The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid residue 325 of human ZnT-8 are associated with altered susceptibility to Type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in Type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analysed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioural changes or differences in body weight were observed between wild-type and ZnT-8−/− mice, and ZnT-8−/− mouse islets were indistinguishable from wild-type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8−/− mouse islets, as evaluated both by Timm's histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16-week-old, 6 h fasted animals of either gender; however, plasma insulin concentrations were reduced in both female (∼31%) and male (∼47%) ZnT-8−/− mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8−/− mice, but glucose-stimulated insulin secretion from isolated islets was reduced ∼33% relative to wild-type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

INTRODUCTION

Zinc homoeostasis is maintained by two types of proteins, namely metallothioneins and zinc transporters [1], the former being involved in the intracellular trafficking and storage of zinc. There are two classes of zinc transporters, the SLC39 (Zip) family that controls the cellular uptake of zinc and the SLC30 (ZnT) family that controls zinc efflux into the extracellular matrix and intracellular vesicles [1]. In mammalian cells the ZnT family comprises ten members that share a similar six transmembrane domain structure with a histidine-rich loop located between helices IV and V, with the exception of ZnT-6 which contains a serine-rich loop and ZnT-10 which contains a basic amino-acid rich loop [1].

Chimienti et al. [2] had suggested that expression of the Slc30a8 gene, which encodes the zinc transporter ZnT-8, is restricted to pancreatic islets, specifically insulin-secreting β-cells, but recent data suggest that it is also expressed in glucagon-secreting α-cells [3]. Recent GWA (genome-wide association) studies have shown that sequence variations in SLC30A8 are linked to an increased susceptibility to the development of Type 2 diabetes [47]. ZnT-8 has also recently been identified as an autoantigen in Type 1 diabetes in humans [8]. Interestingly, the same polymorphic variant in amino acid residue 325 of human ZnT-8 that is associated with altered susceptibility to Type 2 diabetes also affects ZnT-8 autoantibody epitope specificity in Type 1 diabetes [9]. Within β-cells ZnT-8 localizes to insulin secretory granules [10] and because insulin is stored as a hexamer bound to two zinc ions, it has been proposed that ZnT-8 is important for providing zinc to allow for the proper maturation, storage and secretion of insulin [11]. In the present study we examine the effect of a global Slc30a8-null mutation in vivo, a mouse model that is directly relevant to Type 2 diabetes susceptibility in humans. The results are consistent with a role for ZnT-8 in islet function, although surprisingly not in whole-body glucose metabolism.

MATERIALS AND METHODS

For details on the generation of the Slc30a8-targeting vector, generation and PCR genotyping of Slc30a8-knockout mice and the analysis of islet number, size and cellular composition in Slc30a8-knockout mice please see Supplementary Materials and methods (at http://www.BiochemJ.org/bj/421/bj4210371add.htm).

Animal care

The animal housing and surgical facilities used for the mice in these studies meet the American Association for the Accreditation of Laboratory Animal Care standards. All animal protocols were approved by the Vanderbilt University Medical Center Animal Care and Use Committee. Mice were maintained on standard rodent chow (LabDiet 5001; 23% protein and 4.5% fat; PMI Nutrition International) with food and water provided ad libitum.

Breeding strategy for Slc30a8-knockout mice

F1 chimaeric (129SvEvBrd×C57BL/6J) mice were interbred to generate F2 wild-type, heterozygous and homozygous knockout mice. The F2 heterozygous mice were then bred with F1 hybrid (C57BL/6J×129SvEvBrd) mice. Two male and two female heterozygous mice from this breeding, along with their heterozygous offspring, were then interbred to generate a mouse colony used in the phenotypic characterization of the effect of the Slc30a8-null mutation.

Immunohistochemical staining

Pancreas tissue was fixed for 1 h in 4% (w/v) paraformaldehyde in PBS and embedded for paraffin sectioning (8 μm). Primary antisera against insulin (guinea-pig 1:100; Dako), glucagon (mouse 1:100; Sigma) and somatostatin (rat 1:100; Abcam) were combined with a rabbit polyclonal antibody raised against a 102 amino acid C-terminal human ZnT-8 peptide (amino acids 268–369; used at 1:500) and were detected with species-specific secondary antibodies conjugated to Cy2 (carbocyanine), Cy3 (indocarbocyanine), Cy5 (indodicarbocyanine) and AMCA (aminomethylcoumarin) (all from Jackson Immunoresearch Laboratories).

