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SLC30A3 responds to glucose- and zinc variations in beta-cells and is critical for insulin production and in vivo glucose-metabolism during beta-cell stress.

Smidt K, Jessen N, Petersen AB, Larsen A, Magnusson N, Jeppesen JB, Stoltenberg M, Culvenor JG, Tsatsanis A, Brock B, Schmitz O, Wogensen L, Bush AI, Rungby J - PLoS ONE (2009)

Bottom Line: In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression.ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels.Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Aarhus, Arhus, Denmark.

ABSTRACT

Background: Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. beta-cells depend on zinc for both insulin crystallization and regulation of cell mass.

Methodology/principal findings: This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in beta-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a beta-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced beta-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals.

Conclusion/significance: Zinc transporting proteins in beta-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

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Insulin expression, content and secretion after 24 hours of 100 µM DEDTC treatment.INS-1E cells were treated with DEDTC at 5 mM glucose. A) Insulin gene expression normalized to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Insulin secretion related to insulin content in INS-1E cells. Data are mean and SEM (*p<0.01). N = 3.
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pone-0005684-g003: Insulin expression, content and secretion after 24 hours of 100 µM DEDTC treatment.INS-1E cells were treated with DEDTC at 5 mM glucose. A) Insulin gene expression normalized to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Insulin secretion related to insulin content in INS-1E cells. Data are mean and SEM (*p<0.01). N = 3.

Mentions: In order to examine the effects of zinc chelation we treated INS-1E cells grown for 24 hours at 11 mM glucose with a zinc chelator (100 µM DEDTC) and visualized free intracellular zinc ions by autometallography. We found that DEDTC removed most of the detectable zinc in β-cells (Fig. 2). After 24 hours of DEDTC-treatment at 5 mM glucose we examined insulin gene expression, intracellular insulin content and insulin secretion along with the expression of ZnTs. Insulin expression decreased significantly after DEDTC treatment (p<0.01), whereas the ratio between insulin secretion and insulin content was increased in DEDTC treated cells (p<0.01) due to a significantly lower insulin content in DEDTC treated cells (data not shown) (Fig. 3A and Fig. 3B). DEDTC treatment stimulated the expression of ZnT-3 significantly (p<0.05) (Fig. 4A) but decreased the expression of ZnT-8 (p<0.01) (Fig. 4B) (all comparisons relative to untreated cells). We have previously demonstrated that ZnT-3, ZnT-8 and insulin expression varies with DEDTC treatment, and that the results depend on specific housekeeping genes [29]. Accordingly we normalised to HPRT, Cltc and HSP since they were the most stable HKGs.


SLC30A3 responds to glucose- and zinc variations in beta-cells and is critical for insulin production and in vivo glucose-metabolism during beta-cell stress.

Smidt K, Jessen N, Petersen AB, Larsen A, Magnusson N, Jeppesen JB, Stoltenberg M, Culvenor JG, Tsatsanis A, Brock B, Schmitz O, Wogensen L, Bush AI, Rungby J - PLoS ONE (2009)

Insulin expression, content and secretion after 24 hours of 100 µM DEDTC treatment.INS-1E cells were treated with DEDTC at 5 mM glucose. A) Insulin gene expression normalized to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Insulin secretion related to insulin content in INS-1E cells. Data are mean and SEM (*p<0.01). N = 3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683566&req=5

pone-0005684-g003: Insulin expression, content and secretion after 24 hours of 100 µM DEDTC treatment.INS-1E cells were treated with DEDTC at 5 mM glucose. A) Insulin gene expression normalized to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Insulin secretion related to insulin content in INS-1E cells. Data are mean and SEM (*p<0.01). N = 3.
Mentions: In order to examine the effects of zinc chelation we treated INS-1E cells grown for 24 hours at 11 mM glucose with a zinc chelator (100 µM DEDTC) and visualized free intracellular zinc ions by autometallography. We found that DEDTC removed most of the detectable zinc in β-cells (Fig. 2). After 24 hours of DEDTC-treatment at 5 mM glucose we examined insulin gene expression, intracellular insulin content and insulin secretion along with the expression of ZnTs. Insulin expression decreased significantly after DEDTC treatment (p<0.01), whereas the ratio between insulin secretion and insulin content was increased in DEDTC treated cells (p<0.01) due to a significantly lower insulin content in DEDTC treated cells (data not shown) (Fig. 3A and Fig. 3B). DEDTC treatment stimulated the expression of ZnT-3 significantly (p<0.05) (Fig. 4A) but decreased the expression of ZnT-8 (p<0.01) (Fig. 4B) (all comparisons relative to untreated cells). We have previously demonstrated that ZnT-3, ZnT-8 and insulin expression varies with DEDTC treatment, and that the results depend on specific housekeeping genes [29]. Accordingly we normalised to HPRT, Cltc and HSP since they were the most stable HKGs.

Bottom Line: In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression.ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels.Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Aarhus, Arhus, Denmark.

ABSTRACT

Background: Ion transporters of the Slc30A- (ZnT-) family regulate zinc fluxes into sub-cellular compartments. beta-cells depend on zinc for both insulin crystallization and regulation of cell mass.

Methodology/principal findings: This study examined: the effect of glucose and zinc chelation on ZnT gene and protein levels and apoptosis in beta-cells and pancreatic islets, the effects of ZnT-3 knock-down on insulin secretion in a beta-cell line and ZnT-3 knock-out on glucose metabolism in mice during streptozotocin-induced beta-cell stress. In INS-1E cells 2 mM glucose down-regulated ZnT-3 and up-regulated ZnT-5 expression relative to 5 mM. 16 mM glucose increased ZnT-3 and decreased ZnT-8 expression. Zinc chelation by DEDTC lowered INS-1E insulin content and insulin expression. Furthermore, zinc depletion increased ZnT-3- and decreased ZnT-8 gene expression whereas the amount of ZnT-3 protein in the cells was decreased. Zinc depletion and high glucose induced apoptosis and necrosis in INS-1E cells. The most responsive zinc transporter, ZnT-3, was investigated further; by immunohistochemistry and western blotting ZnT-3 was demonstrated in INS-1E cells. 44% knock-down of ZnT-3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion. Streptozotocin-treated mice had higher glucose levels after ZnT-3 knock-out, particularly in overt diabetic animals.

Conclusion/significance: Zinc transporting proteins in beta-cells respond to variations in glucose and zinc levels. ZnT-3, which is pivotal in the development of cellular changes as also seen in type 2 diabetes (e.g. amyloidosis in Alzheimer's disease) but not previously described in beta-cells, is present in this cell type, up-regulated by glucose in a concentration dependent manner and up-regulated by zinc depletion which by contrast decreased ZnT-3 protein levels. Knock-down of the ZnT-3 gene lowers insulin secretion in vitro and affects in vivo glucose metabolism after streptozotocin treatment.

Show MeSH
Related in: MedlinePlus