SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure

In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [ 3 H]NAD+ and [ 14 C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5′-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5′-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1–mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.