Timm's staining analyses in Slc30a8-knockout mice

The determination of zinc content in wild-type and Slc30a8-knockout mouse pancreas was based on further modification of the revised Timm's protocol described by Danscher et al. [12]. Briefly, pancreatic tissue sections (8 μm; frozen and paraffin) fixed in 4% (w/v) paraformaldehyde were placed on glass slides and immersed in 0.1% sodium sulfide in 0.15 M sodium phosphate buffer (pH 7.4) for 1 h in glass jars inside a chemical fume hood. The slides were briefly rinsed in PBS and immersed in AMG (autometallography) developer [pH 3.8; 60 ml of gum arabic, 10 ml of sodium citrate (25.5 g of citric acid monohydrate+23.5 g of sodium citrate dihydrate in 100 ml of de-ionized water), 15 ml of reducing agent (0.056% hydroquinone in de-ionized water at 40 °C) and 15 ml of solution containing silver ions (0.008% silver lactate in de-ionized water at 40 °C) added just before use]. All glassware used for AMG development was rinsed in Farmer's solution (10% sodium thiosulfate/10% potassium ferricyanide; 9:1) and warm water. AMG development was carried out at room temperature (22 °C), in the dark and with gentle shaking. The reaction was stopped after 45 min with 5% sodium thiosulfate solution for 10 min. The slides were rinsed in warm water several times, counterstained with haematoxylin and eosin and permanently mounted.

Measurement of islet zinc content

Freshly isolated islets from wild-type and Slc30a8-knockout mice were washed in Ca2+-free Hank's balanced salt solution and frozen down at −80 °C in 20 islet aliquots. Islet pellets were lysed by re-suspension in 1 ml of lysis buffer [1% Triton X-100 in 10 mM Tris/HCl (pH 7.4)]. The Zn2+ concentration in the lysate was measured using the Zn2+-sensitive fluorescent dye FluoZin-3 (Invitrogen). In the presence of 1.181 μM FluoZin-3 the fluorescent signal at the emission peak (516 nm) was measured in the total sample lysate using a fluorometer (PTI Instruments). The fluorescent signal was compared with a standard curve generated from serial dilutions of ZnSO4 in lysis buffer to obtain the lysate Zn2+ concentration and thus the Zn2+ content per islet. As a normalization factor, the protein content per islet was measured in the total sample lysate using the BCA (bicinchoninic acid) protein assay (Pierce). To minimize contaminating Zn2+, all solutions were made in double-distilled water (18.2 MΩ), avoiding the use of any glassware. Blank samples were also prepared during the islet isolation to quantify any additional Zn2+ contamination.

Phenotypic analysis of Slc30a8-knockout mice

Animals were fasted for 5 h, weighed and then 1 h later anaesthetized using isoflurane before collection of blood samples (∼200 μl) from the retro-orbital venous plexus. Whole-blood glucose concentrations were determined using an Accu-Check Advantage monitor (Roche). EDTA (5 μl; 0.5 M) was then added before centrifugation (16000 g for 10 min at 4 °C) to isolate plasma. Trasylol (aprotinin; 5 μl; Bayer Health Care) was added to the plasma to prevent proteolysis of glucagon. Cholesterol was assayed using the cholesterol reagent kit (Raichem), whereas triacylglycerol and glycerol were assayed using a serum triacylglycerol determination kit (Sigma). Insulin and glucagon levels were quantified using radioimmunoassays by the Vanderbilt Diabetes Center Hormone Assay Core.

Intraperitoneal glucose tolerance tests on overnight fasted conscious mice were performed as previously described [13].

