Limits...
p16(INK4a) suppression by glucose restriction contributes to human cellular lifespan extension through SIRT1-mediated epigenetic and genetic mechanisms.

Li Y, Tollefsbol TO - PLoS ONE (2011)

Bottom Line: The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter.Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling.Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Although caloric restriction (CR) has been shown to increase lifespan in various animal models, the mechanisms underlying this phenomenon have not yet been revealed. We developed an in vitro system to mimic CR by reducing glucose concentration in cell growth medium which excludes metabolic factors and allows assessment of the effects of CR at the cellular and molecular level. We monitored cellular proliferation of normal WI-38, IMR-90 and MRC-5 human lung fibroblasts and found that glucose restriction (GR) can inhibit cellular senescence and significantly extend cellular lifespan compared with cells receiving normal glucose (NG) in the culture medium. Moreover, GR decreased expression of p16(INK4a) (p16), a well-known senescence-related gene, in all of the tested cell lines. Over-expressed p16 resulted in early replicative senescence in glucose-restricted cells suggesting a crucial role of p16 regulation in GR-induced cellular lifespan extension. The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter. GR resulted in an increased expression of SIRT1, a NAD-dependent histone deacetylase, which has positive correlation with CR-induced longevity. The elevated SIRT1 was accompanied by enhanced activation of the Akt/p70S6K1 signaling pathway in response to GR. Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling. Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems.

Show MeSH

Related in: MedlinePlus

GR resulted in p16 repression.A. Graphic presentation of relative mRNA levels of p16 in WI-38 (left), MRC-5 (middle) and IMR-90 (right) cells during early, intermediate and late proliferation of cell growth. Human fibroblasts were cultured in NG or GR medium as indicated previously. Data are in triplicate from three independent experiments and were normalized to GAPDH. p16 mRNA level at PD 0 was calibrated to 1. Columns, mean; Bars, SD; *, P<0.05, significantly different from p16 mRNA level at PD 0. B. The protein levels of p16 were determined by western-blot analysis. WI-38 cell proteins were extracted once every two weeks until cells ceased growth and underwent replicative senescence. C. The protein expression of p16 and phosphorylated Rb (p-Rb) were detected in human fibroblasts, WI-38 (upper), MRC-5 (middle) and IMR-90 (lower), during early, intermediate and late proliferation of cell growth. The definitions of early, intermediate and late passages were described above. Cells were treated with/without GR medium as described above. Membranes were reprobed with anti-GAPDH antibody to ensure for equal loading. Representative photographs are derived from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044759&req=5

pone-0017421-g003: GR resulted in p16 repression.A. Graphic presentation of relative mRNA levels of p16 in WI-38 (left), MRC-5 (middle) and IMR-90 (right) cells during early, intermediate and late proliferation of cell growth. Human fibroblasts were cultured in NG or GR medium as indicated previously. Data are in triplicate from three independent experiments and were normalized to GAPDH. p16 mRNA level at PD 0 was calibrated to 1. Columns, mean; Bars, SD; *, P<0.05, significantly different from p16 mRNA level at PD 0. B. The protein levels of p16 were determined by western-blot analysis. WI-38 cell proteins were extracted once every two weeks until cells ceased growth and underwent replicative senescence. C. The protein expression of p16 and phosphorylated Rb (p-Rb) were detected in human fibroblasts, WI-38 (upper), MRC-5 (middle) and IMR-90 (lower), during early, intermediate and late proliferation of cell growth. The definitions of early, intermediate and late passages were described above. Cells were treated with/without GR medium as described above. Membranes were reprobed with anti-GAPDH antibody to ensure for equal loading. Representative photographs are derived from three independent experiments.

Mentions: As shown in Fig. 3A, we found that p16 mRNA gradually accumulated under the NG condition, whereas GR decreased p16 transcription as cellular passages progressed in all of the three cell lines we tested. In NG cells, p16 mRNA accumulated and reached peak levels at the late passage indicating cellular senescence processes were initiated. In contrast to NG treatment, GR resulted in p16 expression suppression consistently during all cellular passages. However, glucose-restricted IMR-90 cells, which resulted in the longest lifespan extension (66.7%), did not show a maximal reduction of p16 expression compared with that in WI-38 and MRC-5 cells indicating that although p16 may serve a major role in lifespan regulation, other factors could also be involved. To elucidate p16 protein expression in response to NG and GR medium, cellular proteins in WI-38 cells were extracted at every other two week intervals and subjected to western blot assay. As indicated in Fig. 3B, p16 protein gradually accumulated from the early five weeks of proliferation in NG growth medium, whereas the p16 protein signal was only detected at the later 12 weeks of treatment with GR medium when cellular replicative senescence may occur. Consistent with p16 mRNA expression in Fig. 3A, these results suggest that GR-impaired p16 accumulation may contribute to cellular lifespan extension.


p16(INK4a) suppression by glucose restriction contributes to human cellular lifespan extension through SIRT1-mediated epigenetic and genetic mechanisms.

