Select Amino Acids Alter DMT1 Abundance on the BBM of Intestinal Enterocytes and Enhance or Reduce Iron Flux in Duodenal Organ Cultures

Abstract

Approximately 1.6 billion people worldwide are anemic, with iron deficiency (ID) as the most common cause. Moreover, iron overload (IO), most commonly associated with the genetic disease Hereditary Hemochromatosis (HH), affects some 1 million individuals in the US. Both conditions warrant better treatment options, as iron supplementation is not always effective for individuals with ID, and current treatments for IO (phlebotomy and Fe chelation) are non‐specific and have untoward side effects. Our previous studies demonstrated that select amino acids (AA) can influence trafficking of membrane transport proteins in enterocytes and thus influence electrolyte absorption in the gut. We thus hypothesized that certain AAs could also influence trafficking of the predominant intestinal iron transporter, divalent metal‐ion transporter 1 (DMT1). We further surmised, that if true, this could represent a novel therapeutic approach to modulate intestinal iron absorption. This is particularly important since overall body iron levels are determined by assimilation of dietary iron in the intestine since humans and other mammals cannot excrete excess iron (and iron imbalance is common in many diseases). For an initial screen of the effect of AAs on DMT1 trafficking, ex vivo duodenal loops excised from adult mice were filled with a buffer containing individual AAs and incubated at 37° C in an oxygenated bath for 45 min. Subsequently, brush‐border membrane vesicles (BBMVs) were purified from mucosal scrapes and DMT1 protein expression levels were determined by immunoblotting. Amino acids were grouped based on how they influenced DMT1 protein expression on the BBM. Based upon the Western blotting data, two formulations were made that contained mixtures of AAs that either increased or decreased DMT1 protein expression on the BBM. To confirm these observations at a functional level, 59Fe flux studies were next performed with mouse duodenal organ cultures using Ussing chambers after exposure to the AA mixtures. These experiments initially showed that one AA formulation increased 59Fe flux by ≈4‐fold (p<0.05), which was in agreement with the Western blotting data. Interestingly, this same AA formulation decreased conductance (p<0.0001), which indicates a tightening of the epithelial barrier. To test the other AA formulation (which decreased DMT1 BBM protein expression), we utilized a mouse model of HH, hepcidin (Hamp) KO mice, in which intestinal iron absorption is inappropriately elevated. Importantly, 59Fe flux studies with Hamp−/− mice showed an approximate 7‐fold reduction in iron transport (p<0.05) after exposure to the AA mixture, again confirming the immunoblotting data. We thus conclude that select AAs can influence DMT1 trafficking to and from the enterocyte BBM and thus alter vectorial iron flux. Although additional in vivo testing is warranted, these initial findings could lead to the development of specific AA formulations that could be used to mitigate pathological changes in intestinal iron transport associated with common disease states.Support or Funding InformationThis work was supported by NIH grants R01 DK074867 and R01 DK109717 (to JFC).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.