Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells

Aims/hypothesis Hypoxic damage complicates islet isolation for transplantation and may contribute to beta cell failure in type 2 diabetes. Polymorphisms in the SLC30A8 gene, encoding the secretory granule zinc transporter 8 (ZnT8), influence type 2 diabetes risk, conceivably by modulating cytosolic Zn2+ levels. We have therefore explored the role of ZnT8 and cytosolic Zn2+ in the response to hypoxia of pancreatic islet cells. Methods Human, mouse or rat islets were isolated and exposed to varying O2 tensions. Cytosolic free zinc was measured using the adenovirally expressed recombinant targeted zinc probe eCALWY4. Gene expression was measured using quantitative (q)RT-PCR, western (immuno-) blotting or immunocytochemistry. Beta cells were identified by insulin immunoreactivity. Results Deprivation of O2 (1% vs 5% or 21%) for 24 h lowered free cytosolic Zn2+ concentrations by ~40% (p < 0.05) and ~30% (p < 0.05) in mouse and human islet cells, respectively. Hypoxia similarly decreased SLC30A8 mRNA expression in islets, and immunoreactivity in beta cells. Implicating lowered ZnT8 levels in the hypoxia-induced fall in cytosolic Zn2+, genetic ablation of Slc30a8 from mouse islets lowered cytosolic Zn2+ by ~40% (p < 0.05) and decreased the induction of metallothionein (Mt1, Mt2) genes. Cell survival in the face of hypoxia was enhanced in small islets of older (>12 weeks) Slc30a8 null mice vs controls, but not younger animals. Conclusions/interpretation The response of pancreatic beta cells to hypoxia is characterised by decreased SLC30A8 expression and lowered cytosolic Zn2+ concentrations. The dependence on ZnT8 of hypoxia-induced changes in cell survival may contribute to the actions of SLC30A8 variants on diabetes risk in humans. Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3266-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.


Introduction
Pancreatic beta cells are highly dependent on oxidative metabolism for ATP synthesis, particularly at elevated glucose concentrations [1,2]. Correspondingly, hypoxia has been shown to influence islet survival and function during transplantation [3,4]. Moreover, as many as 25% of islets are exposed in vivo to low oxygenation [5], suggesting that hypoxia acts as a regulator of islet function under physiological conditions. Indeed, glucose-induced oxygen consumption creates intracellular hypoxia sufficient to activate hypoxiainducible factors (HIFs) in rat beta cells [6], an effect that is increased in diabetic animals [7] and which may contribute to defective insulin secretion in some forms of type 2 diabetes.
Hypoxic stress induces genes such as metallothionein (MT1/2) as a defence against large changes in free metal ion concentration [8], which may affect the activity of antioxidative enzymes [9]. Whether genetic factors influence the susceptibility of pancreatic beta cells to hypoxia has not previously been explored. Suggesting this as a possibility, genome-wide association studies (GWAS) have revealed that a non-synonymous single nucleotide polymorphism (rs13266634) in the SLC30A8 gene (encoding the secretory granule-resident zinc transporter 8 [ZnT8]) is associated with a~20% increase in disease risk per allele [10][11][12]. SLC30A8 expression is largely confined to pancreatic beta and alpha cells [13], and is required for the accumulation of zinc into the secretory granule, where it binds to insulin [14,15]. Consequently, mice inactivated systemically for Slc30a8 display profound changes in insulin crystal formation and secretory granule morphology [14,15], consistent with the lower zinc-transporting activity of the transporter isoform encoded by the risk allele [15]. Defective insulin secretion is seen in global ZnT8 −/− mice on some [15], but not all, backgrounds [14,16,17], and mice with selectively deleted beta cell Slc30a8 display marked changes in insulin secretion and glucose tolerance [18]. Whether Slc30a8 influences cytosolic, as well as granular, Zn 2+ concentrations has not previously been examined because of the uncertain subcellular targeting of the probes used in earlier work [15].