Li Y, Tollefsbol TO - PLoS ONE (2011)

GR resulted in p16 repression.A. Graphic presentation of relative mRNA levels of p16 in WI-38 (left), MRC-5 (middle) and IMR-90 (right) cells during early, intermediate and late proliferation of cell growth. Human fibroblasts were cultured in NG or GR medium as indicated previously. Data are in triplicate from three independent experiments and were normalized to GAPDH. p16 mRNA level at PD 0 was calibrated to 1. Columns, mean; Bars, SD; *, P<0.05, significantly different from p16 mRNA level at PD 0. B. The protein levels of p16 were determined by western-blot analysis. WI-38 cell proteins were extracted once every two weeks until cells ceased growth and underwent replicative senescence. C. The protein expression of p16 and phosphorylated Rb (p-Rb) were detected in human fibroblasts, WI-38 (upper), MRC-5 (middle) and IMR-90 (lower), during early, intermediate and late proliferation of cell growth. The definitions of early, intermediate and late passages were described above. Cells were treated with/without GR medium as described above. Membranes were reprobed with anti-GAPDH antibody to ensure for equal loading. Representative photographs are derived from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044759&req=5

pone-0017421-g003: GR resulted in p16 repression.A. Graphic presentation of relative mRNA levels of p16 in WI-38 (left), MRC-5 (middle) and IMR-90 (right) cells during early, intermediate and late proliferation of cell growth. Human fibroblasts were cultured in NG or GR medium as indicated previously. Data are in triplicate from three independent experiments and were normalized to GAPDH. p16 mRNA level at PD 0 was calibrated to 1. Columns, mean; Bars, SD; *, P<0.05, significantly different from p16 mRNA level at PD 0. B. The protein levels of p16 were determined by western-blot analysis. WI-38 cell proteins were extracted once every two weeks until cells ceased growth and underwent replicative senescence. C. The protein expression of p16 and phosphorylated Rb (p-Rb) were detected in human fibroblasts, WI-38 (upper), MRC-5 (middle) and IMR-90 (lower), during early, intermediate and late proliferation of cell growth. The definitions of early, intermediate and late passages were described above. Cells were treated with/without GR medium as described above. Membranes were reprobed with anti-GAPDH antibody to ensure for equal loading. Representative photographs are derived from three independent experiments.
Mentions: As shown in Fig. 3A, we found that p16 mRNA gradually accumulated under the NG condition, whereas GR decreased p16 transcription as cellular passages progressed in all of the three cell lines we tested. In NG cells, p16 mRNA accumulated and reached peak levels at the late passage indicating cellular senescence processes were initiated. In contrast to NG treatment, GR resulted in p16 expression suppression consistently during all cellular passages. However, glucose-restricted IMR-90 cells, which resulted in the longest lifespan extension (66.7%), did not show a maximal reduction of p16 expression compared with that in WI-38 and MRC-5 cells indicating that although p16 may serve a major role in lifespan regulation, other factors could also be involved. To elucidate p16 protein expression in response to NG and GR medium, cellular proteins in WI-38 cells were extracted at every other two week intervals and subjected to western blot assay. As indicated in Fig. 3B, p16 protein gradually accumulated from the early five weeks of proliferation in NG growth medium, whereas the p16 protein signal was only detected at the later 12 weeks of treatment with GR medium when cellular replicative senescence may occur. Consistent with p16 mRNA expression in Fig. 3A, these results suggest that GR-impaired p16 accumulation may contribute to cellular lifespan extension.

Bottom Line: The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter.Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling.Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Although caloric restriction (CR) has been shown to increase lifespan in various animal models, the mechanisms underlying this phenomenon have not yet been revealed. We developed an in vitro system to mimic CR by reducing glucose concentration in cell growth medium which excludes metabolic factors and allows assessment of the effects of CR at the cellular and molecular level. We monitored cellular proliferation of normal WI-38, IMR-90 and MRC-5 human lung fibroblasts and found that glucose restriction (GR) can inhibit cellular senescence and significantly extend cellular lifespan compared with cells receiving normal glucose (NG) in the culture medium. Moreover, GR decreased expression of p16(INK4a) (p16), a well-known senescence-related gene, in all of the tested cell lines. Over-expressed p16 resulted in early replicative senescence in glucose-restricted cells suggesting a crucial role of p16 regulation in GR-induced cellular lifespan extension. The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter. GR resulted in an increased expression of SIRT1, a NAD-dependent histone deacetylase, which has positive correlation with CR-induced longevity. The elevated SIRT1 was accompanied by enhanced activation of the Akt/p70S6K1 signaling pathway in response to GR. Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling. Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems.

Show MeSH
Related in: MedlinePlus