An in vitro method for assessment of amino acid bidirectional transport and intracellular metabolic fluxes in mammary epithelial cells

摘要

Understanding uptake of AA by mammary tissue assupply varies is critical for predicting milk componentproduction. Our objective was to develop an in vitromethod to quantify cellular uptake, efflux, and intracellularmetabolism of individual AA that could be implementedfor evaluating these factors when AA supplyand profile are varied. Bovine primary mammary epithelialcells were grown to confluency and exposed tomedium with an AA profile and concentration similarto lactating dairy cow plasma for 24 h. Cells were thenpreloaded in medium enriched with 15N-labeled AA for24 h followed by removal of the 15N-labeled mediumand incubation with medium enriched with 13C-labeledAA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellularfree AA and intracellular free and protein-boundAA were analyzed for concentrations and isotopic enrichmentby gas chromatography-mass spectrometry. Adynamic, 12-pool model was constructed representingextracellular and intracellular free and protein-boundpools of an AA, and their respective 15N and 13C isotopes.Markov chain Monte Carlo simulation (n =5,000) was conducted to evaluate prediction errors byderiving standard errors and posterior distributions forrate constants, fluxes, and pools. Cellular Ala influxand efflux were higher than Leu, reflecting Ala role indriving system L transport and the high capacity ofsodium-dependent transport. The Ala and Leu turnoverrates were 181 and 95, 580 and 857, and 74 and157% per hour for extracellular, intracellular, and fastprotein-bound pools, respectively. The intracellularand extracellular Ala to Leu ratios were quite different,meaning the blood AA profile is not the AA profile providedfor protein translation. The high level of exchangeand rapid turnover of pools provide a mechanism formatching the AA supplies to the precision necessaryfor translation. This also understates the importance ofusing experimental medium similar to what is observedin vivo given that some AA depend on other AA for influx(exchange driven). The average root mean squaredprediction error across the isotope enrichments, pools,and concentrations was 9.7 and 14.1% for Ala and Leu,respectively, and collinearity among parameters waslow, indicating adequate fit and identifiability. The describedmodel provides insight on individual AA transportkinetics and a method for future evaluation of AAtransport and intracellular metabolism when subjectedto varying AA supplies.