Characterization of zinc amino acid complexes for zinc delivery in vitro using Caco-2 cells and enterocytes from hiPSC

Abstract

Zn is essential for growth and development. The bioavailability of Zn is affected by several factors such as other food components. It is therefore of interest, to understand uptake mechanisms of Zn delivering compounds to identify ways to bypass the inhibitory effects of these factors. Here, we studied the effect of Zn amino acid conjugates (ZnAAs) on the bioavailabilty of Zn. We used Caco-2 cells and enterocytes differentiated from human induced pluripotent stem cells from a control and Acrodermatitis enteropathica (AE) patient, and performed fluorescence based assays, protein biochemistry and atomic absorption spectrometry to characterize cellular uptake and absorption of ZnAAs. The results show that ZnAAs are taken up by AA transporters, leading to an intracellular enrichment of Zn mostly uninhibited by Zn uptake antagonists. Enterocytes from AE patients were unable to gain significant Zn through exposure to ZnCl2 but did not show differences with respect to ZnAAs. We conclude that ZnAAs may possess an advantage over classical Zn supplements such as Zn salts, as they may be able to increase bioavailability of Zn, and may be more efficient in patients with AE.

Keywords: Absorption; Acrodermatitis enteropathica; Enterocyte; Gastro-intestinal; Zip4; hIPSC.

Conflict of interest statement

The authors declare that there is no conflict of interests regarding the publication of this paper.

Figures

Fig. 1

Uptake of Zn from ZnAAs in Caco-2 cells. a Blood levels of Zn from mice on different diets for 9 weeks. Whole-blood Zn levels were investigated by AAS in three animals per group. Animals on a Zn deficient diet (Diet 2) show significantly reduced Zn levels compared to mice on the control diet (Diet 1). Mice on the control diet with increased levels of Zn uptake antagonists (Diet 3) similarly show a significant reduction in blood-zinc levels. b–h Zinpyr-1 fluorescence intensity of Caco-2 cells incubated for 30 min with ZnCl2 solution (50 μM) or ZnAAs delivering an equivalent of 50 μM Zn2+. b ZnCl2 solution, ZnGlu, and ZnLys/Glu and ZnLys/Glu/Met significantly increase intracellular Zn. A trend for an increase was seen after application of ZnLys and ZnMet. ZnCl2 solution increases intracellular Zn levels significantly more compared to ZnLys and ZnMet (n = 10 cells per group). c After 120 min, ZnCl2 solution and all ZnAAs lead to a significant increase in intracellular Zn (n = 10 cells per group). d Application of Glu and Lys alone do not lead to differences in Zn compared to untreated controls. The application of Met alone results in a significant increase in intracellular Zn compared to controls, but significantly less than seen with ZnCl2 solution (n = 10). e The significant increase in intracellular Zn levels provided by ZnCl2 solution is not affected by the presence of a surplus of AAs in the medium. f A significant increase in intracellular Zn after application of ZnGlu, ZnLys, and ZnMet was not present when ZnGlu, ZnLys, and ZnMet were applied together with Glu, Lys and Met, respectively. g The increase in intracellular Zn is reduced in a concentration dependent manner by co-application of 10, 50, 100, 150 and 200 μM Lys or Glu, respectively (n = 10). h Exemplary images for Caco-2 cells stained with Zinpyr-1 are shown. The bottom row shows the Zinypr1 signal intensity color-coded

Fig. 2

Effect of antagonistic factors on uptake of Zn from ZnAAs in Caco-2 cells. Zinpyr-1 fluorescence intensity of Caco-2 cells. a Application of Zn uptake antagonists calcium (provided as CaCl2) and copper (provided as CuCl2) together with ZnCl2 solution and ZnAAs. A significant inhibitory effect on Zn uptake from ZnCl2 solution is seen. No significant inhibition of the uptake of Zn delivered by ZnGlu, ZnLys, ZnMet, and ZnLys/Glu/Met was observed. The uptake of ZnLys/Glu was significantly inhibited. b Co-application of Zn uptake antagonist phytic acid (PhytAc). Co-application of ZnCl2 and phytic acid leads to significantly less increase in intracellular Zn levels compared to cell treated only with ZnCl2 solution. No significant antagonistic effect was observed upon co-application of phytic acid and ZnGlu, ZnLys, ZnMet, ZnLys/Glu, and ZnLys/Glu/Met. Uptake of ZnLys/Glu/Met was higher in presence of phytic acid. Phytic acid was used in a molar ratio Zn:PhytAc = 1:50 (n = 10). c Co-application of Folic acid (FolAc). Significantly less Zn uptake via ZnCl2 solution (FolAc was used with 100 μM concentration) is observed. A significant inhibitory effect on the uptake of ZnLys, ZnLys/Glu, and ZnLys/Glu/Met was observed. Only ZnGlu and ZnMet are unaffected, although ZnMet did not significantly increase intracellular Zn levels within 30 min (n.s. not significant)

Fig. 3

Zn uptake in human enterocytes differentiated from healthy controls and patients with Acrodermatitis enteropathica. a Overview of the protocol for generation of enterocytes from hIPSC of a healthy control and AE patient. b, c Enterocytes were exposed to ZnCl2 solution (50 μM) or ZnAAs delivering an equivalent of 50 μM Zn for 30 min. Treatment with the Zn chelator TPEN confirmed the specificity of the Zinpyr-1 signal. b Intracellular Zn levels in enterocytes of the healthy control are significantly increased after treatment with ZnCl2 solution and ZnGlu. ZnLys, ZnMet, ZnLys/Glu and ZnLys/Glu/Met did not significantly increase intracellular Zn within 30 min after application. c Intracellular Zn levels in enterocytes of the AE patient did not show a significant increase after treatment with ZnCl2. Application of ZnLys/Glu/Met (significant) and ZnGlu (as trend) leads to an increase in intracellular Zn levels. d A significant difference in uptake of Zn from ZnCl2 solution between enterocytes from Control and the AE patient was detected. Uptake of ZnAAs is not significantly lower in enterocytes from the AE patient compared to the Control. Uptake from ZnLys/Glu/Met was significantly higher in cells from the AE patient. e Enterocytes differentiated from hIPSC were plated on the filter of a transwell® system. ZnCl2 solution or ZnAAs were applied to the supernatant. After 120 min, the supernatant and the basal medium were removed. Cells on the filter were grown until they were 100% confluent and are visualized by DAPI staining of cell nuclei. f After incubation with ZnCl2 or ZnAAs, a significant increase in Zinpyr-1 staining in cells (n = 10) was detected. g Left: Using cells from the Control, all treatments resulted in a significant increase of Zn concentration in the basal medium (measured by AAS). The absorption of ZnGlu was significantly less compared to ZnCl2 solution and ZnMet. Right: Using cells from the AE patient, incubation with ZnCl2 solution did not result in a significant increase of Zn concentration in the basal medium compared to untreated control cells, while application of all ZnAAs significantly increase the Zn concentration in the basal medium. The absorption of ZnLys was significantly higher compared to ZnCl2 solution, ZnGlu and ZnMet. h Comparing the healthy control and the AE patent, significant differences were detected in the absorption of Zn from ZnCl2 solution and after application of ZnGlu and ZnMet

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)