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. 2022 Feb 11;253(1):1-11.
doi: 10.1530/JOE-21-0271.

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine-induced cell death

Maria Karsai  1 , Richard A Zuellig  1 , Roger Lehmann  1 , Federica Cuozzo  2   3 , Daniela Nasteska  2   3 , Edlira Luca  1 , Constanze Hantel  1   4 , David J Hodson  2   3 , Giatgen A Spinas  1 , Guy A Rutter  5   6   7 , Philipp A Gerber  1
Affiliations

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine-induced cell death

Maria Karsai et al. J Endocrinol. .

Abstract

Pancreatic β-cells depend on the well-balanced regulation of cytosolic zinc concentrations, providing sufficient zinc ions for the processing and storage of insulin, but avoiding toxic effects. The zinc transporter ZnT8, encoded by SLC30A8,is a key player regarding islet cell zinc homeostasis, and polymorphisms in this gene are associated with altered type 2 diabetes susceptibility in man. The objective of this study was to investigate the role of ZnT8 and zinc in situations of cellular stress as hypoxia or inflammation. Isolated islets of WT and global ZnT8-/- mice were exposed to hypoxia or cytokines and cell death was measured. To explore the role of changing intracellular Zn2+ concentrations, WT islets were exposed to different zinc concentrations using zinc chloride or the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). Hypoxia or cytokine (TNF-α, IFN-γ, IL1-β) treatment induced islet cell death, but to a lesser extent in islets from ZnT8-/- mice, which were shown to have a reduced zinc content. Similarly, chelation of zinc with TPEN reduced cell death in WT islets treated with hypoxia or cytokines, whereas increased zinc concentrations aggravated the effects of these stressors. This study demonstrates a reduced rate of cell death in islets from ZnT8-/- mice as compared to WT islets when exposed to two distinct cellular stressors, hypoxia or cytotoxic cytokines. This protection from cell death is, in part, mediated by a reduced zinc content in islet cells of ZnT8-/- mice. These findings may be relevant for altered diabetes burden in carriers of risk SLC30A8 alleles in man.

Keywords: beta cell; hypoxia; inflammation; pancreatic islet; zinc; zinc transporter.

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Figures

Figure 1
Figure 1
Loss of ZnT8 protects against hypoxia-mediated islet cell death despite impaired glucose control. (A). The proportion of dead cells in islets of WT and ZnT8−/− mice at the age of ~25 weeks is depicted after incubation of islets for 24 h at normoxia (21% O2) or hypoxia (1% O2, Hx), n (islet number) ≥ 100 islets per condition. (B) A glucose tolerance test was performed after i.p. administration of glucose (2 g/kg body weight) in 48-week-old mice, n  = 4 per group. Corresponding areas under the curve (AUC) are shown. (C) Glucose control was assessed by HbA1c determination in WT and ZnT8−/− mice at the age of ~25 weeks, n (number of mice) = 4 in each group. Data are expressed as mean ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Generalized multivariate analysis (A), Student’s t-test (B) and Mann–Whitney U test (C) were performed.
Figure 2
Figure 2
ZnT8-mediated changes of hypoxia-induced islet cell death rate depend on Zn2+. (A) Cell death in pancreatic islets from WT mice is induced by ZnCl2 in a dose-dependent manner. n (islet number) ≥ 30 per condition. (B) The proportion of dead cells was assessed in islets of WT and ZnT8−/− mice exposed to 600 µM ZnCl2 in the presence of normoxia (21% O2, Nx) or hypoxia (1% O2, Hx) for 24 h. n (islet number) ≥ 50 per condition. (C) Islets of WT mice were exposed to normoxia or hypoxia for 24 h with or without 50 µM of the zinc chelator TPEN. n (islet number) ≥ 80 per condition. Representative microscopy images of pancreatic islets are depicted: staining of viable cells (calcein-AM, green) and cell death (PI, red). Data are expressed as mean ± s.d. ns, not significant; * P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Generalized multivariate analysis was performed.
Figure 3
Figure 3
Cytokine-mediated cell death in pancreatic islets is dependent on the level of ZnT8 expression and Zn2+ concentrations. The proportion of dead cells in islets of WT and ZnT8−/− mice at age of (A) ~14 weeks and (B) ~29 weeks is depicted after incubation of islets for 24 h with or without a mixture of cytokines (1000 U/mL TNF-α + 1000 U/mL IFN-γ + 50 U/mL IL1-β, Mix). n (islet number) ≥ 250 per condition. (C) The cytosolic zinc concentration was estimated applying FluoZin-3AM staining in islets of WT and ZnT8−/− mice with and without a mixture of cytokines (Mix). Measurements were corrected for autofluorescence (no FluoZin condition). n (islet number) ≥ 80 per condition. (D) The proportion of dead cells in islets of WT and ZnT8−/− mice is depicted after incubation of islets for 24 h with a mixture of cytokines (Mix), 600 µM ZnCl2 or both. n (islet number) ≥ 80 per condition. (E) The proportion of dead cells in WT islets is depicted after 24 h incubation with cytokine mixture (Mix) and/or the zinc chelator TPEN. n (islet number) ≥ 100 per condition. (F) Fold change of positively stained cells for apoptosis (TUNEL, vs control) and (G) for proliferation (PCNA, vs control) in WT pancreatic islets is depicted after incubation (24 h) with different combinations of cytokines (Mix), ZnCl2 or TPEN. Representative microscopy images of pancreatic islets are depicted: Staining of apoptosis (TUNEL, green), proliferation (PCNA, red) and DAPI (blue). n (islet number) ≥ 12 per condition. Data are expressed as mean ± s.d. ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Generalized multivariate analysis (A, B, D, E), Mann–Whitney U test (C) and ANOVA (F, G) were performed.
Figure 4
Figure 4
The effect of cytokine treatment on GSIS in pancreatic islets from ZnT8−/− and WT mice. Mouse islets were incubated for 24 h with a cytokine mixture (1000 U/mL TNF-α + 1000 U/mL IFN-γ + 50 U/mL IL1-β, Mix). The insulin concentration (fmol/min/IEQ) in the supernatant was determined following incubation for 1 h in each 3.3 mM, 16.7 mM and again 3.3 mM glucose. Experiments were performed with three islet pools per condition (≥150 islets in each pool). Data are represented as mean ± s.d.**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; Mann–Whitney U test was performed.
Figure 5
Figure 5
The effect of cytokine treatment on the expression of genes encoding zinc transporters in pancreatic islets. Mouse WT islets were incubated for 24 h with a cytokine mixture (Mix). (A) Slc30a (ZnT) mRNA expression, (B) Slc39a (ZiP) mRNA expression. (C) EndoC-βH5 cells were incubated for 24 h with a cytokine mixture (Mix) and protein expression was assessed by Western blot analysis. Data are expressed as mean ± s.d.n (replicates number) = 4; *P≤ 0.05 **P ≤ 0.01, ***P ≤ 0.001; Student’s t-test was performed.
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