Fig. 4.

Physiological responses to Zn uptake in Caco-2 cells. a Quantification of gene expression levels using qRT-PCR. The average mRNA expression (Δct value) normalized to HMBS is shown compared to untreated (control) cells. Cells were treated with ZnCl₂ (50 μM), ZnGlu (delivering an equivalent of 50 μM), ZnGlu and Glu (200 μM), or Glu (200 μM) alone. No significant changes were detected for the Zn transporters ZIP2 and ZnT1. Expression of ZnT1 shows high dynamics and a clear trend towards an up-regulation. ZIP4 levels are significantly different between cells treated with Glu compared to ZnCl₂ and ZnGlu treatment. Amino acid transporters (SLC1A1, SLC7A6, SLC7A8 and SLC6A14) are unaffected by the treatments. SLC36A1 is significantly up-regulated after treatment with ZnGlu + Glu and shows significantly different expression in response to ZnGlu vs. Glu alone. SLC36A2 shows significantly increased expression after treatment with ZnCl₂ and ZnGlu (as a trend) (n = 3 per condition). b–d Expression of Zip2, Zip4 and ZnT1 on protein level using Western Blot. The integrated density of 3 immunoreactive bands per condition was measured. b 30 min after application of ZnCl₂ and ZnGlu, no significant regulation can be detected on protein level. c After 120 min, no alterations are detected regarding total Zip2 and Zip4 levels. A significant decrease in total cell ZnT1 concentration is observed after treatment with ZnCl₂ or ZnGlu. d Exemplary western blot bands for the evaluation of Zip2, Zip4, ZnT1 and SLC36A1, SLC36A2, and SLC6A14 protein levels after 120 min. e The AA transporter SLC6A14 shows an up-regulation on total protein level after 120 min only after treatment with ZnGlu. No significant difference is detected for SLC36A1 and SLC36A2. f Immunocytochemistry was performed on Caco-2 cells (n = 10 per condition) and the fluorescence intensity of SLC6A14, Zip2, Zip4 and ZnT1 signals measured 120 min after treatment. A significant decrease of cell surface SLC6A14 signals after treatment with ZnLys but not the other ZnAAs is detected. Application of Lys alone does not elicit these alterations. Glu treatment slightly enhances SLC6A14 surface localization. For Zip2, no significant differences are detected in cell surface location, although a trend towards a reduction after ZnCl₂ treatment and treatment with ZnAAs is seen (significant for ZnCl₂ vs. Glu). No significant differences are detected in cell surface location of Zip4. The levels of ZnT1 at the cell surface were significantly increased after treatment with both ZnCl₂ solution and ZnAAs, despite the decrease in total protein levels (c)