Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity

Abstract

Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.

Figure 1. Global Slc39a8 deletion leads to systemic Mn deficiency.

(A) qPCR analysis of Slc39a8 expression in male Slc39a8^fl/fl and ZIP8-iKO mice injected with tamoxifen at 8 weeks of age and sacrificed 5 weeks after the injection (n = 3–6). (B) ICP-OES analysis of Mn levels in male Slc39a8^fl/fl and ZIP8-iKO mice injected with tamoxifen at 8 weeks of age and sacrificed 5 weeks after injection (n = 5–6). (C) qPCR analysis of Slc39a8 expression in 12- to 14-week-old male WT and ZIP8 Het mice (n = 4–6). (D) ICP-OES analysis of Mn levels in 12- to 14-week-old male WT and ZIP8 Het mice (n = 4–5). qPCR results were normalized to Gapdh. ICP-OES results were normalized to wet tissue weight. Mn levels in the tissues were normalized to the average of the control group. Data on the absolute Mn content can be found in the Supplemental Table. All data represent the mean ± SD. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, by Student’s t test.

Figure 2. Hepatic ZIP8 regulates whole-body Mn homeostasis.

(A) qPCR analysis of Slc39a8 expression in 12- to 14-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice (n = 3–4). (B) ICP-OES analysis of Mn levels in 12- to 14-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice (n = 4). (C) ICP-MS analysis of Mn levels in the whole blood of 14- to 16-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice (n = 7 and 6, respectively). (D) ICP-OES analysis of Mn levels in 12- to 14-week-old male Slc39a8^fl/fl mice injected with AAV-null and in ZIP8-LSKO mice injected with AAV-null or AAV-ZIP8 (n = 3–6). (E) Western blot analysis of ZIP8 in liver lysates of male B6 mice injected with AAV-null or AAV-ZIP8 at 10 weeks of age and sacrificed 4 weeks after injection. Arrows indicate the ZIP8 bands. (F) ICP-OES analysis of Mn levels in male B6 mice injected with AAV-null or AAV-ZIP8 at 10 weeks of age and sacrificed 4 weeks after injection (n = 6). (G) ICP-MS analysis of Mn levels in the whole blood of male B6 mice injected with AAV-null or AAV-ZIP8 at 8 weeks of age and sacrificed 4 weeks after injection (n = 5 and 4, respectively). qPCR results were normalized to Gapdh. ICP-OES results were normalized to wet tissue weight. Mn levels were normalized to the average of the control group. Data on the absolute Mn content can be found in the Supplemental Table. (A–C, F, and G) Comparisons between 2 groups were performed using Student’s t test. Multiple comparisons in D were performed using 1-way ANOVA and Tukey’s multiple comparisons test. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05. (H) Correlation analysis between hepatic Mn and Mn levels in the kidney, brain, and heart of WT and ZIP8-LSKO mice and AAV-null– and AAV-ZIP8–injected B6 mice (n = 19). (I) Correlation analysis between hepatic Mn and Mn levels in the whole blood of WT and ZIP8-LSKO mice and AAV-null– and AAV-ZIP8–injected B6 mice (n = 22). Mn levels were normalized to the average of the control group. The results in H and I were analyzed by Pearson’s test. All data are shown as the mean ± SD.

Figure 3. ZIP8 reclaims Mn from the bile.

(A and B) Immunofluorescence analysis of ZIP8 (green) and MDR1 (magenta) localization in Slc39a8^fl/fl and ZIP8-LSKO mouse liver sections. Arrows indicate bile ducts. Results are representative of 3 independent experiments. (C) ⁵⁴Mn uptake study of HEK293T cells overexpressing human ZIP8 (n = 3). (D) ICP-MS analysis of Mn levels in the bile of 12- to 14-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice (n = 4). (E) ICP-MS analysis of Mn levels in the bile of male B6 mice injected with AAV-null or AAV-ZIP8 at 10 weeks of age and sacrificed 4 weeks after injection (n = 6 and 5, respectively). Scale bars: 10 μm. All data are shown as the mean ± SD. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤0.05, by Student’s t test.

Figure 4. ZIP8 acts through Mn to quantitatively modulate arginase activity.

(A) Arginase activity in the livers of 8- to 10-week-old male Slc39a8^fl/fl mice (WT) injected with AAV-null and ZIP8-LSKO mice injected with AAV-null or AAV-ZIP8 and sacrificed 4 weeks after injection (n = 7, 4, and 4, respectively). (B) Arginase activity in the livers of 10-week-old male B6 mice injected with AAV-null or AAV-ZIP8 and sacrificed 4 weeks after injection (n = 5 and 6, respectively). (C) Western blot analysis of arginase protein in the liver lysates of mice depicted in A and B. (D and E) Arginase activity in the livers of 10-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice after preincubation with increasing concentrations of MnCl₂. Lysates from 3 mice of the same genotype were pooled, and 3 technical replicates were performed. (F) Arginase activity normalized to the average of the Slc39a8^fl/fl liver lysate at each MnCl₂ concentration. (G) Arginase activity in the livers of individual 10-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice, with or without preincubation with 250 μM MnCl₂ (n = 6 and 7, respectively). (H) Correlation analysis of hepatic Mn levels and arginase activity in all 4 mouse models. Mn levels and arginase activity were normalized to the average of the control groups (n = 41). (I). ICP-OES analysis of kidney Mn levels in 10- to 12-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice (n = 5). ICP-OES results were normalized to wet tissue weight. Mn levels were normalized to the average of the control group. (J) Arginase activity in the kidneys of mice described in I (n = 5). (K) Correlation analysis of kidney Mn levels and arginase activity in mice depicted in I and J (n = 10). Mn levels and arginase activity were normalized to the average of the control group. All data are shown as the mean ± SD. Comparisons between 2 groups were performed by Student’s t test. Multiple comparisons in A were performed using 1-way ANOVA and Tukey’s multiple comparisons test, and multiple comparisons in G were performed using 2-way ANOVA and Bonferroni’s post-hoc test. ***P ≤ 0.001, **P ≤ 0.01. Correlation analyses were performed using Pearson’s test.

Figure 5. Slc39a8 loss of function results in protein N-glycosylation defects.

(A and B) MALDI-TOF analysis of the N-glycan profile for serum obtained 5 weeks after male Slc39a8^fl/fl and ZIP8-iKO mice were injected with tamoxifen at 8 weeks of age. (C and D) MALDI-TOF analysis of the N-glycan profile for serum obtained from 10- to 12-week-old male Slc39a8^fl/fl and ZIP8-LSKO mice. Each sample was pooled from 5 mice of the same genotype. White diamonds, sialic acid; yellow circles, galactose; blue squares, N-acetyl-glucosamine; green circles, mannose. The numbers above the peaks indicate the mass-to-charge ratios of the N-glycan species. 2853, disialo-biantennary glycans; 2448, monosialo-digalacto-biantennary glycans; 2257, monosialo-monogalacto-biantennary glycans; 1852, asialo-monogalacto-biantennary glycans; 1661, asialo-agalacto-biantennary glycans; 1416, asialo-agalacto-mono-GlcNAc-biantennary N-glycan. (E) Abundance of monosialo-monogalacto-biantennary glycans in the plasma of rs13107325 major and minor allele homozygotes. N = 11 and 12, respectively. Data are shown as the mean ± SD. Comparisons were performed using Student’s t test. *P ≤ 0.05.