (G3), 16.7mM glucose (G16.7), 16.7mM glucose plus 20nM GLP-1 (GLP-1), or 3mM glucose + 30mM KCl (KCl) for 30 minutes at 37°C. Secreted insulin was determined and, after normalization to total insulin, expressed as fold change vs the 3mM glucose condition (A). [Ca2+]i dynamics were assessed using Nipkow spinning disk microscopy. No significant differences were seen in either the amplitude or the AUC of glucose-evoked (17mM; G17) whole-islet Ca2+ rises (ns vs Cre−; Student's t test) (B). The number of glucose-responsive cells was unchanged between Cre− and Cre+ mice (data not shown). Likewise, there were no differences seen in KCl-induced (30mM KCl) [Ca2+]i rises (ns vs Cre−; Student's t test) (C). Correlation analyses of glucose-evoked Ca2+ traces (32) showed no difference in β-cell-β-cell connectivity in Cre+ islets vs Cre− islets at both low (3mM) and high (11mM) glucose, indicating maintained cell quiescence and synchronicity, respectively. D, Connectivity map depicting location, number, and strength (color coded; 0 [blue] = lowest, 1 [red] = highest) of significantly correlated cell pairs (E). Values represent mean ± SEM.

We next used the recombinant Förster resonance energy transfer (FRET)-based probe eCALWY4 (32, 33) to measure cytosolic Zn2+ concentrations. Consistent with findings in global ZnT8 null mice (34), Ins1Cre+/−:ZnT8f/lfl β-cells showed a significant reduction in free cytosolic Zn2+ concentration (920 0.3 ± 261pM vs 212.8 ± 32.5pM; **, P < .01, Cre− vs Cre+, Student's t test, n = 20 and 11 islets, respectively) (Figure 4, A–C). Furthermore, use of the cell surface-targeted Zn2+ binding probe zinc indicator for monitoring induced exocytotic release (ZIMIR) (35), to measure Zn2+ cosecreted from insulin granules, demonstrated that Ins1Cre+/−:ZnT8fl/fl islets secreted substantially less Zn2+ compared with control islets after stimulation with glucose (Figure 4D).

Figure 4.

Intracellular zinc dynamics and secretion in control in Ins1CreZnT8KO mouse β-cells. To measure cytosolic free Zn2+ levels, isolated islets were dispersed and infected with an adenovirus expressing the Zn2+-sensitive FRET probe eCALWY4 (34) (A). Steady-state fluorescence intensity ratio (citrine to cerulean) was first measured 1) before obtaining the Rmax, 2) under perifusion with Krebs-Henseleit Buffer buffer containing the zinc chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (50μM; zinc-free condition). Finally, 3) the Rmin was obtained under perfusion with Krebs-Henseleit Buffer buffer containing 5μM pyrithione and 100μM Zn2+ (zinc-saturated condition), providing saturating intracellular Zn2+ concentrations (B). The free cytosolic concentration of Zn2+ (C) was calculated using the next formula: [Zn2+] = Kd (Rmax − R)/(R − Rmin), revealing significant decreases in cytosolic free zinc levels in Ins1Cre:ZnT8fl/fl animals compared with littermate controls (**, P < .01, Cre+ vs Cre−, respectively, n = 20 Cre+ and n = 11 Cre− islets). Zinc secretion from isolated islets, using the zinc binding probe zinc indicator for monitoring induced exocytotic release (ZIMIR), was decreased in Cre+ islets as shown by significant decreases in both the amplitude (****, P < .0001, Student's t test) and AUC (*, P < .05, Student's t test) of glucose-evoked ZIMIR responses; n = 22 Cre− islets and n = 17 Cre+ islets (D).

Ins1Cre+/−:ZnT8f/f mice show altered insulin granule morphology but preserved β-cell mass

The above metabolic and cellular disturbances were accompanied by a drastic change in secretory granule morphology (Figure 5A), with a near-complete loss of dense core granules in islets from Ins1Cre+/−:ZnT8fl/fl mice, and the emergence of “atypical” granules possessing abnormal “rod-like” (0.65 ± 0.28% vs 31.9 ± 3.42%, Cre− vs Cre+, P < .0001, n = 8 β-cells/genotype) or “empty” (36.3 ± 2.48% vs 85.6 ± 1.37%, Cre− vs Cre+, P < .0001, n = 8 β-cells/genotype) cores (Figure 5B). The total number of granules was unchanged (129 ± 9.25 vs 128 ± 11.9; Cre− vs Cre+; ns; Student's t test; n = 8 β-cells/genotype) (Figure 5C), but average granule diameter was increased (377.47 ± 5.12 vs 328.09 ± 5.46 nm, Cre+ vs Cre−, P < .001, n = 204/226 granules) (Figure 5D), likely reflecting increased osmotic stress resulting from free electrolytes in the ZnT8 null granule (18). Staining pancreatic slices for insulin and glucagon (Figure 5E) showed no changes in total β-cell mass (0.513 ± 0.07% vs 0.334 ± 0.17%; Cre− vs Cre+; ns; Student's t test; n = 3 animals per genotype) (Figure 5F), α-cell mass (0.055 ± 0.015% vs 0.051 ± 0.019%; Cre− vs Cre+; ns; Student's t test; n = 3 animals per genotype) (Figure 5G) or α- to β-cell ratio (0.151 ± 0.011 vs 0.206 ± 0.025; Cre− vs Cre+; ns; Student's t test; n = 3 animals per genotype) (Figure 5H). These data are in line with previous findings using alternative deleter strains to eliminate ZnT8 from the β-cell (14, 18, 36).

Figure 5.

Ins1CreZnT8KO mice exhibit abnormal granule morphology but unchanged β-cell mass. Transmission electron microscopic images of β-cells from islets isolated from Cre− (upper) or Cre+ (lower) animals show altered dense core granule structure. Scale bar, 1 μm. A, Insulin granules were grouped into 3 categories according to morphological structure and counted. Cre+ animals showed a significant reduction in granules containing a dense core coupled with significant increases in granules showing a rod like structure or a gray interior (***,

Ins1CreZnT8KO mice exhibit abnormal granule morphology but unchanged β-cell mass. Transmission electron microscopic images of β-cells from islets isolated from Cre− (upper) or Cre+ (lower) animals show altered dense core granule structure. Scale bar, 1 μm. A, Insulin granules were grouped into 3 categories according to morphological structure and counted. Cre+ animals showed a significant reduction in granules containing a dense core coupled with significant increases in granules showing a rod like structure or a gray interior (***,