(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.