| Summary: | 碩士 === 中州科技大學 === 保健食品系 === 100 === Caloric restriction (CR) can extend the lifespan of organisms. Nicotinamide adenine dinucleotide (NAD+) is one of bioactive from of vitamin B3. Sirtuins is a protein family called NAD+-dependent deacetylase, which is considered as a longevity protein. FK866 is an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway of NAD+ synthesis from nicotinamide and has ability to decrease intracellular NAD+ level. In this study, we examined whether CR can increase intracellular NAD+ levels, and then activate SIRT1 and extend replicative lifespan of human Hs68 cells. Using cells cultured at lower levels of glucose than normal level of glucose in medium (glucose restriction; GR) to mimic CR, we compared the effects of GR, NAD+ and FK866 on cell lifespan, intracellular NAD+ levels, SIRT1 activity and senescence associated -galactosidase (SA-G) activity. We also examined whether the effects of FK866 on Hs68 cells can be antagonized by GR and adding NAD+ into the culture medium. The lifespan of cells was measured as the cumulated growth curve of population doubling levels. The biomarker of cell senescence was detected by determination of senescence associated -galactosidase (SA-G) activity using X-Gal staining. Intracellular NAD+ was determined by an acid extraction method and followed by an enzymatic cycling method. SIRT1 activities were determined by SIRT1 Direct Fluorescent Screening Assay Kit or used p53 acetylation to represent cellular activities of SIRT1. The results showed that GR (>5day) could significantly increase NAD+ levels in cells, activated SIRT1 activity, and extended the lifespan of Hs68 cells. Add NAD+ in to the medium showed with similar effects as GR. Oppositely, FK866 has abilities to reduce intracellular NAD+ levels, shorten the cells lifespan, decrease SIRT1 activity, and increase SA-βG activity. Importantly, all of these effects of FK866 on Hs68 cells can be effectively blocked by GR or adding NAD+ into the medium. The results demonstrated that increase of intracellular NAD+ level plays a crucial role in the mechanism of CR-extended lifespan of human Hs68 cells.
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