Kinetic isotope tracing of glycerol and de novo proteogenic amino acids in Human Lung Carcinoma cells using [U-¹³C₆]glucose

Abstract

In vitro studies involving cell lines or primary cells, provide critical insights into their physiology under normal and perturbed conditions like cancer and infection. Given that there are multiple sources of carbon, nitrogen, and other nutrients available in routinely used standard media (such as DMEM, RPMI), it is vital to quantify their contribution to cellular metabolism. 13C based Isotopic tracers of the media components can be used to kinetically track their oxidation by the cell systems such as Human Lung Carcinoma (A549) cells. In this study, a universally labelled glucose tracer ([13C6]glucose) was used to quantify its metabolic contribution that provided further insights into the central carbon metabolism of A549 cells. Gas chromatography and mass spectrometry (GC-MS) based mass isotopomer analysis (average 13C) of methanolic extracts (glycerol: 5.46±3.53 % and lactate: 74.4±2.65 %), amino acids derived from acid hydrolysates of protein (Serine: 4.51±0.21 %, Glycine: 2.44±0.31 %, Alanine: 24.56±0.59 %, Glutamate: 8.81±0.85 %, Proline: 6.96±0.53 % and Aspartate: 10.72±0.95 %) and the metabolites of the culture filtrate (glycerol: 43.14±1.45 % and lactate: 81.67±0.91 %), allowed to capture the relative contribution of glucose. We observed the Warburg effect and a significant amount of lactate contributed from glucose, was released to the media. 13C glycerol of glucogenic origin was kinetically released to the culture filtrate and might be playing a critical role in metabolic reprogramming of A549 cells. Part of the protein biomass contributed from amino acids were of glucogenic origin. Besides, the workflow adopted for 13C analysis and derived average 13C of each metabolite provided a standard methodology that could be useful in defining the metabolic phenotypes of cells in normal and perturbed conditions. Understanding precisely the altered cellular metabolism to meet the biomass demand under a range of physiological conditions, kinetically, may identify pathways for targeted and effective therapeutic interventions.