uptake of TBI by the liver was 40% lower in Dmt1liv/liv mice, compared with Dmt1flox/flox mice (Fig. 4B), revealing that hepatocyte DMT1 is partially required for hepatic uptake of iron from plasma transferrin. The effect was specific for the liver because TBI uptake was unaffected in kidneys, pancreas, or hearts of Dmt1liv/liv mice. To determine whether lower hepatic TBI uptake by Dmt1liv/liv mice represents a delay in clearance of plasma TBI, which may resolve at a later time, we measured the percentage of 59Fe in plasma 2 and 24 hours after injection. By 2 hours, the percentage of 59Fe in plasma did not differ between Dmt1flox/flox and Dmt1liv/liv mice (P = 0.11) (Fig. 4C), and by 24 hours, very little 59Fe was detectable in plasma. These data indicate Selleckchem Galunisertib that lower hepatic TBI uptake by Dmt1liv/liv mice does not represent a delay in clearance of plasma TBI. The percentage of 59Fe in the blood and spleen also did
not differ at either time point, suggesting that iron uptake into developing erythroid cells was unaffected in Dmt1liv/liv mice. Although lower hepatic TBI uptake in Dmt1liv/liv mice appears to directly result from inactivation of Dmt1, it is possible that it results from a secondary effect on other proteins implicated in TBI uptake. It is equally possible that the lack of an effect of hepatic Dmt1 inactivation on NTBI uptake is the result of compensatory responses in other proteins involved in NTBI uptake. MG-132 datasheet Therefore, we measured levels Demeclocycline of TfR1, TfR2, and ZIP14, which may also participate in TBI/NTBI uptake.[28, 29] Western blotting analysis revealed that levels of these proteins did not differ between Dmt1flox/flox and Dmt1liv/liv mice (Fig. 5A-C). To determine whether hepatocyte DMT1 is required to maintain iron status during iron deficiency, we compared iron status parameters of
Dmt1flox/flox and Dmt1liv/liv mice that were fed iron-deficient diets. After 3 weeks, mice became iron deficient, as compared to control (Dmt1flox/flox) mice fed a standard diet (Fig. 6A-D). However, no differences were observed between iron-deficient Dmt1flox/flox and Dmt1liv/liv mice. TBI uptake by livers of Dmt1flox/flox mice was higher in iron-deficient animals, compared to controls (36% versus 30%, respectively; P < 0.05) (Fig. 6E). By contrast, TBI uptake by livers of Dmt1liv/liv mice was not higher in iron-deficient animals, compared to controls, suggesting that DMT1 is required for enhanced TBI uptake into an iron-deficient liver. Confocal immunofluorescence (IF) microscopy was used to localize DMT1 in the liver. Human liver was used instead of mouse liver because IF staining of mouse tissue was too weak to allow for reliable localization. In hepatocytes, DMT1 displayed intracellular punctate staining with little, if any, staining of plasma membrane (Supporting Fig.