Islet isolation and GSIS (glucose-stimulated insulin secretion) assays

Islets were isolated from ∼5-month-old male mice as described previously [14]. After isolation, islets were rinsed in three 12 ml changes of RPMI-1640 medium containing 10% (v/v) FBS (foetal bovine serum), 100 units/ml penicillin, 100 μg/ml streptomycin and 11 mM glucose, and then cultured in 10 cm non-treated plates overnight at 37 °C. The next day islets were transferred into medium with 5 mM glucose and allowed to equilibrate for 1 h at 37 °C. Following the equilibration period, 50 IEQs (islet equivalents) were incubated in 5 ml of RPMI-1640 medium with 5 or 11 mM glucose for 30 min at 37 °C. At the end of the static incubations, islets were collected into 1.5 ml tubes, washed three times with 1 ml of 1×PBS and insulin was extracted in 0.2 ml of acid alcohol for 48 h at 4 °C. The medium from the static incubations was harvested into 15 ml conical tubes and centrifuged at 600 g for 1 min at 4 °C. Islet insulin extracts and static incubation medium were stored at −80 °C until assayed for insulin by radioimmunoassay.

Statistical analyses

Data were analysed using a Student's t test: two sample assuming equal variance. The level of significance was as indicated (two-sided test).

RESULTS

Biochemical characterization of ZnT-8−/− mice

The human and mouse ZnT-8 genes contain eight exons ([2] and results not shown). A modified mouse Slc30a8 allele, in which 135/147 bp of exon 3 and the first 10 bp of intron 3 were replaced by a LacZ/Neo cassette, was generated by homologous recombination in 129/SvEvBrd (Lex-2) ES (embryonic stem) cells (Figure 1A). Deletion of exon 3 disrupts two putative ZnT-8 transmembrane domains [2]. Correct gene targeting was confirmed by Southern blot (Figure 1B) and PCR (results not shown) analysis prior to injection of ES cells into C57BL/6 (albino) blastocysts and subsequent generation of ZnT-8−/+ mice on a mixed 129/SvEvBrd×C57BL/6 background.

Generation and biochemical characterization of ZnT-8−/− mice

Figure 1
Generation and biochemical characterization of ZnT-8−/− mice

(A) Strategy used to generate ZnT-8−/− mice by homologous recombination in ES cells. A schematic representation of the wild-type murine Slc30a8 locus and the targeting construct are shown. Exon 3 was replaced with a cassette containing an IRES (internal ribosome entry site), the LacZ gene and a TK (thymidine kinase)-neomycin selectable marker. Correctly targeted clones were identified by Southern blot analysis using the indicated probes and were confirmed by PCR using the primers indicated. The primers represented sequences in exon 3 (primer 1), intron 2 (primer 2), intron 3 (primer 4) and the Neo gene (Neo3a primer). (B) Southern blot analysis of the Slc30a8 locus using genomic DNA extracted from the indicated targeted ES cell lines, or wild-type ES cell genomic DNA, designated Lex-2, as a control, using 5′ and 3′ diagnostic probes (A). The sizes of the wild-type locus, targeted allele and DNA markers are indicated. Clone 2H8 was used to achieve germline transmission. (C) Immunohistochemical staining of wild-type and ZnT-8−/− mouse pancreas with antisera raised against insulin, glucagon, somatostatin and ZnT-8 was performed as described in the Materials and methods section. Representative pictures (200×magnification) are shown. KO, knockout; WT, wild-type.

Figure 1
Generation and biochemical characterization of ZnT-8−/− mice

(A) Strategy used to generate ZnT-8−/− mice by homologous recombination in ES cells. A schematic representation of the wild-type murine Slc30a8 locus and the targeting construct are shown. Exon 3 was replaced with a cassette containing an IRES (internal ribosome entry site), the LacZ gene and a TK (thymidine kinase)-neomycin selectable marker. Correctly targeted clones were identified by Southern blot analysis using the indicated probes and were confirmed by PCR using the primers indicated. The primers represented sequences in exon 3 (primer 1), intron 2 (primer 2), intron 3 (primer 4) and the Neo gene (Neo3a primer). (B) Southern blot analysis of the Slc30a8 locus using genomic DNA extracted from the indicated targeted ES cell lines, or wild-type ES cell genomic DNA, designated Lex-2, as a control, using 5′ and 3′ diagnostic probes (A). The sizes of the wild-type locus, targeted allele and DNA markers are indicated. Clone 2H8 was used to achieve germline transmission. (C) Immunohistochemical staining of wild-type and ZnT-8−/− mouse pancreas with antisera raised against insulin, glucagon, somatostatin and ZnT-8 was performed as described in the Materials and methods section. Representative pictures (200×magnification) are shown. KO, knockout; WT, wild-type.

