Up-regulation of nicotinamide phosphoribosyltransferase and increase of NAD+ levels by glucose restriction extend replicative lifespan of human fibroblast Hs68 cells

Calorie restriction (CR) extends lifespan in a remarkable range of organisms. However, the mechanisms of CR related to the longevity effects are not fully elucidated to date. Using human fibroblast Hs68 (Hs68) cells cultured at a lower level of medium glucose (i.e., glucose restriction; GR) to mimic CR, we investigated the crucial role of nicotinamide phosphoribosyltransferase (Nampt), nicotinamide adenine dinucleotide (NAD+), and nicotinamide (NAM) in GR-extended replicative lifespan of Hs68 cells. We found that GR extended the lifespan of Hs68 cells, in parallel to significantly increased expression of Nampt, intracellular NAD+ levels, and SIRT1 activities, and to significantly decreased NAM levels. The lifespan-extending effects of GR were profoundly diminished by FK866 (a noncompetitive inhibitor of Nampt) and blocked by sirtinol (a noncompetitive inhibitor of sirtuins). However, the steady-state intracellular NAM level (averaged 2.5 μM) was much lower than the IC50 of NAM on human SIRT1 (about 50 μM). All these results suggest that up-regulation of Nampt play an important role in GR-extended lifespan of Hs68 cells by increasing the intracellular NAD+ levels followed by activating SIRT1 activity in Hs68 cells. In contrast, the role of NAM depletion is limited.