Monitoring cytosolic Zn 2+ with the molecularly targeted recombinant probe eCALWY4 [19], the present study aimed to explore the impact of hypoxia on Zn 2+ homeostasis, and the expression of SLC30A8/ZnT8 and other zinc transporters and importers, in human and rodent beta cells.
Mouse and rat strains and maintenance Female CD1 mice and male Wistar rats were purchased from Harlan (Bicester, UK/Itingen, Switzerland). Global ZnT8 −/− mice [15] on a mixed SV129/C57BL/6 background and 129S7/ SvEvBrd-Mt1 tm1Bri Mt2 tm1Bri /J mice (purchased from Jackson Laboratory, Bar Harbor, ME, USA) have previously been described [20]. Mice were killed at 10-12 weeks (−15 weeks for cell death analysis) of age by cervical dislocation as approved by the UK Home Office Animal Scientific Procedures Act, 1986 (PPL 70/7349), with ethics approvals.
Islet isolation, culture, infection and dissociation Human islets were isolated from six beating-heart donors (electronic supplementary material [ESM] [21] or in Geneva, Switzerland [22], and maintained in RPMI medium containing 5.5 mmol/l glucose and 10% (vol./vol.) FCS. Mouse and rat islets were prepared as in Ravier and Rutter [23]. Islets were dissociated by 10 min incubation in Hanks'-based enzyme-free cell dissociation buffer (Gibco, Invitrogen, Paisley, UK), plated onto 24 mm sterile coverslips treated with 0.1% poly-L-lysine and allowed to recover overnight in RPMI medium containing 11 mmol/l glucose.
RNA extraction and qRT-PCR Total RNA from~50 islets was obtained using TRIzol reagent and reverse-transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems). cDNA (equivalent to 10-20 ng of RNA) was subject to qRT-PCR using Power SYBR Green master mix (Applied Biosystems/Life Technologies) in a 7500 fast real-time PCR system (Applied Biosystems/Life Technologies) and analysed by the comparative C t method (primers are shown in ESM Table 2). The expression of target genes was normalised to the expression of cyclophilin A.
Dead:live cell assay Islets were incubated for 15 min in PBS containing 3 μmol/l calcein-AM (Life Technologies) and 2.5 μmol/l propidium iodide (PI; Sigma-Aldrich) before detection of absorbance/emission at 491/525 nm and 561/620 nm, respectively. The islet area occupied by dead cells (PI) was calculated and expressed as a unitary ratio vs that occupied by all (live and dead) cells (PI plus calcein) using ImageJ (http://imagej.nih.gov/ij/) [25].  Transmission electron microscopy Isolated islets were fixed and analysed as previously described [26].
Insulin secretion and content Total and secreted insulin were measured after batch incubation of islets in KHB buffer as previously described [23].
Statistics Data are given as mean ± SE or relative frequency. For comparison of continuous variables in two independent groups, Student's two-tailed t test was used. Bonferroni correction was applied for multiple comparisons. Multiple linear regression was used to assess the effect of different factors on islet cell death. A value of p<0.05 was considered significant. Statistical analyses were performed using SPSS 18.0 software (SPSS, Chicago, IL, USA) and Excel 2010 software (Microsoft, Redmond, WA, USA).

