Bioluminescent Assays for Glucose and Glutamine Metabolism: High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites

Abstract

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1-5 pmol/sample), broad linear range (0.1-100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z' = 0.6-0.9) and could be multiplexed with a real-time viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production.

Regulation of Cancer Stem Cell Metabolism by Secreted Frizzled-Related Protein 4 (sFRP4)

“…For metabolite analysis, 4.5 μL of thawed sample was transferred to respective 96 well white luminescent plates for glutamine and glutamate detection. Samples were then assayed as described in Glutamine/Glutamate-Glo Assay (#J8021, Promega) standard protocols [ 77 ]. Luminescence was read on a luminometer (EnSpire Multilabel Plate Reader, Perkin-Elmer).…”

Treating Cells as Reagents to Design Reproducible Assays

“…Mass spectrometry or biochemical assays are available to measure the levels of energy source molecules and metabolites such as glucose, lactate, glutamine, and glutamate. 10 Care should be taken to not overcompensate for depletion of glucose by using what is commonly called “high-glucose medium,” which contains nonphysiological levels of glucose (~25 mM) that mimic a diabetic environment, which could interfere with the cell-based assay model system.…”

Metabolic therapy and bioenergetic analysis: The missing piece of the puzzle

“…Overall turnover of glucose, lactate, as well as glutamine and glutamate, can be measured using specialized biochemistry analyzers [ 201 , 202 ]. Bioluminescent assays can be adapted for high-throughput extracellular and intracellular detection of metabolites [ 203 ]. Focusing strictly on ATP production, the contribution of glycolysis and OXPHOS can be measured by conventional techniques, including enzymatic methods and targeted luciferase measurements [ 204 ].…”