To confirm that the targeting strategy had abolished ZnT-8 expression, immunohistochemical staining was performed on pancreas sections prepared from a ZnT-8−/− mouse and a wild-type littermate. Figure 1(C) shows that ZnT-8 was detected in both α- and β-cells in wild-type, but not ZnT-8-knockout, mouse islets.

The size and number of islets in ZnT-8−/− animals were indistinguishable from wild-type littermates, as were the relative numbers of α- and β-cells (Supplementary Figure S1 at http://www.BiochemJ.org/bj/421/bj4210371add.htm). Histological analysis of zinc content on frozen pancreatic sections using a modified Timm's staining procedure that involves silver enhancement of metal sulfide precipitation showed that, in wild-type mouse pancreas, islets contained abundant zinc relative to the exocrine tissue (Figure 2A). This contrasted with ZnT-8−/− mouse pancreas in which no difference was observed in Timm's staining between islets and exocrine tissue, although gross islet morphology was preserved (Figure 2A). These results are consistent with analyses of zinc content in isolated islets using an assay that detects free and loosely bound zinc (Figure 2B). Figure 2(B) shows that zinc content was markedly reduced in islets isolated from Slc30a8-knockout mice relative to those isolated from wild-type mice. The concentration of zinc detected in wild-type islets was similar to that previously reported in the islet-derived INS1 cell line [10].

Analysis of islet zinc content in ZnT-8−/− mice

Figure 2
Analysis of islet zinc content in ZnT-8−/− mice

(A) A modified Timm's staining protocol was used to assess zinc content in both frozen and paraffin pancreas sections prepared from male wild-type and ZnT-8-knockout mice as described in the Materials and methods section. Representative pictures (100×magnification) are shown. (B) The zinc content in isolated islets was determined as described in the Materials and methods section. Results are the means±S.E.M. (n=12). *P<0.05 compared with wild-type. BG, background; KO, knockout; WT, wild-type.

Figure 2
Analysis of islet zinc content in ZnT-8−/− mice

(A) A modified Timm's staining protocol was used to assess zinc content in both frozen and paraffin pancreas sections prepared from male wild-type and ZnT-8-knockout mice as described in the Materials and methods section. Representative pictures (100×magnification) are shown. (B) The zinc content in isolated islets was determined as described in the Materials and methods section. Results are the means±S.E.M. (n=12). *P<0.05 compared with wild-type. BG, background; KO, knockout; WT, wild-type.

Phenotypic characterization of ZnT-8−/− mice

Genotype analysis of 383 3-week-old pups generated by cross-breeding heterozygous ZnT-8−/+ mice demonstrated that 83 mice were ZnT-8+/+, 203 were ZnT-8−/+ and 97 were ZnT-8−/−, a distribution close to the expected pattern for Mendelian inheritance. The ratio of male to female mice was 206:177. Cross-breeding experiments revealed that both male and female homozygous ZnT-8−/− mice are fertile.

The activity and behaviour of ZnT-8−/− mice were indistinguishable from their wild-type and heterozygous littermates at all ages, from birth up to 1 year in age. No gross anatomical changes were observed either externally or to major internal organs, and no differences were seen in the weights or lengths of ZnT-8−/− compared with wild-type mice (Table 1).

Table 1
Phenotypic characterization of ZnT-8-knockout mice

At 16 weeks of age mice were fasted for 5 h and then weighed. Mice were anaesthetized 1 h later, their length was measured and blood isolated. Blood glucose and plasma cholesterol, triacylglycerol, glycerol, insulin and glucagon levels were determined as described in the Materials and methods section. Results are means±S.E.M. obtained from the number of animals indicated in parentheses. KO, knockout; WT, wild-type; −/+, heterozygote.

