Zinc proteinate with moderate chelation strength enhances zinc absorption by upregulating the expression of zinc and amino acid transporters in primary cultured duodenal epithelial cells of broiler embryos

Recent study showed that zinc (Zn) and amino acid transporters may be involved in enhancing Zn absorption from Zn proteinate with moderate chelation strength (Zn-Prot M) in the duodenum of broilers. However, the specific mechanisms by which Zn-Prot M promotes the above Zn absorption are unknown. Therefore, in this study, 3 experiments were conducted to investigate specific and direct effects of Zn-Prot M and Zn sulfate (ZnS) on Zn absorption and expression of related transporters in primary duodenal epithelial cells of broiler embryos so as to preliminarily address possible mechanisms. In experiment 1, cells were treated with 100 μmol Zn/L as ZnS or Zn-Prot M for 20, 40, 60, 80, 100, or 120 min. Experiment 2 consisted of 3 sub-experiments. In experiment 2A, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 100 or 200 μmol Zn/L as ZnS or Zn-Prot M for 60 min; in experiment 2B, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 200 μmol Zn/L of as the ZnS or Zn-Prot M for 120 min; in experiment 2C, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 or 800 μmol Zn/L as ZnS or Zn-Prot M for 120 min. In experiment 3, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 μmol Zn/L as ZnS or Zn-Prot M for 120 min. The results of experiment 1 indicated that the minimum incubation time for saturable Zn absorption was determined to be 50.83 min using the best fit line. The results in experiment 2 demonstrated that a Zn concentration of 400 μmol/L and an incubation time of 120 min were suitable to increase the absorption of Zn from Zn-Prot M compared to ZnS. In experiment 3, Zn absorption across cell monolayers was significantly increased by Zn addition (P < 0.05), and was significantly greater with Zn-Prot M than with ZnS (P < 0.05). Compared to the control, Zn addition significantly decreased Zn transporter 10 and peptide-transporter 1 mRNA expression levels and increased y + L-type amino transporter 2 (y + LAT2) protein abundance (P < 0.05). Moreover, protein expression levels of zrt/irt-like protein 3 (ZIP3), zrt–irt-like protein 5 (ZIP5), and y + LAT2 were significantly greater for Zn-Prot M than for ZnS (P < 0.05). These findings suggest that Zn-Prot M promote Zn absorption by increasing ZIP3, ZIP5 and y + LAT2 protein expression levels in primary duodenal epithelial cells.Zinc proteinate with moderate chelation strength increased the zinc absorption in primary duodenal epithelial cells of broiler embryos by promoting the protein expression levels of ZIP3, ZIP5, and y + LAT2 compared to zinc sulfate.Our previous studies demonstrated that zinc (Zn) proteinate with moderate chelation strength (Zn-Prot M) exhibited the greatest bioavailability compared to the inorganic Zn sulfate (ZnS) and other organic Zn sources with either weak or strong chelation strength in broilers. Our recent study further showed that Zn and amino acid transporters could be potentially involved in promoting the absorption of Zn as Zn-Prot M in the duodenum of broilers. Nevertheless, further in vitro experiments are required to reveal the specific mechanisms by which Zn-Prot M promotes Zn absorption, where it is necessary first to investigate the specific and direct effect of Zn-Prot M on Zn absorption and the expression of Zn and amino acid transporters compared to that of ZnS. Therefore, we performed an in vitro study and found that Zn-Prot M increased Zn absorption and protein expression levels of the zrt–irt-like protein 3 (ZIP3), zrt–irt-like protein 5 (ZIP5), and y + L-type amino transporter 2 (y + LAT2) compared to ZnS, suggesting that ZIP3, ZIP5, and y + LAT2 might be involved in promoting the absorption of Zn from Zn-Prot M in the primary cultured duodenal epithelial cells of broiler embryos.