Results
Exposure of pancreatic beta cells to hypoxia decreases cytosolic free Zn 2+ concentrations and Slc30a8/ZnT8 expression Given the previously described role of Zn 2+ ions in the responses of other tissues to hypoxia and ischaemia [27], we first determined whether lowered oxygen tensions might affect cytosolic free Zn 2+ concentrations ([Zn 2+ ] cyt ) in mouse or human islet cells by using eCALWY4. This recombinantly expressed probe is confined exclusively to the cytosolic compartment, and is expected chiefly to report changes in beta cells which predominate in the islet preparations used (>70% for rodent islets [28],~60% in human islets [29] [30] families) are involved in the hypoxia-induced changes in [Zn 2+ ] cyt , their expression was measured in control or hypoxia-exposed islets. Whereas exposure to hypoxia for 24 h exerted no significant effect on the expression of members of the latter family in both mice and rats (ESM Fig. 1a, b), marked decreases were observed in levels of mRNA encoding Slc30a8 in both mouse and human islets (Fig. 2a, b), as well as rat islets (ESM Fig. 1c). These changes were accompanied by the expected increase in the expression of the major islet metallothionein genes, MT1 (also known as MT1A) (Mt1) and MT2 (also known as MT2A) (Mt2) (Fig. 2b, c). Western blot analysis revealed a significant decrease in ZnT8 protein abundance during hypoxia exposure in mouse islets (Fig. 2d). Confirming that the above hypoxiainduced changes were unlikely to require the more severely hypoxic conditions that may exist in the islet core [3], or paracrine intra-islet signalling (e.g. by inflammatory signals) [31], similar changes in the expression of Slc30a8 and Mt1/2 were apparent in dissociated mouse and human islet cells after exposure to hypoxia for 24 h (Fig. 3a, b). Correspondingly, a hypoxia-induced decrease in the expression of ZnT8 at the protein level was readily revealed by immunocytochemical analysis of individual insulinpositive mouse beta cells (Fig. 3c). To exclude hyperoxiainduced Slc30a8 overexpression, we compared the effects of 1%, 5% and 21% oxygen concentrations. Exposure to 1%, but not 5%, ambient oxygen altered the expression of Slc30a8 compared with 21% ambient oxygen (ESM Fig. 2a). The hypoxia-induced changes in Slc30a8 mRNA levels, as well as cytosolic free Zn 2+ concentrations in mouse islets/islet cells, were largely reversible after 24 h of re-oxygenation (ESM Fig. 2b, c).
To exclude a decrease in cell viability as the chief reason for the reduced Slc30a8/ZnT8 expression, cell death was quantified 24 h after exposure to hypoxia. This analysis revealed a dead:live cell ratio of 12.4±1.9% and 3.9±1.2% in mouse and human islets, respectively, maintained under hypoxic conditions, compared with values of 0.7±0.2 and 0.4±0.1% in normoxia (ESM Fig. 3a, b). Correspondingly, hypoxia did not cause major changes in the morphology or ultrastructure of surviving islet cells as examined by electron microscopy (ESM Fig. 3c). As expected [32,33], characterisation of hypoxic islets revealed reduced glucose-stimulated insulin secretion: insulin secretion ratio (release at high [16.7 mmol/l] glucose vs low [3.3 mmol/l] glucose) was only 4% of the ratio in normoxia (ESM Fig. 3d). Similarly, KCl-induced insulin secretion was also reduced in hypoxia (ESM Fig. 3d).
We sought next to determine whether the hypoxia-induced changes in the expression of Slc30a8/ZnT8 might reflect more global cellular dedifferentiation and/or alterations in the ratio of different islet cell types. Arguing against these possibilities, the expression of beta cell-enriched genes including insulin 1 (Ins1) and glucokinase (Gck) (also present in alpha cells) [34] did not change significantly during hypoxia and reoxygenation treatment of mouse islets (Fig. 4a, b). By contrast, and as expected, large changes in the expression of the HIF1α targets GLUT1 (Slc2a1) and solute carrier family 16 (monocarboxylic acid transporters), member 3 (MCT4) (Slc16a3) were observed (Fig. 4c, d). Similar to the expression of Slc30a8 (Fig. 4e), expression of pancreatic duodenum homeobox-1 (Pdx1), a key transcription factor involved in the regulation of insulin genes and of beta cell differentiation [35], was markedly decreased by hypoxia (Fig. 4f), and this change was not as rapidly reversed as those for the mainly HIF1α-controlled genes (Slc2a1 and Slc16a3) or the metallothioneins (Mt1, Mt2) (Fig. 4g, h).
To further explore a possible role for HIF1α in controlling Slc30a8/ZnT8 expression, pancreatic islets were exposed to the HIF1α-stabilising agent DMOG for 24 h. DMOG treatment did not significantly affect Slc30a8 expression (ESM Fig. 4).
