Bioluminescent Assays for Glucose and Glutamine Metabolism: High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites.
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.
1 Research and Development, Promega Corporation, Madison, WI, USA.
Donna
D
Leippe
1 Research and Development, Promega Corporation, Madison, WI, USA.
Mary
M
Sobol
1 Research and Development, Promega Corporation, Madison, WI, USA.
Gediminas
G
Vidugiris
1 Research and Development, Promega Corporation, Madison, WI, USA.
James J
JJ
Cali
1 Research and Development, Promega Corporation, Madison, WI, USA.
Jolanta
J
Vidugiriene
D045744:Cell Line, Tumor; D005260:Female; D005947:Glucose / Q000032:analysis* / Q000378:metabolism; D018698:Glutamic Acid / Q000032:analysis* / Q000378:metabolism; D005973:Glutamine / Q000032:analysis* / Q000378:metabolism; D006019:Glycolysis; D057166:High-Throughput Screening Assays; D006801:Humans; D019344:Lactic Acid / Q000032:analysis* / Q000378:metabolism; D008163:Luminescent Measurements; D010053:Ovary / Q000378:metabolism / Q000473:pathology; D012680:Sensitivity and Specificity
D005973:Glutamine; D019344:Lactic Acid; D018698:Glutamic Acid; D005947:Glucose
bioluminescence; cell-based assays; extracellular metabolites; glucose; glutamate; glutamine; lactate