Gender Genotype Weight (g) Length (mm) Glucose (mg/dl) Cholesterol (mg/dl) Triacylglycerol (mg/dl) Glycerol (mg/dl) Insulin (ng/ml) Glucagon (pg/ml) 
Female WT 23.8±0.4 (29) 98.8±0.5 (28) 110.1±3.9 (29) 75.1±4.0 (26) 49.2±2.5 (27) 2.5±0.1 (27) 0.45±0.05 (19) 70.5±5.6 (23) 
Female −/+ 23.3±0.3 (67) 98.7±0.3 (65) 110.0±1.8 (66) 87.8±2.4 (64)* 49.5±1.5 (62) 2.6±0.1 (63) 0.34±0.03 (38) 71.8±3.4 (56) 
Female KO 23.5±0.4 (32) 98.8±0.4 (31) 116.9±3.7 (32) 82.5±2.8 (30) 46.1±1.7 (30) 2.6±0.1 (30) 0.31±0.05 (20)† 74.2±6.3 (26) 
Male WT 32.4±0.5 (35) 105.5±0.4 (34) 135.7±3.9 (35) 100.7±4.4 (34) 68.4±2.7 (32) 2.5±0.1 (33) 1.50±0.23 (12) 63.2±5.6 (29) 
Male −/+ 32.4±0.4 (63) 104.7±0.3 (60) 135.7±3.0 (63) 103.6±3.2 (58) 73.2±2.0 (58) 2.7±0.1 (59) 1.07±0.22 (15) 66.4±3.5 (54) 
Male KO 31.6±0.7 (34) 105.3±0.6 (32) 137.2±3.4 (34) 111.7±3.6 (33) 70.6±2.9 (32) 2.5±0.1 (31) 0.79±0.11 (16)‡ 52.0±4.7 (26)§ 
Gender Genotype Weight (g) Length (mm) Glucose (mg/dl) Cholesterol (mg/dl) Triacylglycerol (mg/dl) Glycerol (mg/dl) Insulin (ng/ml) Glucagon (pg/ml) 
Female WT 23.8±0.4 (29) 98.8±0.5 (28) 110.1±3.9 (29) 75.1±4.0 (26) 49.2±2.5 (27) 2.5±0.1 (27) 0.45±0.05 (19) 70.5±5.6 (23) 
Female −/+ 23.3±0.3 (67) 98.7±0.3 (65) 110.0±1.8 (66) 87.8±2.4 (64)* 49.5±1.5 (62) 2.6±0.1 (63) 0.34±0.03 (38) 71.8±3.4 (56) 
Female KO 23.5±0.4 (32) 98.8±0.4 (31) 116.9±3.7 (32) 82.5±2.8 (30) 46.1±1.7 (30) 2.6±0.1 (30) 0.31±0.05 (20)† 74.2±6.3 (26) 
Male WT 32.4±0.5 (35) 105.5±0.4 (34) 135.7±3.9 (35) 100.7±4.4 (34) 68.4±2.7 (32) 2.5±0.1 (33) 1.50±0.23 (12) 63.2±5.6 (29) 
Male −/+ 32.4±0.4 (63) 104.7±0.3 (60) 135.7±3.0 (63) 103.6±3.2 (58) 73.2±2.0 (58) 2.7±0.1 (59) 1.07±0.22 (15) 66.4±3.5 (54) 
Male KO 31.6±0.7 (34) 105.3±0.6 (32) 137.2±3.4 (34) 111.7±3.6 (33) 70.6±2.9 (32) 2.5±0.1 (31) 0.79±0.11 (16)‡ 52.0±4.7 (26)§ 
*

P<0.01, female wild-type compared with female heterozygote.

P  =0.05, female wild-type compared with female knockout.

P<0.01, male wild-type compared with male knockout.

§

P<0.05, male heteozygote compared with male knockout.