Regulation of Slc30a8/ZnT8 by hypoxia does not require changes in metallothionein gene expression The expression of Slc30a8/ZnT8 has previously been shown to be regulated by changes in intracellular free Zn 2+ [36], which in turn are expected to be influenced by alterations in metallothionein expression. We therefore determined whether the regulation of Slc30a8 expression by hypoxia might be diminished in islets from mice null for Mt1 and Mt2 [20]. Hypoxia still resulted in a marked decrease in Slc30a8 expression in Mt1 −/− :Mt2 −/− mice, to a similar extent to that observed in wild-type (WT) mouse islets (Fig. 5a). Further supporting a metallothioneinindependent effect of hypoxia on Slc30a8 expression in CD1 mouse islets, Slc30a8 mRNA levels tended to be decreased as early as 5 h after the initiation of hypoxia (significant decrease after 10 h) (Fig. 5b).

Hypoxia-induced metallothionein gene induction and cell death is modulated by ZnT8
To further explore the signalling pathway(s) involved in regulating metallothionein expression (c) Dispersed CD1 mouse islet cells were incubated for 24 h at normoxia (21%) or hypoxia (1%). Scale bar, 10 μm. The level of ZnT8 was quantified by calculating the fluorescence ratio of ZnT8 vs insulin (white bar, 21%; black bar, 1%). Bars represent mean ± SE. * p<0.05, ** p<0.01 and *** p<0.001 and free cytosolic Zn 2+ in response to hypoxia, we next asked whether inactivation of ZnT8 affected these responses. Induction of Mt1 and Mt2 by hypoxia was significantly impaired in ZnT8 −/− mouse islets, consistent with the lower [Zn 2+ ] cyt in the latter vs WT mouse islet cells (Fig. 6a, b). As expected, Mt1/ Mt2 expression was induced by exogenously added Zn 2+ ions in hypoxia (Fig. 6c). Similar to the observations in hypoxia, [Zn 2+ ] cyt was lower in islet cells from mice lacking ZnT8 vs WT islets (Fig. 6d). This difference between WT and ZnT8 −/− islets was no longer observed in hypoxia (Fig. 6e). Conversely, overexpression of Slc30a8 in the clonal INS1 (832/13) beta cell line increased [Zn 2+ ] cyt (Fig. 6f). ZnCl 2 was able to raise the expression of the most abundant metallothionein, Mt2, in hypoxic ZnT8 −/− mouse islets to levels similar to those of hypoxic WT islets (Fig. 6g). In addition to the regulation of metallothioneins, the expression of other genes important in the response of the beta cell to hypoxia was assessed (ESM Fig. 5). In contrast to metallothionein, there was a tendency for enhanced expression of these genes in ZnT8 −/− islets, reaching statistical significance for Slc16a3 (MCT4) when compared in WT and ZnT8 −/− islets during hypoxia. Insulin content did not significantly differ between WT and ZnT8 −/− mouse islets in normoxia or hypoxia (ESM Fig. 6).
In contrast to Mt1 −/− :Mt2 −/− islets, cell survival was not affected in a large sample of ZnT8 −/− mouse islets (Fig. 6h) exposed to hypoxia. Multiple linear regression revealed a significant effect of mouse age ( p=0.001) and islet size ( p<0.001), but not genotype, on islet cell survival. However, when assessed in a defined population of smaller islets (<120 μmol/l) from older mice (12-15 weeks), we observed a significant difference in islet cell survival with a survival advantage of ZnT8 −/− mouse islets after 24 h ( p<0.001) or 48 h of hypoxia exposure (p<0.01) (Fig. 6i).

Discussion
We demonstrate here that hypoxia strongly, but reversibly, regulates the expression of Slc30a8/ZnT8 in islets and beta cells from human and two rodent species. These findings thus extend the list of pathophysiological factors, currently including cytokines [31,37] and fatty acids [36], which regulate the expression of this type 2 diabetes risk gene in islets. Hypoxia might therefore contribute to the downregulation of Slc30a8 previously observed in human type 2 diabetes islets [38].
