Beta cell-specific Znt8 deletion in mice causes marked defects in insulin processing, crystallisation and secretion
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Zinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues.
We generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis.
Znt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis.
This is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.
KeywordsAlpha cell Beta cell Glucagon Insulin Pancreatic islets SLC30A8 Zinc ZnT8
HOMA for insulin resistance
Quantitative real-time PCR
Rat insulin promoter
Yellow fluorescent protein
Zinc transport family member 8
Alpha cell-specific Znt8 knockout
Beta cell-specific Znt8 knockout
Global Znt8 knockout
Conditional Znt8 mice
Zinc transport family member 8 (ZnT8) is a member of the cation diffusion facilitator family and a zinc efflux transporter recently linked to type 2 diabetes [1, 2, 3, 4, 5]. Since then, ZnT8 has also been found to be targeted by autoantibodies in type 1 diabetic patients . Interestingly, studies suggest that the risk allele of ZnT8 (also known as SLC30A8) is associated with reduced insulin secretion in type 2 diabetic patients [7, 8]. The important role of zinc in modulating insulin biosynthesis and secretion , and the unique localisation of ZnT8 with insulin [10, 11] have focused much interest on determining the function of ZnT8 in controlling glucose homeostasis.
The generation of global Znt8 knockout (Znt8KO) mice has provided an excellent tool to better study the link between diabetes and ZnT8. These mice are glucose-intolerant with abnormalities in zinc accumulation, insulin granule morphology and insulin secretion from beta cells [12, 13, 14], although some differences in phenotypes exist between groups. Interestingly, on a high-fat diet, Znt8KO mice were severely insulin-resistant and more obese than controls, suggesting that ZnT8 may play a role in peripheral tissues. Thus, glucose intolerance may not necessarily result from the absence of ZnT8 in beta cells alone. Although ZnT8 was originally thought to be pancreas-specific [11, 15], studies now suggest it is also present in human adipose tissue , blood lymphocytes  and the cubical epithelium that lines thyroid follicles and adrenal cortex . We have also shown that ZnT8 is present in alpha cells [13, 19]. However, its function there remains unknown as although zinc is localised to alpha cell granules , zinc is not thought to be required for glucagon processing and packaging. Interestingly, a recent study showed that overexpression of Znt8 inhibits, while knockdown stimulates, glucagon secretion in cultured mouse alpha cells , suggesting that glucagon secretion may be regulated directly by ZnT8, thus indirectly affecting insulin secretion. It is therefore important to devise ways to understand the role of ZnT8 in beta cells without the problem of confounding effects from other tissues including alpha cells. The insights gained from such studies might also begin to explain the marked phenotypic differences between different colonies of Znt8KO mice [12, 13, 14]. Finally, as some [19, 22, 23, 24], though not all [13, 25] previous studies suggest that glucagon secretion might be regulated by zinc released from beta cells, it is also important to study the impact of ZnT8 on alpha cell function.
In this study, we generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8. We show that Znt8BKO mice are glucose-intolerant with reduced beta cell zinc accumulation, abnormal beta cell granule morphology and impaired insulin processing. Reduced first-phase insulin response to glucose was also evident compared with controls, suggesting impaired insulin secretion. In contrast, Znt8AKO mice showed no detectable phenotypic differences compared with control mice.
Generation of cell-specific Znt8 knockout mice
Chimeric males were bred with C57Bl/6J wild-type mice to generate heterozygous mice carrying the floxed allele. These animals were then mated with pCAG-Flp-expressing C57Bl/6J mice to excise the FRT flanked neomycin cassette and generate neomycin-excised conditional ZnT8 mice (Znt8loxP). The loxP sites flank exon 1, which contains the start codon for Znt8. Homozygous mice crossed to cell-specific Cre mice therefore lack Znt8 in that cell type. Znt8loxP mice were obtained from Genoway, Lyon, France. To generate Znt8BKO mice, Znt8loxP mice were crossed to rat insulin promoter (RIP)-Cre mice. Since RIP promoter can also drive deletion in the hypothalamus , Znt8 expression was determined in this tissue and found to be low, but detectable in quantitative real-time PCR (qPCR) analysis (Fig. 2c). To generate Znt8AKO mice, Znt8loxP mice were crossed to glucagon promoter (Gcg)-Cre-ROSA26EYFP (Gcg-Cre-YFP) mice . This cross allows labelling of alpha cells with YFP where Cre is expressed. Mice were genotyped using tail DNA and standard multiplex PCR using flox and Cre primers (Electronic supplementary material [ESM] Table 1). Mice expressing Cre transgene alone were used as controls (RIP-Cre mice were controls for Znt8BKO mice and Gcg-Cre-YFP mice were controls for Znt8AKO mice) and 6- to 8-week-old male mice were used for all experiments, unless otherwise mentioned. All experiments were approved by the Animal Care Committee at the University of Toronto and animals were handled according to the guidelines of the Canadian Council of Animal Care.
