ABSTRACT
Complex regulatory mechanisms control the expression of folate transporters within cells. Liver is the primary reserve of the folate stores within the body. As excessive alcohol consumption or inefficient dietary folate intake are known to create folate deficiency, so therefore the current study was designed to explore various regulatory mechanisms controlling the expression of folate transport in liver cells in conditions of ethanol exposure and folate deficiency. In order to see whether the effects mediated by the treatments are reversible or not, ethanol removal, and folate repletion was done after ethanol exposure and folate deficiency treatment respectively. Folate deficiency resulted an increase, whereas ethanol treatment decreased the folic acid uptake within the cells. The alterations in folic acid uptake were in agreement with the observed changes in the expression of folate transporters. Ethanol exposure resulted an increase in promoter methylation of reduced folate carrier; however, folate deficiency had no effect. The effects produced by ethanol exposure and folate deficiency were found to be reversible in nature as depicted in case of ethanol removal and folate repletion group. Rate of synthesis of folate transporters was found to be increased whereas half lives of mRNA of folate transporters was found to be decreased on folate deficiency treatment and reverse was the case on ethanol treatment. Overall, alteration in the expression of folate transporters under ethanol exposure and folate deficient conditions can be attributed to those regulatory mechanisms which work at the mRNA level.
Subject(s)
Ethanol/pharmacology , Folic Acid/pharmacology , Gene Expression Regulation/drug effects , Proton-Coupled Folate Transporter/genetics , RNA, Messenger/metabolism , Reduced Folate Carrier Protein/genetics , Biological Assay , Biological Transport , Cell Survival/drug effects , DNA Methylation/drug effects , Folate Receptor 1/agonists , Folate Receptor 1/antagonists & inhibitors , Folate Receptor 1/genetics , Folate Receptor 1/metabolism , Folic Acid Deficiency/genetics , Folic Acid Deficiency/metabolism , Folic Acid Deficiency/pathology , Hep G2 Cells , Humans , Lacticaseibacillus casei/growth & development , Lacticaseibacillus casei/metabolism , Models, Biological , Promoter Regions, Genetic , Proton-Coupled Folate Transporter/agonists , Proton-Coupled Folate Transporter/antagonists & inhibitors , Proton-Coupled Folate Transporter/metabolism , RNA, Messenger/agonists , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , Reduced Folate Carrier Protein/agonists , Reduced Folate Carrier Protein/antagonists & inhibitors , Reduced Folate Carrier Protein/metabolism , Signal Transduction , TritiumABSTRACT
5-Aminoimidazole-4-carboxamide riboside (AICAR), an agent with diverse pharmacological properties, augments transport of folates and antifolates. This report further characterizes this phenomenon and defines the mechanism by which it occurs. Exposure of HeLa cells to AICAR resulted in augmentation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate initial rates and net uptake in cells that express the reduced folate carrier (RFC). This did not occur in cells that express only the proton-coupled folate transporter and accumulated folates by this mechanism. Transport stimulation correlated with the accumulation of 5-aminoimidazole-4-carboxamide ribotide monophosphate (ZMP), the monophosphate derivative of AICAR, within cells as established by liquid chromatography. When ZMP formation was blocked with 5-iodotubercidin, an inhibitor of adenosine kinase, folate transport stimulation by AICAR was absent. When cells first accumulated ZMP and were then exposed to 5-iodotubercidin or AICAR-free buffer, the ZMP level markedly decreased and folate transport stimulation was abolished. Extracellular ZMP inhibited RFC-mediated folate influx, and the presence of intracellular ZMP correlated with inhibition of folate efflux. The data indicate that intracellular ZMP trans-stimulates folate influx and inhibits folate efflux, which, together, produce a marked augmentation in the net cellular folate level. This interaction among ZMP, folates, and RFC, a folate/organic phosphate antiporter, is consistent with a classic exchange reaction. The transmembrane gradient for one transport substrate (ZMP) drives the uphill transport of another (folate) via a carrier used by both substrates, a phenomenon intrinsic to the energetics of RFC-mediated folate transport.