The present findings extend to the islet beta cell those of a recent report [39] showing that hypoxia lowers the expression of Slc30a8/ZnT8 in the retinal pigment epithelium of the eye. The latter studies [39] provided evidence for control of ZnT8 levels via HIF1 stabilisation. By contrast, in our hands, the HIF1α-stabilising agent DMOG tended only slightly to reduce ZnT8 mRNA levels. Instead, our results suggest that the effects of hypoxia on Slc30a8/ZnT8 expression in pancreatic islets are more complex and may conceivably involve changes in the expression of Pdx1, recently shown to control Slc30a8 expression in clonal beta cells [40].
Recently, Lefebvre and colleagues [36] reported that depletion of intracellular zinc reduced Slc30a8 expression in human islets, confirming findings in INS-1E cells [41]. These earlier observations thus raise the possibility that the lowered cytosolic Zn 2+ concentrations reported here during hypoxia may contribute to the lowering of Slc30a8/ZnT8 expression. Arguing against this view, addition of extracellular Zn 2+ (30 μmol/l ZnCl 2 ) did not rescue the lowering of Slc30a8 mRNA levels after hypoxia (ESM Fig. 7). Moreover, any direct or indirect (e.g. by alterations in cytosolic Zn 2+ ) effect of hypoxia-driven metallothionein gene induction also seems unlikely, as hypoxia-induced changes in Slc30a8 mRNA levels were not different in Mt1/Mt2 −/− mice compared with control animals (Fig. 5a).
We propose instead a model by which hypoxia initially depresses Slc30a8 transcription and/or mRNA stability by mechanisms which remain to be elucidated (as discussed below). This then leads to a lowering of cytosolic free Zn 2+ , and a consequent drop in Mt1/Mt2 expression. The above sequence of events is supported by the observation of reduced cytosolic Zn 2+ in ZnT8 −/− mice compared with controls (Fig. 6d), and by an increased cytosolic Zn 2+ concentration in clonal pancreatic beta cells when Slc30a8 was overexpressed (Fig. 6f ).
The observation that cytosolic free Zn 2+ is lowered by ablating Slc30a8, a mediator of Zn 2+ uptake into granules, may at first glance appear surprising. We would stress that the use here of a molecularly targeted cytosolic Zn 2+ probe excludes uncertainties over the subcellular compartment in which Zn 2+ concentrations are interrogated. So how might this apparent paradox be explained? First, our data suggest that ZnT8 may, under normal circumstances, catalyse Zn 2+ efflux from granules, in line with bidirectional transport by ZnT5 [42]. Alternatively, gradual release of Zn 2+ from granules by exocytosis may elevate the extracellular Zn 2+ concentration in the medium surrounding WT, but not ZnT8 −/− , cells for which granule Zn 2+ content is near zero (see Chimienti et al and Li et al [13,43]). The released Zn 2+ may then be recaptured, at least in part, and taken into the cytosol, by plasma-membrane-located Zn 2+ -uptake systems (e.g. ZIP1, voltage-gated Ca 2+ channels) [30]. However, we calculate that release of Zn 2+ ions into the medium is likely to increase total external Zn 2+ concentration to only a miniscule extent (<50 nmol/l) vs a total extracellular concentration of >6 μmol/l [44], although local concentrations at the cell surface [14,43] may be higher.
One of the effects of ZnT8 inhibition and consequently lowered cytosolic Zn 2+ levels was impaired metallothionein induction in response to hypoxia (Fig. 6a, b). However, this did not affect islet cell survival under the conditions examined, despite the fact that islets lacking metallothionein-1 and -2 exhibited a twofold higher frequency of beta cell death compared with control islets (Fig. 5c). The latter finding is consistent with earlier data showing that overexpression of metallothionein protects islets against hypoxia, leading to improved islet cell survival [45]. We assume, therefore, that residual levels of metallothionein (~50%) in ZnT8 −/− mouse islets are sufficient to prevent an increase in cell death.
