Influence of cytokines on Dmt1 iron transporter and ferritin expression in insulin-secreting cells

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   Figure 1

   Expression of the different Dmt1 isoforms in insulin-producing cells, primary rat islets, and PC12 cells. Total RNA was isolated 24 h after culture under control conditions from RINm5F cells, INS-1 cells, primary rat islets, and PC12 cells. The gene expression of all investigated Dmt1 isoforms was analyzed by qRT-PCR with Dmt1 1A, 1B (grey bars), +IRE and −IRE (white bars)-specific primers and with a primer pair detecting a Dmt1 consensus sequence. The expression levels were normalized to the housekeeping gene β-actin. The gene expression of total Dmt1 in each cell type was set as 100%. Data are mean±s.e.m. from 6 to 14 individual experiments.
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   Figure 2

   Effects of hypoxia on Dmt1 gene expression in insulin-producing RINm5F cells. RINm5F cells were incubated for up to 24 h with 1% O₂, for 24 h under hypoxic conditions followed by a 6 h normoxic incubation (24+6 h) or for 24 h under control conditions. After incubation, total RNA was isolated and the Dmt1 isoform expression and total Dmt1 expression was analyzed by qRT-PCR with specific primers. The expression levels were normalized to the housekeeping gene β-actin and the expression level of each Dmt1 isoform or the total Dmt1 expression under control conditions was set as 100%. Data are mean±s.e.m. from 4 to 14 individual experiments. *P<0.05, **P<0.01 compared with the control incubation.
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   Figure 3

   Effects of pro-inflammatory cytokines on the Dmt1 gene expression in insulin-producing RINm5F cells. RINm5F cells were incubated for 24 h with IL1β (600 U/ml) alone (grey bars) or a cytokine mixture (IL1β 60 U/ml, TNFα 185 U/ml, IFNγ 14 U/ml, black bars). After incubation, total RNA was isolated and the Dmt1 isoform expression and total Dmt1 expression was analyzed by qRT-PCR with specific primers. The expression levels were normalized to the housekeeping gene β-actin and the expression level of each Dmt1 isoform or the total Dmt1 expression under control conditions was set as 100%. Data are mean±s.e.m. from five to six individual experiments. *P<0.05, **P<0.01, ***P<0.005 compared with the control incubation.
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   Figure 4

   Effects of different pro-inflammatory cytokines on Dmt1 gene expression in insulin-producing INS-1E cells. INS-1E cells were incubated for 24 h under control conditions (white bars), with IL1β (600 U/ml), IFNγ (140 U/ml), or TNFα (1850 U/ml) alone (light grey bars), with IFNγ (14 U/ml) together with TNFα (185 U/ml, dark grey bars) or with the cytokine mixture (IL1β 60 U/ml, TNFα 185 U/ml, IFNγ 14 U/ml, black bars). After incubation total RNA was isolated and the Dmt1 isoform expression and total Dmt1 expression were analyzed by qRT-PCR with specific primers. The expression levels were normalized to the housekeeping gene β-actin and the expression level of each Dmt1 isoform or the total Dmt1 expression under control conditions was set as 100%. Data are mean±s.e.m. from three individual experiments. *P<0.05, **P<0.01 compared with the control incubation.
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   Figure 5

   Effects of pro-inflammatory cytokines on Dmt1 protein expression in insulin-producing INS-1E cells. INS-1E cells were incubated for 48 h with IL1β (600 U/ml) alone (grey bars) or a cytokine mixture (IL1β 60 U/ml, TNFα 185 U/ml, IFNγ 14 U/ml, black bars). After incubation protein expression of Dmt1 was quantified by western blot analysis and normalized to the housekeeping protein β-actin. Protein expression of untreated INS-1E cells was set as 100%. Data are mean±s.e.m. from eight to ten individual experiments. *P<0.05 compared with the control incubation.
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   Figure 6

   Effects of pro-inflammatory cytokines on Ft gene expression in insulin-producing RINm5F cells. RINm5F cells were incubated for 24 h under control conditions (white bars) or for 8 and 24 h with IL1β (600 U/ml) alone (grey bars) and with the cytokine mixture (IL1β 60 U/ml, TNFα 185 U/ml, IFNγ 14 U/ml, black bars). After incubation total RNA was isolated and Ft-H (A), Ft-L (B), and Mtft expression (C) was analyzed by qRT-PCR with specific primers. The expression levels were normalized to the housekeeping gene β-actin and the expression level of each Ft subunit under control conditions was set as 100%. Data are mean±s.e.m. from six individual experiments. *P<0.05, **P<0.01 compared with the control incubation; ^#P<0.05, ^##P<0.01 compared with IL1β incubation.

• © 2014 Society for Endocrinology