Cellular Iron Imbalance Alters Metabolite Profile in Gut Epithelial Cells
Authors: Navazesh Shya , Ji Peng 
Published: May 2022

ObjectiveThe study aims to investigate the metabolic profile and proliferative activity of small intestinal enterocytes in response to dietary iron deficiency and excess. We hypothesize that iron imbalance affects cellular iron, energy and fatty acid metabolism and alters cell proliferation.MethodsA porcine jejunal epithelial cell line (IPEC‐J2) was used as an in vitro model and cultured in DMEM/F12 containing 10% fetal bovine serum and 1% antibiotics. Cells were treated with the complete growth media for as control (CON), or the growth media supplemented with 500 µM Deferiprone (DFP) to induce iron deficiency (ID), 10 µM ferric ammonium citrate (FAC) to induce iron overload, or 500 µM DFP followed by 10 µM FAC in treatment for iron deficiency and replenishment (IDIR). An XTT assay determined doses of 500 µM of DFP and 10 µM of FAC as non‐cytotoxic. The relative expression of genes encoding iron regulatory proteins and zinc transporters were determined at 24, 48, 72, and 96 hours of treatment using RT‐qPCR. A BrdU assay was used to assess the proliferative activity of cells cultured after 48 hours of IE, ID or control treatments. Cell lysate was extracted after 48 hours of treatment media and analyzed for untargeted metabolomics using GC coupled to time‐of‐flight MS. Data were subjected to two‐way ANOVA mixed‐effect analysis with Geisser‐Greenhouse correction and statistical significance was determined at p<0.05.ResultsThe relative expression of transferrin receptor 1 was significantly higher in IE compared to ID (P < 0.05) regardless of the time of exposure. Iron treatment did not affect the expression of cytochrome B reductase (CYBRD1), divalent metal transporter 1 (DMT1), Solute Carrier Family 39 (ZIP14), or ferritin light chain (FTL). FPN expression increased significantly at 96 hour post DFP treatment compared to 24 hours. (P <0.05). Iron deficiency gradually increased the expression of CYBRD1, DMT1 and FTL with the increase of exposure time (Ptime < 0.05). The results suggested a compensatory induction of genes associated with iron uptake in IPEC‐J2 cells in response to extended exposure to iron deficiency. BrdU assay revealed that ID significantly reduced the rate of DNA replication compared to IE (P < 0.05), which did not alter cell proliferation rate in comparison with IR. Metabolome analysis revealed that iron treatment altered 71 metabolites (FDR < 0.1), among which 19 were mapped in the user database. In comparison with CON, the most drastic changes in metabolite profile were observed in ID‐treated cells and 33 altered metabolites changed to an opposite direction in IE‐ and ID‐treated cells. Iron replenishment dampened or reversed changes of metabolites affected by ID. The profile of altered metabolites highlighted reduction of cytidine‐5‐monophosphate and changes in glucose metabolism (e.g. glucuronic acid and glucose‐1‐phosphate), the Kreb’s cycle (e.g. citric acid and aconitic acid) suggesting a significant impact of ID on cellular energy metabolism.ConclusionIntracellular iron status can have a significant impact on the cellular metabolism in enterocytes. Given the absorptive function of the enterocyte, whether and how ID and IE affect nutrient absorption in the intestinal epithelium and proliferative activity of crypt‐based intestinal stem cells warrants future study.