Protocols for analyzing metabolic derangements caused by increased NADH/NAD⁺ ratio in cell lines and in mice

Mitochondrial electron transport chain (ETC) dysfunction elevates the NADH/NAD⁺ ratio to cause metabolic derangements. Here we describe a protocol to measure the NADH/NAD⁺ ratio and analyze the rewiring of glucose metabolism using [4-²H]-glucose, [3-²H]-glucose, and [U-¹³C]-glucose in ETC-inhibited human cancer cells. We also describe a protocol to analyze the NADH/NAD⁺ ratio-sensitive metabolites in mouse plasma and mouse liver following phenformin treatment. These protocols comprehensively analyze the metabolic derangements resulting from increased NADH/NAD⁺ ratio in in vitro and in vivo models. For complete details on the use and execution of this profile, please refer to Liu et al. (2021).