Table 1 summarizes metabolic parameters in these animals assayed at 16 weeks of age following a 6 h fast. No marked changes in plasma cholesterol, triacylglycerol or glycerol were observed in either male or female ZnT-8−/− mice relative to wild-type animals (Table 1). Blood glucose and glucagon concentrations were also unchanged in both male and female ZnT-8−/− mice relative to wild-type animals; however, a statistically significant difference in plasma insulin concentrations was observed (Table 1). This result suggests that, although the absence of ZnT-8 might affect islet function, it has a limited effect on whole-body glucose metabolism. In addition, since a statistically significant difference in plasma insulin concentrations was not observed between male or female ZnT-8−/+ mice relative to wild-type animals, this suggests that loss of a single Slc30a8 allele is insufficient to affect islet function (Table 1). ZnT-8−/− mice showed gender-related variation in the majority of these metabolic parameters that were in the same direction and of similar magnitude to the gender-related differences in wild-type mice. Thus in males compared with females, insulin, triacylglycerols, cholesterol and glucose were all higher, whereas glucagon was lower (Table 1).

Since some metabolic disturbances only become readily apparent under stimulatory rather than basal conditions intraperitoneal glucose tolerance tests were used to provide a measurement of dynamic islet function in vivo. Following glucose injection (2 g/kg of body weight) blood glucose was assessed over a 120 min period (Figure 3). The data show no impairment in glucose clearance between wild-type and ZnT-8−/− mice (Figure 3). This result again suggests that, although the absence of ZnT-8 might affect islet function it has a limited impact on whole-body glucose metabolism, at least under the conditions examined.

Analysis of glucose tolerance in ZnT-8−/− mice in vivo

Figure 3
Analysis of glucose tolerance in ZnT-8−/− mice in vivo

Intraperitoneal glucose tolerance tests were performed on overnight fasted conscious wild-type (closed symbols) and ZnT-8−/− (open symbols) male mice as described in the Materials and methods section. Results show the mean glucose concentrations ± S.E.M. in wild-type (n=30; mean age 20 weeks) and ZnT-8−/− (n=36; mean age 20 weeks) animals. KO, knockout; WT, wild-type.

Figure 3
Analysis of glucose tolerance in ZnT-8−/− mice in vivo

Intraperitoneal glucose tolerance tests were performed on overnight fasted conscious wild-type (closed symbols) and ZnT-8−/− (open symbols) male mice as described in the Materials and methods section. Results show the mean glucose concentrations ± S.E.M. in wild-type (n=30; mean age 20 weeks) and ZnT-8−/− (n=36; mean age 20 weeks) animals. KO, knockout; WT, wild-type.

Insulin secretion from ZnT-8−/− mouse islets

To directly assess the impact of ZnT-8 on islet function, GSIS was compared in islets isolated from male wild-type and ZnT-8−/− mice in static incubations. Figure 4(A) shows that insulin content did not differ between wild-type and ZnT-8−/− mouse islets, whereas Figure 4(B) shows that GSIS from ZnT-8−/− mouse islets was reduced ∼33% relative to that from wild-type mouse islets.

Analysis of insulin content and GSIS in isolated ZnT-8−/− mouse islets

Figure 4
Analysis of insulin content and GSIS in isolated ZnT-8−/− mouse islets

Islets were isolated from wild-type (WT) and ZnT-8 knockout (KO) mice and then insulin content (A) and GSIS (B) were assayed as described in the Materials and methods section. Results are the means ± S.E.M. from three to four islet preparations. *P<0.05 compared with wild-type 11 mM glucose.

Figure 4
Analysis of insulin content and GSIS in isolated ZnT-8−/− mouse islets

Islets were isolated from wild-type (WT) and ZnT-8 knockout (KO) mice and then insulin content (A) and GSIS (B) were assayed as described in the Materials and methods section. Results are the means ± S.E.M. from three to four islet preparations. *P<0.05 compared with wild-type 11 mM glucose.

