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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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Knock-down of ZnT-3 in INS-1E cells.A) Relative gene expression of ZnT-3 in ZnT-3 knock-downed INS-1E cells compared to mismatch controls. ZnT-3 gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05) N = 3. B) Relative gene expression of insulin in ZnT-3 knock-down INS-1E cells. Insulin gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05). N = 3. C) Glucose stimulated insulin secretion in ZnT-3 knock-down INS-1E cells. Cells were stimulated with either 2, 6.6 or 16.6 mM glucose for 2 hours. Insulin secretion was normalised to insulin content. Data are mean and SEM (p<0.01 for AUC). N = 4.
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pone-0005684-g007: Knock-down of ZnT-3 in INS-1E cells.A) Relative gene expression of ZnT-3 in ZnT-3 knock-downed INS-1E cells compared to mismatch controls. ZnT-3 gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05) N = 3. B) Relative gene expression of insulin in ZnT-3 knock-down INS-1E cells. Insulin gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05). N = 3. C) Glucose stimulated insulin secretion in ZnT-3 knock-down INS-1E cells. Cells were stimulated with either 2, 6.6 or 16.6 mM glucose for 2 hours. Insulin secretion was normalised to insulin content. Data are mean and SEM (p<0.01 for AUC). N = 4.

Mentions: To examine the in vitro effect of ZnT-3 knock-down we transfected INS-1E with ZnT-3 siRNA. The ZnT-3 mRNA level was knocked down by 44% (p<0.05) and insulin expression was reduced by 41% (p<0.05) (Fig. 7A and Fig. 7B). AUC for insulin secretion/insulin content was decreased in ZnT-3 siRNA transfected INS-1E cell compared to mock-transfected INS-1E cells (p<0.05) (Fig. 7C).


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)

Knock-down of ZnT-3 in INS-1E cells.A) Relative gene expression of ZnT-3 in ZnT-3 knock-downed INS-1E cells compared to mismatch controls. ZnT-3 gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05) N = 3. B) Relative gene expression of insulin in ZnT-3 knock-down INS-1E cells. Insulin gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05). N = 3. C) Glucose stimulated insulin secretion in ZnT-3 knock-down INS-1E cells. Cells were stimulated with either 2, 6.6 or 16.6 mM glucose for 2 hours. Insulin secretion was normalised to insulin content. Data are mean and SEM (p<0.01 for AUC). N = 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683566&req=5

pone-0005684-g007: Knock-down of ZnT-3 in INS-1E cells.A) Relative gene expression of ZnT-3 in ZnT-3 knock-downed INS-1E cells compared to mismatch controls. ZnT-3 gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05) N = 3. B) Relative gene expression of insulin in ZnT-3 knock-down INS-1E cells. Insulin gene expression normalised to β-actin and HSP. Data are mean and SEM (*p<0.05). N = 3. C) Glucose stimulated insulin secretion in ZnT-3 knock-down INS-1E cells. Cells were stimulated with either 2, 6.6 or 16.6 mM glucose for 2 hours. Insulin secretion was normalised to insulin content. Data are mean and SEM (p<0.01 for AUC). N = 4.
Mentions: To examine the in vitro effect of ZnT-3 knock-down we transfected INS-1E with ZnT-3 siRNA. The ZnT-3 mRNA level was knocked down by 44% (p<0.05) and insulin expression was reduced by 41% (p<0.05) (Fig. 7A and Fig. 7B). AUC for insulin secretion/insulin content was decreased in ZnT-3 siRNA transfected INS-1E cell compared to mock-transfected INS-1E cells (p<0.05) (Fig. 7C).

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