Regulation of the Metal Transporters ZIP14 and ZnT10 by Manganese Intake in Mice

Abstract

The metal transporters ZIP14 and ZnT10 play key physiological roles in maintaining manganese (Mn) homeostasis. However, in vivo regulation of these two transporters by Mn is not understood. Here, we examined how dietary Mn intake regulates ZIP14 and ZnT10 by feeding mice a low-Mn diet, a control diet, or a high-Mn diet for 6 weeks. Inductively coupled plasma mass spectrometry was used to measure Mn and iron (Fe) levels. ZIP14 and ZnT10 protein levels were measured by western blot analysis. While mice on the high-Mn diet exhibited significantly higher levels of Mn in the blood, liver, and brain, the low-Mn diet group did not display matching reductions, indicating that high Mn intake is more effective in disrupting Mn homeostasis in mice. Additionally, Fe levels were only slightly altered, suggesting independent transport mechanisms for Mn and Fe. In the high-Mn diet group, ZIP14 and ZnT10 were both upregulated in the liver, as well as in the small intestine, indicating a coordinated role for these transporters in Mn excretion. Unexpectedly, this upregulation only occurred in male mice, with the exception of hepatic ZIP14, providing new insight into mechanisms behind widely observed sex differences in Mn homeostasis.

Keywords: ZIP14; ZnT10; homeostasis; intestine; liver; manganese; transporters.

Figure 1

Relative body weights of mice upon initiation of 0.1, 20, and 2000 ppm Mn diets. (A) Male mice. (B) Female mice. Body weights expressed as percent increase from baseline during 6-week diet period (n = 6/sex). Data expressed as mean ± SEM.

Figure 2

Whole blood Mn levels in mice on 0.1, 20, and 2000 ppm Mn diets. Mn content measured by inductively coupled plasma mass spectrometry (ICP-MS) in (A) male and (B) female mice at 12 weeks of age (n = 6/sex). Data expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Tissue Mn levels in mice on 0.1, 20, and 2000 ppm Mn diets. Mn content measured by ICP-MS in male and female mice at 12 weeks of age (n = 6/sex). (A and B) Liver. (C and D) Brain. Data expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Whole Blood Fe levels in mice on 0.1, 20, and 2000 ppm Mn diets. Fe content measured by ICP-MS in (A) male and (B) female mice at 12 weeks of age (n = 6/sex). Data expressed as mean ± SEM. * p < 0.05.

Figure 5

Tissue Fe levels in mice on 0.1, 20, and 2000 ppm Mn diets. Fe content measured by ICP-MS in male and female mice at 12 weeks of age (n = 6/sex). (A and B) Liver. (C and D) Spleen. (E and F) Brain. Data expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Comparison of transporter protein levels in the liver. Expression of (A and B) ZIP14 and (C and D) ZnT10 in male and female mice on 0.1, 20, and 2000 ppm Mn diets (n = 6/sex). Liver samples were obtained at 12 weeks of age and analyzed via western blot. Quantifications of protein levels were performed using GAPDH as a loading control. ZIP14 blots also include results from an age-matched Zip14 knockout (KO) mouse as a control (fed traditional rodent diet). Data expressed as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 7

Comparison of transporter protein levels in the small intestine. Expression of (A and B) ZIP14 and (C and D) ZnT10 in male and female mice on 0.1, 20, and 2000 ppm Mn diets (n = 4–6/sex). Proximal sections of the small intestine were obtained during sacrifice at 12 weeks of age and samples that did not degrade during homogenization were analyzed via western blot. Protein quantifications were performed using GAPDH as a loading control. ZIP14 blots also include results from an age-matched Zip14 knockout (KO) mouse as a control (fed traditional rodent diet). Data expressed as mean ± SEM. * p < 0.05, *** p < 0.001.

Figure 8

Proposed model for the regulation of ZIP14 and ZnT10 under high-Mn conditions. The transporters required for dietary Mn absorption across the apical (AP) and basolateral (BL) membranes of enterocytes are not fully established. In the intestine, ZIP14 transports blood Mn into the enterocyte, while ZnT10 secretes Mn from the enterocyte into the intestinal lumen, both limiting absorption. In the liver, ZIP14 takes up Mn from the blood into the hepatocyte, while ZnT10 secretes Mn into the bile. In male mice, when dietary intake of Mn is high, intestinal ZIP14 and ZnT10 are upregulated to reduce absorption, and hepatic ZIP14 and ZnT10 are upregulated to increase biliary excretion. In female mice, however, only hepatic ZIP14 is significantly upregulated during high dietary Mn conditions.