Islet isolation and dispersion
Mouse islets were isolated by collagenase digestion of the mouse pancreas and dispersed as described . Human islets isolated following the Edmonton Protocol  from healthy donors were provided by J. Shapiro and the ABCC Human Islet Distribution Program (Clinical Islet Laboratory, University of Alberta, Edmonton, AB, Canada). Human islets were dispersed as described .
Transmission electron microscopy
Islets were prepared and images were acquired as described . Dense core, empty, light core (grey) and atypical (rod-shaped) granules were manually counted and quantified.
Immunogold-cryo electron microscopy
Cryo-electron microscopy and immunogold labelling of ZnT8 was performed as described . Based on insulin and glucagon immunogold labelling and literature on granule characterisation , cells within the centre of the mouse islet with granules containing a dense core and a large translucid halo were identified as beta cells, while peripheral cells of the mouse islet with granules containing a dense core and a thin rim halo were identified as alpha cells. Human beta cells were identified strictly by the large translucid halo.
Western immuno-blot analysis
Western blot analysis was performed as described . Lysates were resolved by 10% (wt/vol.) SDS-PAGE and immunoblotted with polyclonal anti-ZnT8 (1:500; Mellitech, Grenoble, France) or anti-beta actin (1:2,000) antibodies followed by anti-rabbit secondary antibody (both from Sigma, St Louis, MO, USA). Immunoblots were scanned on a Kodak imager (Molecular Imaging Systetms, Carestream Health Inc., Rochester, NY, USA) within the linear range of intensity.
Quantitative real-time PCR
Immunostaining and confocal microscopy
Dispersed islet cells were fixed in 4% (vol./vol.) paraformaldehyde for 15 min, permeabilised with 2% (vol./vol.) Triton-X-100 for 10 min and blocked overnight in 5% (wt/vol.) BSA at 4°C. Cells were then labelled overnight at 4°C with polyclonal anti-ZnT8 (1:300) and anti-insulin (0.05 mol/l; DAKO, Glostrup, Denmark), and monoclonal anti-glucagon (1:300; Sigma) and anti-green fluorescent protein (GFP; 1:300, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. Cells were then stained at room temperature for 30 min with Cy5-conjugated anti-rabbit, FITC-conjugated anti-guinea pig or FITC-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) respectively [13, 19]. Finally, cells were washed and mounted on glass slides, and images acquired using a software package (LSM510; Zeiss, Thornwood, NY, USA) on a confocal microscope with 488/633 nm excitation laser line and 545 nm beam-splitter.
Oral glucose tolerance test
Following a 6 h fast, glucose (1.5 g/kg body weight) was given by oral gavage and blood glucose measured from tail vein blood at 0, 10, 20, 30, 60, 120 min using a glucometer. For insulin and proinsulin quantification, blood from the tail vein was collected in EDTA-coated microvettes (Sarstedt, Nümbrecht, Germany) at 0, 10, 30, 60 and 120 min. Area under the curve was calculated using a software package (Graph Pad Prism, La Jolla, CA, USA). HOMA for insulin resistance (HOMA-IR) index was calculated in the fasting state .
Glucagon, insulin and proinsulin measurements
Blood was collected from a tail vein of 16 h fasted mice for glucagon measurements. Plasma was separated from whole blood by centrifugation for 10 min at 5,900×g. Plasma insulin, and islet and plasma proinsulin were measured using ELISA kits (ALPCO Diagnostics, Salem, NH, USA; Mercodia, Uppsala, Sweden respectively). Proinsulin detection was performed according to manufacturer's instructions: Proinsulin in the sample reacts with anti-proinsulin antibodies bound to microtitration wells and to peroxidase-conjugated antibodies in the solution. The bound conjugate is detected by reaction with 3,3′,5,5′ tetramethylbenzidine. The kit detects proinsulin I and II. Islet insulin content and plasma glucagon were measured by radioimmunoassay kits (Linco Research, Millipore, Billerica, MA, USA).
Islet morphological analysis
Isolated pancreases were prepared and labelled for insulin and glucagon as described . Slides were digitised on a bright-field scanner at magnification of ×20 and analysed with a software package (ImageScope; Aperio Technologies, Vista, CA, USA) using the positive-pixel count algorithm. Results were normalised to whole-slice area (total pixel count). Beta and alpha cell mass was determined using the total pancreatic weight. Islet number per slice was manually counted and normalised to whole-slice area. For islet size measurements, images were acquired on a confocal microscope using a software package (LSM510; Zeiss) at magnification of ×20, after which the area of each islet was calculated using ImageScope (Aperio).
