NAD+ level modulation by complex I and NAD+ synthesis and recycling pathways regulate AKT/mTORC1 activity, autophagy, and metastasis.
(A) Ndi1 expression enhanced NAD+/NADH balance. NAD+/NADH ratios in whole-cell or mitochondrial extracts of MDA-MB-435 or MDA-MB-231 control versus Ndi1-expressing cells. Ndi1 stabilized NAD+/NADH ratios, especially under metabolic stress induced by glucose deprivation and hypoxia. NAD+/NADH ratios under stress were measured in whole-cell extracts after 48 hours of culture. (B) Interference with NAD+ synthesis and recycling pathways reduced NAD+/NADH ratios. Knockdown of NAMPT (shNAMPT) in MDA-MB-435 and MDA-MB-231 cells decreased NAD+/NADH ratios (whole-cell extracts after 48 hours growth in 5 mM glucose and normoxia). (C) NAMPT knockdown increased lung colonization activity in MDA-MB-435 and MDA-MB-231 cells (n = 6 per group). (D) NAMPT knockdown affected mTORC1 activity and p62 elimination. Western blot analysis for p62, phospho-AKT substrates, phospho-S6Ser240/244, and phospho-4EBPThr37/46 in MDA-MB-435 and MDA-MB-231 NAMPT-knockdown versus control cells. β-Tubulin served as protein loading control. Signal quantification, measured by infrared imaging (total of detectable bands) and expressed relative to control, is shown below. Lanes were run on the same gel but were noncontiguous (white lines). Results are representative of 3 independent experiments. (A–C) Data are mean ± SEM. *P < 0.05, **P < 0.01, unpaired 2-tailed Student’s t test (A and B) or nonparametric Mann-Whitney test (C).