Unexpectedly, in small islets from older mice, we observed a significantly lower rate of cell death in ZnT8 −/− compared with WT islets. This finding is in line with our previous report on glucose homeostasis in these mice, where a compromising effect of ZnT8 deficiency disappeared with age [15], and suggests that, with ageing, a deleterious effect of ZnT8 deficiency may revert to one of protection. On the other hand, the lack of a genotype effect in large islets may be due to the variance of oxygen concentration in these islets, where the  The percentage of dead cells within intact islets after exposure for 24 h to normoxia (white bars, 21%) or hypoxia (black bars, 1% ambient oxygen). Scale bar, 100 μm. Bars represent mean ± SE. * p<0.05 and ** p<0.01. Mt1/2 −/− , 129S7/SvEvBrd-Mt1 tm1Bri Mt2 tm1Bri /J core is relatively anoxic as a consequence of larger diffusion distance when exposed to hypoxia in vitro. Nonetheless, the expression of hypoxia-inducible genes (with the exception of Mt1/Mt2) tended to be enhanced in ZnT8 −/− mice, possibly reflecting regulation of HIF1α by Zn 2+ [46]. Importantly, the current results may provide an explanation for the recent finding that rare loss-of-function mutations in the SLC30A8 gene in man are associated with protection against type 2 diabetes [47].
Reduced expression of ZnT8 in hypoxia may thus reflect an 'adaptive' response of beta cells to permit survival under a hypoxic/oxidative stress in a less differentiated state (as previously described after partial pancreatectomy-induced hyperglycaemia [48]). The mechanisms behind this observation remain to be elucidated, but may suggest that reduced zinc levels are beneficial in the situation of increased hypoxic stress. Indeed, it has previously been shown that high concentrations of Zn 2+ are able to induce islet cell death in a dose-  Fig. 6 The effect of deficiency for the zinc transporter ZnT8 on the expression of Mt1 and Mt2 and [Zn 2+ ] cyt as well as cell death rate in mouse pancreatic islets. Islets from ZnT8 +/+ (WT) and ZnT8 −/− mice were incubated for 24 h at normoxia (white bars) or hypoxia (black bars). qRT-PCR analysis of (a) Mt1 and (b) Mt2 was performed. (c) CD1 mouse islets were incubated for 24 h at normoxia or hypoxia, with or without 30 μmol/l extracellular ZnCl 2 (white bars, normoxia without ZnCl 2 ; grey bars, normoxia with ZnCl 2 ; dark grey bars, hypoxia without ZnCl 2 ; black bars, hypoxia with ZnCl 2 ); qRT-PCR analysis of Mt1 and Mt2 was performed. (d, e) [Zn 2+ ] cyt was measured in dissociated islets of ZnT8 +/+ (WT) and ZnT8 −/− (knockout) mice after incubation for 24 h at (d) normoxia or (e) hypoxia. (f) Analysis of free [Zn 2+ ] cyt in INS-1(832/13) cells expressing plasmid constructs encoding ZnT8 carrying either the at-risk R325 or the protective W325 polymorphism, compared with control (Ctrl). (g) Islets from ZnT8 +/+ (WT, white bars) and ZnT8 −/− mice (black bars) were incubated for 24 h at hypoxia, with or without 30 μmol/l extracellular ZnCl 2 (Zn). qRT-PCR of Mt2 was performed. (h, i) The proportion of dead cells in islets of ZnT8 +/+ (white bars) and ZnT8 −/− mice (black bars) is depicted (h) after incubation of islets of all sizes for 24 h at normoxia or hypoxia in mice aged 10-15 weeks, and (i) for islets <120 μm in older mice (>12 weeks). Bars represent mean ± SE. * p<0.05, ** p<0.01 and *** p<0.001. Hypox, hypoxia; normox, normoxia; Zn, extracellular ZnCl 2 dependent manner [49,50]. A similar rate of cell death after 24 h and 48 h of hypoxia, as observed here, suggests that, once adapted to hypoxia, islets are better able to survive. Further studies will be required, however, to determine whether pharmacological modification of ZnT8 function may be beneficial in islet transplantation or type 2 diabetes mellitus.