Zinc content measurement
Insulin secretion was assessed by islet perifusion as described . Area under the curve was calculated using Graph Pad Prism.
One-way repeated measures ANOVA with Tukey–Kramer or Bonnferroni’s post hoc test was used for the oral glucose tolerance test (OGTT) and islet perifusion experiments, to determine significant variation in blood glucose levels and insulin secretion. In all other experiments, Student’s t test was used. A value of p < 0.05 was considered statistically significant. Data are expressed as mean ± SEM. The n values represent the number(s) of animals used.
ZnT8 localized to granules in beta and alpha cells
ZnT8 eliminated in Znt8BKO mouse islets
Znt8BKO mice are glucose-intolerant
Znt8BKO mice show abnormal beta cell morphology, reduced islet insulin processing and beta cell zinc accumulation
Znt8BKO mice show a reduced first-phase insulin response
When isolated islets were perifused with low (2.8 mmol/l) and high (11 mmol/l) glucose or 10 mmol/l arginine (Fig. 6c), we observed reduced first-phase insulin secretion from Znt8BKO islets compared with controls (as measured by the area under the OGTT curve; Fig. 6d). Second-phase insulin secretion and the response to arginine were not significantly different between groups.
Znt8AKO mice maintain normal plasma glucagon and glucose homeostasis
Zinc transport family member 8 has recently attracted much attention after the identification in genome-wide array studies of an association between a non-synonymous polymorphism in the Znt8 gene and increased risk of type 2 diabetes [1, 2, 3, 4, 5]. Although immunofluorescent co-staining has localised ZnT8 with insulin [10, 11], our study is the first to demonstrate this at the level of the granule, using transmission electron microscopy. Furthermore, although ZnT8 has been observed in alpha cells using immunostaining [12, 13, 14, 19] and qPCR analysis of highly purified primary mouse islet alpha cells , the subcellular localisation of the protein has remained uncertain. We provide evidence here that ZnT8 also resides within glucagon granules. In support of a role for zinc in glucagon processing or packaging, recent work by Egefjord and colleagues showed zinc concentrated in the alpha cell secretory granule periphery .
Until its link to diabetes was established, only a couple of studies had explored the role of ZnT8 in beta cells. These suggested that Znt8 overexpression increases, while knockdown decreases zinc accumulation and insulin secretion in cultured beta cells [10, 35]. Since then, three independent groups have studied Znt8KO mice in order to understand the molecular link between ZnT8 and glucose homeostasis [12, 13, 14]. Although a significant loss of islet or beta cell zinc content was observed in all three studies, only Nicolson et al. reported significant glucose intolerance at most ages and in both sexes of Znt8KO mice, and decreased insulin secretion during intraperitoneal glucose challenge . More importantly, the impact of Znt8 deletion on in vitro insulin secretion was variable [12, 13, 14], with Lemaire et al.  showing no effect, Pound et al.  showing a decrease and Nicolson et al.  showing enhanced insulin secretion from Znt8KO mouse islets under the conditions studied. While pancreatic or islet insulin content was unchanged in all three studies, Pound et al.  showed reduced fasting plasma insulin in Znt8KO mice; this was in contrast to the other two studies, which showed no change [12, 13]. These differences have been attributed to possible variations in genetic background, age and diet of the mice .
To provide a more appropriate model to understand ZnT8 specifically in the beta cell, we have now created Znt8BKO mice. The key findings in Znt8BKO mice are glucose intolerance, reduced beta cell zinc accumulation and atypical insulin granules. In vitro, we demonstrated reduced first-phase glucose-stimulated insulin secretion in Znt8BKO mice, although plasma insulin was similar between groups during OGTT. Reasons for these differences remain unclear and require further investigation.
Our findings are in part consistent and partly inconsistent with the Znt8KO phenotype [12, 13, 14]. Since Znt8 expression is not restricted to the beta cell [16, 17, 18], differences in glucose homeostasis between Znt8KO and Znt8BKO mice could be due to Znt8 deletion in other cell types and compensatory changes at these sites. ZnT8 is present in the alpha cell, adipose tissue and the adrenal cortex, which are well-known sites for regulation of glucose homeostasis [37, 38, 39]. It is also possible that low but significant expression of Znt8 centrally, including the hypothalamus, also influences the activity of neurons involved in control of feeding and satiety, or other aspects of energy homeostasis.
