James F. Collins

Mechanisms of heme-iron (HI) absorption remain uncertain. Heme is probably absorbed by receptor-mediated endocytosis; iron is then enzymatically liberated from heme within endosomes, or in the cytosol. If heme is catabolized in endosomes, a transporter, such as DMT1, would be required to export ionic iron into the cytosol. Here, we tested the hypothesis that intestinal DMT1 functions in the HI absorption pathway. The first experimental approach utilized Belgrade (b) rats, expressing dysfunctional DMT1 protein, and the second used conditional, intestine-specific DMT1 KO mice. HI absorption experiments were carried out with radiolabeled, donor rat RBCs, containing ~98% of iron as HI and with >94% of activity in 59Fe-heme. Iron-deficient, anemic +/b (control) rats absorbed 10.8% of a peroral dose of 59Fe-heme within 24 hours, while similarly anemic b/b rats absorbed 1.4% (an 8-fold reduction). Notably, serum hepcidin levels were invariable between both groups of rats (and 10x lower than controls). Moreover, ablation of intestinal DMT1 led to iron-restricted erythropoiesis and severe anemia in adolescent mice weaned on to nonheme iron (NHI) or HI diets. DMT1 was also required to upregulate 59Fe-heme absorption in iron-deficient, anemic mice. DMT1 thus facilitates the absorption of both main forms of dietary iron, NHI and HI.

Dietary iron exists mainly as non-heme iron (NHI) and heme iron (HI). Mechanisms of NHI absorption have been recently revealed, but details of HI absorption remain uncertain. Heme is likely absorbed by receptor-mediated endocytosis, but to date, no receptor (or transporter) has been identified. Within enterocytes, heme may be catabolized by heme oxygenase in the ER, thus liberating ionic iron that then joins the labile cytosolic iron pool. One limiting factor in HI absorption research has been the lack of tractable experimental models. We addressed this shortcoming by establishing a nutritional paradigm that enables investigation of this process using pre-clinical rat and mouse models (JCI Insight. 2025;10(11):e184742). The approach utilized specially formulated diets containing lyophilized porcine RBCs that contained ~ 85% HI and 15% NHI. We have expanded upon these studies here to include heme iron polypeptide (HIP) as an iron source (Proferrin; Colorado Biolabs). HIP, a partially proteolyzed form of hemoglobin, is highly enriched for HI (~ 93% HI, 7% NHI). We again utilized a classic depletion/repletion experimental design in SD rats and C57BL/6 mice to compare outcomes when using three AIN-93-based test diets with ~ 50 ppm total iron: a HIP diet, a NHI diet containing FeSO₄ and a HI-enriched diet formulated with porcine RBCs. Outcomes notably showed that all three experimental diets similarly corrected the anemia and normalized erythropoietic biomarkers in iron-depleted rats and mice within the 24-28-day test periods. These results solidify the concept that laboratory rodents are useful in vivo models to study intestinal HI absorption and utilization.

Iron is an essential nutrient for most organisms; however, it can also be toxic when present in excess. For this reason, life has evolved complex pathways to tightly control the amount of iron contained within body tissues. Unlike most other nutrients, mammals do not have a regulated excretory mechanism for iron and, therefore, must regulate body iron levels at the point of dietary iron absorption in the proximal small intestine. In this chapter, we will describe the molecules and pathways involved in this important process, including our current understanding of the mechanisms of both heme and non-heme iron absorption. The regulation of this process will also be discussed, with particular emphasis on local and systemic factors that affect how much iron is absorbed from the diet. We also highlight areas where our knowledge is incomplete and where more research is required to fully understand the molecular mechanisms responsible for this essential process.

Heme iron (HI), derived principally from hemoglobin (Hb) in animal foods, is a highly bioavailable source of dietary iron for humans. Despite several decades of focused research, however, molecular mechanisms governing HI absorption remain undefined. Previous studies in mice and rats have not produced a consensus, definitive model of efficient HI absorption/utilization. We hypothesized that a nutritional approach, using semipurified, HI-containing diets, could be utilized to establish a tractable rodent model of HI absorption that could ultimately be employed to test the roles of receptors, transporters, and enzymes using genetic engineering technology. Experiments were designed to assess HI utilization by feeding animals AIN-93G–based, HI-enriched experimental diets formulated with lyophilized porcine RBCs, containing approximately 85% HI and 15% nonheme iron (NHI). Total iron was within the physiological range (50–75 ppm) and precisely matched NHI control diets containing ferrous sulfate were utilized as comparators. Notably, in Sprague-Dawley (S-D) rats and C57BL/6 (B6) mice, dietary HI effectively (a) resolved iron-deficiency anemia; (b) supported normal pregnancy, lactation, and neonatal development; and (c) contributed to iron loading in Hamp -KO mice and rats (modeling hereditary hemochromatosis). A nutritional paradigm has thus been established that facilitates investigation into mechanisms of HI absorption by S-D rats and B6 mice.

β-thalassemia is an iron-loading anemia caused by homozygous mutation of the hemoglobin subunit β (HBB) gene. In β-thalassemia intermedia (βTI), a non–transfusion-dependent form of the disease, iron overload is caused by excessive absorption of dietary iron due to inappropriately low production of the iron-regulatory hormone hepcidin. Low hepcidin stabilizes the iron exporter ferroportin (FPN) on the basolateral membrane of enterocytes. High FPN activity may deplete intracellular iron and enhance expression of the predominant iron importer divalent metal-ion transporter 1 (DMT1). In mice, DMT1 mediates normal iron absorption under physiological conditions and excessive iron absorption in pathological iron overload (eg, hereditary hemochromatosis). Here, we hypothesized that DMT1 drives elevated iron absorption in βTI. Accordingly, we crossed Hbbth3/+ mice, a preclinical model of βTI, with intestine-specific DMT1-knockout mice. Ablation of intestinal DMT1 in Hbbth3/+ mice caused a pathophysiological shift from iron overload to an iron-deficiency phenotype with exacerbated anemia. DMT1 is thus required for iron absorption and iron loading in Hbbth3/+ mice. Based upon these outcomes, we further logically postulated that in vivo knockdown of intestinal DMT1 would mitigate iron loading in Hbbth3/+ mice. Ginger-derived, lipid nanoparticles carrying DMT1-specific (or control) small interfering RNAs (siRNAs) were administered by oral, intragastric gavage to 4-week-old Hbbth3/+ mice daily for 16 days. siRNA treatment reduced DMT1 expression by >80% and blunted iron loading, as indicated by significant reductions in liver iron and serum ferritin (which reflect body iron stores). These notable experimental outcomes establish intestinal DMT1 as a plausible therapeutic target to mitigate iron overload in βTI.