Because of the central role of energy metabolism in health and disease, and its effect on other cellular processes, assays to monitor changes in cellular metabolic state have wide application in both basic research and drug discovery. In the Webinar “Tools for Cell Metabolism: Bioluminescent NAD(P)/NAD(P)H-Glo™ Assays” Jolanta Vidurigiene, a Senior Research Scientist at Promega, introduces three new metabolism assays for measuring oxidized and reduced forms of NAD and NADP.
In this webinar, Jolanta provides background information on why it is important to be able to accurately measure metabolites such as NAD/NADH and NADP/NADPH. She outlines the roles of each, and highlights some of the challenges involved in developing assays that can accurately measure these metabolites. She discusses key considerations for successful NAD(P)/NAD(P)H assays and provides examples of how to use these assays to measure either total (both oxidized and reduced) forms of NAD and NADP, or to measure oxidized and reduced forms individually in a single assay plate.
The metabolic requirements of differentiated and proliferating cells differ. Differentiated cells primarily rely on catabolic pathways where complex organic molecules are broken down by glycolysis and oxidative phosphorylation, generating ATP. Proliferating cells rely on both anabolic pathways, which enable processes such as fatty acid biosynthesis and protein synthesis, as well as catabolic energy-generating pathways.
Cancer cells are in a state of uncontrolled proliferation, and their metabolic pathways are rewired to support changing energy requirements, balancing energy production with increased biomolecule synthesis and the need for protection against reactive oxygen species. These cancer cell-specific metabolic signatures include increased glucose uptake, a shift from oxidative phosphorylation to aerobic glycolysis, increased lactate production, activation of the pentose phosphate pathway, and increased anabolic reactions.
NAD+, NADH, NADP+ and NADPH are fundamental co-factors of cellular energy metabolism, and as such are essential for macromolecule biosynthesis and maintenance of redox potential. NAD-dependent signaling pathways are also involved in regulating other processes linked to cancer development, including epigenetic regulation, cell cycle progression, DNA repair, and circadian rhythm. This central role of NAD(P)/NAD(P)H in cellular energy metabolism and signaling makes these dinucleotides important target-independent molecules that link the metabolic state of cells with energy homeostasis and gene regulation. Therefore, rapid, easy-to-use assays for measuring NAD(P)/NAD(P)H provide convenient tools for examining metabolic processes in both health and disease.
Specific uses for each of the three assays described in the webinar are as follows:
The NAD/NADH-Glo™ Assay can be used to monitor cellular or biochemical levels of total oxidized and reduced nicotine adenine dinucleotide. It can also be used to determine NAD+/NADH ratios.
The NADP/NADPH-Glo™ Assay can be used to monitor cellular or biochemical levels of total oxidized and reduced nicotine adenine dinucleotide phosphates. It can also be used to determine NADP+/ NADPH ratios.
The NAD(P)H-Glo™ Assay can be used in biochemical screening to measure the reduced forms of nicotine adenine dinucleotides. It does not discriminate between NADH and NADPH.
Listen to the webinar (available here) for complete details on the how these bioluminescent assays can be used to elucidate the role of nicotinamide adenine dinucleotides in cellular energy metabolism and cell signaling more rapidly and with higher precision.