The significant reduction in insulin granule crystallisation leading to fewer dense-core granules is reminiscent of beta cells from animals with deficiencies in insulin-processing enzymes [46, 47]. In addition to crystallisation, the increased presence of empty granules seen in Znt8BKO beta cells suggests a general defect in insulin packaging. Increased plasma and islet proinsulin, and reduced production of key transcription factors and insulin-processing enzyme genes in Znt8BKO mice suggest defects in both insulin transcription and processing (Fig. 8b). As zinc is an important co-factor for gene transcription, this reduction in expression of Pdx1 and Mafa, as well as that of processing enzymes could be a general phenomenon of zinc deficiency in beta cells. In fact, several zinc-dependent transcription factors are known to regulate Pdx1 and Mafa expression, possibly affecting insulin gene expression [48, 49]. As such, we observed a trend towards reduced insulin gene expression, but surprisingly failed to observe a corresponding decrease in plasma insulin. This may have been due to the inability of current methods to differentiate between proinsulin, insulin or other inactive forms. However, the more remarkable reduction in expression of genes encoding proinsulin-converting enzymes indicates that the principal impact is on post-translational modifications in the insulin biosynthetic pathway. The impaired conversion of proinsulin to insulin has also been observed in human carriers of the ZnT8 risk allele . In vitro, Znt8BKO islets displayed reduced first-phase insulin secretion, which could have been due to reduced cargo or to the reduced or delayed exocytosis of insulin granules (Fig. 8b). Both scenarios are supported in the present study. First, indiscriminate exocytosis of granules regardless of maturity or crystallisation status including empty granules would lead to reduced secretion of biologically active insulin. In addition, Znt8BKO beta cells contain fewer granules per given area compared with control cells. Second, a zinc deficiency in Znt8BKO islets could affect the activity of zinc-dependent transcription factors and proteins associated with glucose sensing and the exocytotic machinery. For example, a decrease in expression of Kcnj11, as observed here, could affect glucose responsivity. Future studies will investigate these possibilities further.
Previously it has been shown that zinc regulates glucagon secretion from alpha cells [19, 22, 23, 24]; however, this is not a universal finding [13, 25, 39] and the mechanism of inhibition is still a matter of debate. Based on evidence that zinc inhibits glucagon secretion and that zinc and ZnT8 are localised to the alpha cell granule , we would predict an increase in glucagon secretion from alpha cells lacking ZnT8 specifically. Alternatively, since ZnT8 is localised to the glucagon granules, deletion of the gene encoding it might cause zinc accumulation within the cytosol, thus suppressing glucagon secretion in this way. Regardless of the possible method, in the present study, we observed no differences in fasting plasma glucagon levels or glucose homeostasis in Znt8AKO and control mice. Under the conditions studied, therefore, ZnT8 appears largely dispensable for alpha cell function. However, further examination of Znt8AKO islets and isolated alpha cells may reveal important physiological functions of zinc and ZnT8 in this cell type.
In conclusion, we provide the first report of specific beta and alpha cell deletion of Znt8. Our study suggests that ZnT8 does not have a major role in glucagon biosynthesis and secretion. In contrast, we demonstrate that ZnT8 is required for proper insulin processing, crystallisation and packaging, and that this in turn is required to maintain normal insulin secretion and glucose homeostasis (Fig. 8b). The similarities between Znt8KO and Znt8BKO mice make it apparent that the phenotype of the former is indeed primarily a consequence of Znt8 deletion in beta cells and not in alpha cells or at other sites of ZnT8 activity, where the differences may have arisen due to compensation at these other sites in Znt8KO mice. Interestingly, the Znt8BKO phenotype has remarkably similar characteristics to human carriers of the risk allele of ZnT8 [7, 8, 50], suggesting that it is a useful model to facilitate understanding of the molecular mechanisms linking Znt8 variants to the development of type 2 diabetes.
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR; MOP-102588) to M. B. Wheeler. A doctoral award from CIHR supported N. Wijesekara. G. A. Rutter was supported by Programme and Project grants from the Wellcome Trust, National Institutes of Health, Medical Research Council (UK), Juvenile Diabetes Research Foundation International, Diabetes UK and the European Union. We thank P. Herrera (University of Geneva Medical School, Department of Cell Physiology and Metabolism, Geneva, Switzerland) and P. Gilon (Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, Brussels, Belgium) for kindly providing Gcg-Cre-YFP mice. RIP-Cre mice were generously given by M. Woo (Ontario Cancer Institute/Princess Margaret Hospital, Toronto, ON, Canada). We also thank R. Temkin (Advanced Bioimaging Centre, Mount Sinai Hospital, Pathology and Laboratory Medicine, Toronto, ON, Canada), S. Doyle and B. Calvieri (Microscopy Imaging Lab, University of Toronto, Toronto, ON, Canada), Y. Liu, L. Li and S. Sultan (Department of Physiology, University of Toronto, Toronto, ON, Canada) for their technical support, and E. Allister (Department of Physiology, University of Toronto, Toronto, ON, Canada) for a critical review of the manuscript.
Duality of interest
G. A. Rutter has received grant support from Servier IdS (Suresnes, France). F. Chimienti is employed by Mellitech (Grenoble, France). All other authors declare that there is no duality of interest associated with this manuscript.
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