DISCUSSION

In the present study we examine the effect of a global Slc30a8-null mutation in vivo, a mouse model that is directly relevant to Type 2 diabetes susceptibility in humans. The study addresses the hypothesis, based on GWA data, that changes in the activity or stability of the ZnT-8 protein may result in islet dysfunction, which contributes to the pathogenesis of Type 2 diabetes. The results indicate that deletion of the Slc30a8 gene results in a mild metabolic phenotype on a mixed 129SvEvBrd×C57BL/6 background. Plasma insulin is reduced in both male and female ZnT-8−/− mice following a 6 h fast (Table 1). Consistent with this observation, GSIS from isolated islets is impaired (Figure 4) and islet zinc content is markedly reduced (Figure 2), although islet size, number and cellular composition are unaffected (Supplementary Figure S1). These observations in ZnT-8−/− mice are consistent with the demonstration that overexpression of ZnT-8 in INS-1 cells has the opposite effect, stimulating zinc accumulation and GSIS [10]. Although the loss of ZnT-8 function only has a mild effect presumably accounts for the small contribution of Slc30a8 mutations to Type 2 diabetes, as reflected in the odds ratio of 1.12 [47]. Future studies will be designed to examine whether the absence of ZnT-8 affects glucose metabolism under conditions more favourable for the development of glucose intolerance, such as following high-fat feeding or in older animals.

Although the results indicate that ZnT-8 is important for normal islet function, surprisingly whole-body glucose metabolism appears unaltered based on an assessment of fasting glucose levels (Table 1) and glucose tolerance tests (Figure 3). These observations suggest that ZnT-8 is not necessary for glucose homoeostasis, at least under the conditions examined. There appear to be two possible explanations for the decrease in plasma insulin without a concomitant increase in blood glucose (Table 1). There is a statistically significant decrease in plasma glucagon (∼20%) between male ZnT-8−/+ and ZnT-8−/− mice, although not between ZnT-8+/+ and ZnT-8−/− mice (Table 1). If the latter simply reflects a lack of power to detect a small change in plasma glucagon, the former would imply that glucagon secretion is also impaired in male ZnT-8−/− mice. In this event an offsetting decrease in both insulin and glucagon secretion could result in normal blood glucose. Indeed, the insulin/glucagon ratios in individual animals were not statistically different between male wild-type and ZnT-8−/− mice (results not shown). Future experiments studying glucagon secretion from isolated islets will address this possibility. In contrast, in female mice there are no statistically significant differences in glucagon levels between groups (Table 1). This suggests that there may be differences in insulin sensitivity between female wild-type and ZnT-8−/− mice. This would be consistent with the results of post-hoc QUICKI calculations [15] suggesting a statistically significant difference in insulin sensitivity between female wild-type and ZnT-8−/− mice (results not shown). Such a difference could have arisen as an adaptive change during development to compensate for low plasma insulin. Alternatively, a difference in insulin sensitivity could arise if ZnT-8 were expressed in other tissues, specifically ones which directly or indirectly modulate insulin-dependent glucose disposal. Interestingly, Murgia et al. [16] have recently demonstrated that ZnT-8 is expressed at low levels in tissues other than islets. Future experiments will directly compare insulin sensitivity in female wild-type and ZnT-8−/− mice using hyperinsulinaemic clamps. Finally, zinc is required not only for stabilizing the insulin crystal within the insulin storage granule, but may also be essential for the conversion of pro-insulin into insulin [17]. Increased circulating pro-insulin is a feature of early Type 2 diabetes and impaired glucose tolerance [18,19] and in a group genetically at risk of developing Type 2 diabetes, the SLC30A8 allele was associated with reduced pro-insulin into insulin conversion, although not insulin secretion [20]. Since pro-insulin has only ∼3% of the potency of insulin [21], if pro-insulin secretion was markedly increased in the female ZnT-8-knockout mice this could also explain the reduced plasma insulin levels associated with unchanged blood glucose levels. Unfortunately, commercially available assays for murine pro-insulin are unavailable at the current time.

Chimienti et al. [2] had previously reported that ZnT-8 is only expressed in pancreatic islet β-cells, but using antiserum raised against a 102-amino-acid C-terminal human ZnT-8 peptide (amino acids 268–369) we found that ZnT-8 is also clearly expressed in α-cells (Figure 1C). This observation is consistent with the results of Gyulkhandanyan et al. [3] who examined ZnT-8 expression in dispersed islet cells. The specificity of our antiserum was confirmed by the absence of staining in sections prepared from ZnT-8−/− mouse pancreas (Figure 1C). The hypothesis that glucagon secretion may be impaired in male ZnT-8−/− mice would therefore be consistent with the expression of ZnT-8 in α-cells (Figure 1C). However, the possibility also exists that glucagon secretion from α-cells has been indirectly affected by the absence of ZnT-8 in β-cells. Indeed, although the mechanism(s) involved are disputed, zinc release from β-cells inhibits glucagon secretion from α-cells [3,22,23].

