Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice

Abstract

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules," which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Fig. 1.

ZnT8 expression is absent in ZnT8^−/− mice. (A) mRNA expression of the 10 known efflux zinc transporters (Slc30a family) analyzed via microarray (n = 3). (B) ZnT8 protein expression was analyzed via western blots. Please note complete absence of the expected MW for ZnT8 monomers (M) and SDS-resistant dimers (D), * = non-specific protein. (C) Immunohistochemistry of pancreatic sections from ZnT8^−/− and ZnT8^+/+ mice. In ZnT8^+/+ mice ZnT8 protein is strongly expressed in all insulin-positive cells and weakly expressed in a minority of glucagon cells. Nuclei are stained blue by 4′,6-diamidino-2-phenylindole (DAPI). (Scale bar, 10 μm.)

Fig. 2.

ZnT8 is required for islet dithizone staining and glucose-regulated granular zinc release from beta cells. (A) Dithizone staining of pancreata from ZnT8^+/+ and ZnT8^−/− mice, 15 min after i.p. injection of the dye. Islets can be seen at the pancreatic surface of ZnT8^+/+ mice but not of ZnT8^−/− mice. (B) Dithizone staining of isolated islets is positive in ZnT8^+/+ mice (red color) but negative in ZnT8^−/− mice. (C) Zinc release from clusters of islet cells imaged by TIRF microscopy during 7 min. Cluster size was 3.7 ± 0.4 vs. 3.8 ± 0.3 cells, for ZnT8^+/+ and ZnT8^−/−, respectively (ns). Medium contained 8 μM FluoZin-3 and exocytosis was stimulated by 15 mM glucose and 1 μM forskolin. One zinc exocytotic event from a ZnT8^+/+ four-cell cluster is illustrated, boundaries between cells are shown as dotted lines (left panel). Images were taken every 30 ms in the region delimited by a square (middle panel), and the intensity of fluorescence for this release event is plotted on the right panel. Arrows show fluorescence intensity for the indicated time points illustrated in the middle panel, (D) Quantification of zinc exocytotic events in clusters of ZnT8^+/+ and ZnT8^−/− islet cells. The horizontal line shows the average of 18 clusters from three individual mice.

Fig. 3.

ZnT8 is required for zinc-insulin crystal formation. (A) Transmission electron microscopy of representative ZnT8^+/+ and ZnT8^−/− beta cells (ZnT8^+/+, n = 4; ZnT8^−/−, n = 7). Secretory granules of ZnT8^−/− beta cells lack the electron dense cores and halo that are typical for wild-type mice. Furthermore, the size of the secretory granules is larger in ZnT8^−/− mice. (Scale bar, 0.5 μm.) (B) Quantification of % dense core granules in ZnT8^+/+ and ZnT8^−/− beta cells (n = 622 and 740 granules, respectively,), (C and D) Morphometric quantification of granule diameter (n = 126 and 83 granules, respectively), (C) and relative cytoplasmic volume of the granule pool (D) in ZnT8^+/+ and ZnT8^−/− beta cells. Data are mean ± SEM, statistical analysis via unpaired Student's t test: *, P < 0.05; **, P < 0.001; ***, P < 0.0001. (E) Bright light reflection of islolated ZnT8^+/+ islets contrasts with dull gray appearance of ZnT8^−/− islets under a dissection microscope.

Fig. 4.

Insulin biosynthesis, processing and release are not affected in ZnT8^−/− mice. (A) Insulin content from isolated pancreata of ZnT8^−/− and ZnT8^+/+ mice. Data are mean ± SEM, n = 4). (B) Radiometric quantification of pro-insulin synthesis and conversion to insulin. Isolated islets from ZnT8^−/− and ZnT8^+/+ mice were labeled for 30 min with ³⁵S-amino acids and chased for 30 min (upper panel) or 1 h (lower panel) in the presence of 10 mM glucose. Radioactive (pro)-insulin was quantified after immunoprecipitation, gel electrophoresis and autoradiography. Similar amounts of pro-insulin synthesis and conversion were found [30 min (≈20%) and 1 h (≈40%), (n = 3)] in both strains. (C) Glucose-induced insulin release. After overnight culture in 10 mM glucose (G), islets from adult ZnT8^+/+ (black circle) and ZnT8^−/− (open circle) mice were perifused for 30 min with a medium containing 3 mM glucose. When indicated by the arrows, the glucose concentration was increased to 15 mM, the K_ATP channel opener diazoxide (Dz) was added, and the KCl concentration was increased from 4.8 to 30 mM. Data are mean ± SEM of four experiments.

Fig. 5.

Glucose and insulin tolerance in ZnT8^+/+ and ZnT8^−/− mice fed a control diet. ZnT8^+/+ (black circles) and ZnT8^−/− (open circles) mice were given a control chow and were tested between 6 and 52 weeks of age. Blood glucose levels were measured at the indicated time points after i.p. injection of 2.5 mg glucose/g BW glucose in overnight (16 h) fasted mice aged 6 weeks (A), 12 weeks (B), 25 weeks (C), and 1 year (D). (E) Insulin tolerance test after injection of 0.75 mU/g BW insulin i.p. in 6 h fasted mice aged 12 weeks, expressed as % over baseline. Data are mean ± SEM, statistical analysis via unpaired Student's t test: *, P ≤ 0.05.