The zinc transporter Zip14 (SLC39a14) affects Beta-cell Function: Proteomics, Gene expression, and Insulin secretion studies in INS-1E cells

Abstract

Insulin secretion from pancreatic beta-cells is dependent on zinc ions as essential components of insulin crystals, zinc transporters are thus involved in the insulin secretory process. Zip14 (SLC39a14) is a zinc importing protein that has an important role in glucose homeostasis. Zip14 knockout mice display hyperinsulinemia and impaired insulin secretion in high glucose conditions. Endocrine roles for Zip14 have been established in adipocytes and hepatocytes, but not yet confirmed in beta-cells. In this study, we investigated the role of Zip14 in the INS-1E beta-cell line. Zip14 mRNA was upregulated during high glucose stimulation and Zip14 silencing led to increased intracellular insulin content. Large-scale proteomics showed that Zip14 silencing down-regulated ribosomal mitochondrial proteins, many metal-binding proteins, and others involved in oxidative phosphorylation and insulin secretion. Furthermore, proliferation marker Mki67 was down-regulated in Zip14 siRNA-treated cells. In conclusion, Zip14 gene expression is glucose sensitive and silencing of Zip14 directly affects insulin processing in INS-1E beta-cells. A link between Zip14 and ribosomal mitochondrial proteins suggests altered mitochondrial RNA translation, which could disturb mitochondrial function and thereby insulin secretion. This highlights a role for Zip14 in beta-cell functioning and suggests Zip14 as a future pharmacological target in the treatment of beta-cell dysfunction.

Figure 1

Measurements of Zip14, Znt8, and insulin mRNA by PrimeFlow RNA Assay. INS-1E cells were incubated with 5, 11, 16, or 24 mM glucose for 24 h. Zip14, Znt8, and insulin (Ins) mRNA levels were measured by flow cytometry using specific RNA probes incorporated in PrimeFlow RNA assays. (A) Zip14 mRNA probe, (B) Znt8 mRNA probe, and (C) Insulin mRNA probe. (A–C) Mean median fluorescence value ± SEM of indicated mRNA probe at 5, 11, 16, and 24 mM glucose. n = 4–6 in each group. *p < 0.05, **p < 0.01 compared with 5 mM glucose as reference.

Figure 2

Transfection efficiency. Transfection efficiency was assessed by measurement of mRNA expression level by real-time PCR and at the protein level by selected reaction monitoring mass spectrometry. Zip14 siRNA-treated cells were compared with non-targeting siRNA-treated cells (Control siRNA). (A) Zip14 mRNA expression (n = 6). Results are expressed as the mean starting quantity of (Zip14/three house-keeping genes) ± SEM. (B) ZIP14 protein expressed as a mean ratio of an endogenous ZIP14-specific peptide/a heavy labeled standard peptide with the same sequence ± SEM. (Endogen Zip14/Internal standard) (n = 4–5). *p < 0.05.

Figure 3

mRNA expression levels of zinc transporters. Measurements of zinc transporter mRNAs by real-time PCR in Zip14 siRNA- and non-targeting siRNA-treated samples (Control siRNA). Results are expressed as the mean starting quantity of (gene of interest/three house-keeping genes) ± SEM. (A) ZnT transporters; Znt1, Znt3, Znt5, and Znt8. (B) Zip transporters; Zip6, Zip9, and Zip13.

Figure 4

mRNA expression levels of metallothioneins. Measurements of metallothionein mRNA expression by real-time PCR in Zip14 siRNA- and non-targeting siRNA-treated (Control siRNA) cells. Results are expressed as the mean starting quantity of (gene of interest/three house-keeping genes) ± SEM. (A) Mt1A mRNA expression. (B) Mt3 mRNA expression. *p < 0.05.

Figure 5

Western blotting of Chga (Chromogranin a) and B-actin (internal control). Sample 1–5 from left; Zip14 siRNA-treated samples. Sample 6–9 from left; non-targeted siRNA-treated samples (Control). 5 μg of protein were used.

Figure 6

Insulin secretion and intracellular insulin content of INS-1E cells treated with Zip14 siRNA or non-targeting siRNA at diverse glucose concentrations. Cells were treated with Zip14 siRNA or non-targeting siRNA (Control siRNA) and incubated for 24 h in medium containing 5 mM, 16 mM, or 24 mM glucose (n = 6 replicates, except for 16 mM Control siRNA, where n = 5). Insulin measurements were performed using Rat Insulin Elisa kits and normalized to the protein concentration. Data are mean ± SEM. *p < 0.05, **p < 0.01. (A) Insulin secretion, expressed as µIU insulin/µg of protein. (B) Intracellular insulin content, expressed as µIU insulin/µg of protein. (C) Insulin secretion/intracellular insulin content ratio. (D) Insulin (Ins) mRNA expression at 11 mM glucose, expressed as mean starting quantity of (insulin/three house-keeping genes) ± SEM, measured by real-time PCR.

Figure 7

Overall viability, apoptosis, necrosis, and Bax/Bcl-2 index in INS-1E cells treated with Zip14 siRNA or non-targeting siRNA after 24 h incubation in diverse glucose concentrations. INS-1E cells were transfected with Zip14 siRNA or non-targeting siRNA (Control siRNA) and incubated for 24 h in medium containing 5 mM, 11 mM, 16 mM, or 24 mM glucose. Viability measurements were performed using Via1-cassettes (Chemometec, Denmark) in a Nucleocounter NC-3000 (experiment performed twice, n = 9 in total). A Cell Death Detection ELISAPLUS kit was used for the measurement of apoptosis or necrosis. Detection was performed using an ELISA Reader at 405 nm. n = 6 in each group, except for the 16 mM Control siRNA group, where n = 4. Data are mean ± SEM. (A) Percentage of viable cells, assessed using acridine orange and DAPI staining. (B) Apoptosis, measured as the amount of intracellular histone-associated DNA fragments. (C) Necrosis, measured as the amount of histone-associated DNA fragments in the culture medium. *p < 0.05. (D) The Bax/Bcl-2 index, measured by real-time PCR. Gene expression was measured as mean starting quantity (gene of interest/three house-keeping genes) ± SEM.