The SNP (single nucleotide polymorphism) that linked the human SLC30A8 gene to increased Type 2 diabetes susceptibility [47] is located in the C-terminus of ZnT-8 and represents a non-synonymous polymorphism that changes the sequence of amino acid residue 325 [2]. In theory this ZnT-8 variant could represent either a gain- or loss-of-function, but because overexpression of ZnT-8 enhances GSIS [10] and because deletion of the Slc30a8 gene in mice impairs GSIS (Figure 4) we would predict that this human sequence variant impairs ZnT-8 function. Future experiments will address this hypothesis.

We thank Dr K. Platt (Lexicon Pharmaceuticals, The Woodlands, Texas, U.S.A.) and Ms K. Rufus (Vanderbilt University, Nashville, TN, U.S.A.) for assistance with this project. We also thank Ms W. Snead, Mr G. Poffenberger and Ms B. Trivedi (all from Vanderbilt University, Nashville, TN, U.S.A.) for performing insulin and glucagon assays and Dr M. Brissova and Ms A. Golovin (both from Vanderbilt University, Nashville, TN, U.S.A.) for performing islet isolations and GSIS analyses. We would also like to thank Dr C. J. Easley (Vanderbilt University, Nashville, TN, U.S.A.) for helpful discussions regarding Zn2+ measurements.

Abbreviations

     
  • AMG

    autometallography

  •  
  • ES cell

    embryonic stem cell

  •  
  • GSIS

    glucose-stimulated insulin secretion

  •  
  • GWA

    genome-wide association

  •  
  • IEQ

    islet equivalent

  •  
  • ZnT

    zinc transporter

AUTHOR CONTRIBUTION

Lynley Pound generated and maintained the ZnT-8-knockout mouse colony, performed mouse genotyping and metabolic phenotyping and the intraperitoneal glucose tolerance test assays. Suparna Sarkar performed Timm's staining, and quantitation of mouse islet size, number and composition. Richard K. P. Benninger performed quantification of islet zinc content. Yingda Wang performed LacZ staining and mouse genotyping. Adisak Suwanichkul was involved with the generation and analysis of ZnT-8-knockout mice. Melanie K. Shadoan was involved with the generation and analysis of ZnT-8 knockout mice. Richard Printz performed real-time PCR primer design and data analysis. James Oeser performed mouse genotyping and metabolic phenotyping. Catherine E. Lee performed Timm's staining, and quantitation of mouse islet size, number and composition. David Piston performed quantification of islet zinc content. Owen McGuinness analysed the mouse phenotyping data. John Hutton performed Timm's staining, quantification of mouse islet size, number and composition, and data analysis. David R. Powell was involved with the generation and analysis of ZnT-8-knockout mice. Richard O'Brien was involved in the mouse metabolic phenotyping and data analysis.

FUNDING

This work was supported by the National Institutes of Health [grant numbers DK076027 (to R. O'B.), DK076027 (to J. C. H.), DK064877 (to O. M.), DK53434 (to D. W. P.), GM72048 (to D. W. P.)]; the Juvenile Diabetes Research Foundation Autoimmunity Prevention Center (to J. C. H.); the Barbara Davis Center Diabetes and Endocrinology Research Center [grant number P30 DK57516 (to J. C. H.)]; and the Department of Defence Medical Free-Electron Laser Program (to D. W. P.). The Vanderbilt Hormone Assay and Analytical Services Core and the Vanderbilt Islet Procurement and Analysis Core are both supported by the National Institutes of Health [grant number P60 DK20593 (to the Vanderbilt Diabetes Research Training Center), grant number DK59637 (to the Vanderbilt Mouse Metabolic Phenotyping Center)].

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Supplementary data