A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation.

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.

Effects of 2-deoxyglucose and dehydroepiandrosterone on intracellular NAD(+) level, SIRT1 activity and replicative lifespan of human Hs68 cells.

2-Deoxy-D-glucose (2-DG) and dehydroepiandrosterone (DHEA) have been hypothesized to extend lifespan via mimicking calorie restriction (CR). Activation of sirtuins has been proposed to contribute to life extension of CR by increasing intercellular levels of NAD(+) in several organisms. However, it is unclear whether 2-DG and DHEA may affect intracellular NAD(+) levels and human sirtuin 1 (SIRT1) activities. Here, using human fibroblast Hs68 cells we showed that 2-DG increased intracellular NAD(+) levels in both time- and concentration-dependent manners. 2-DG also dose-dependently increased SIRT1 activities and the lifespan (measured as the cumulated growth curve of population doubling levels) of Hs68 cells. In contrast, DHEA at non-cytotoxic concentrations (≤50 µM) did not significantly affect NAD(+) levels, SIRT1 activities or the lifespan of Hs68 cells. These results suggest that 2-DG extends the lifespan of Hs68 cells by increased NAD(+) levels and SIRT1 activities, and that 2-DG has a potential as a CR mimetic.