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Short-term calorie restriction in male mice feminizes gene expression and alters key regulators of conserved aging regulatory pathways.

Estep PW, Warner JB, Bulyk ML - PLoS ONE (2009)

Bottom Line: CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis.Using western analysis we confirmed post-translational inhibition of the TOR pathway.Our data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of females.

View Article: PubMed Central - PubMed

Affiliation: Longenity Inc, Lincoln, MA, USA. pestep@post.harvard.edu

ABSTRACT

Background: Calorie restriction (CR) is the only intervention known to extend lifespan in a wide range of organisms, including mammals. However, the mechanisms by which it regulates mammalian aging remain largely unknown, and the involvement of the TOR and sirtuin pathways (which regulate aging in simpler organisms) remain controversial. Additionally, females of most mammals appear to live longer than males within species; and, although it remains unclear whether this holds true for mice, the relationship between sex-biased and CR-induced gene expression remains largely unexplored.

Methodology/principal findings: We generated microarray gene expression data from livers of male mice fed high calorie or CR diets, and we find that CR significantly changes the expression of over 3,000 genes, many between 10- and 50-fold. We compare our data to the GenAge database of known aging-related genes and to prior microarray expression data of genes expressed differently between male and female mice. CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis. Among the genes showing the largest and most statistically significant CR-induced expression differences are Ddit4, a key regulator of the TOR pathway, and Nnmt, a regulator of lifespan linked to the sirtuin pathway. Using western analysis we confirmed post-translational inhibition of the TOR pathway.

Conclusions: Our data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of females.

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CR-induced phosphorylation changes in Eif4ebp1.Western blots of unphosphorylated Eif4ebp1 (4EBP1) and Thr69 phosphorylated Eif4ebp1 (p4EBP1) for calorie restricted (CR) and high-calorie-fed (HIGHCAL) mice. Labels at top indicate individual mouse sample codes. CR, Calorie Restriction; FHC, Fixed High Calorie feeding; TAL, True Ad Libitum feeding. TAL1, TAL3, and TAL4 are from livers used to make the RNA pool TAL-P. CR1, CR7, and CR8 are from livers used to make the RNA pool CR-P1. CR4, CR5, and CR6 are from livers used to make the RNA pool CR-P2. FHC3 and FHC3B are separate samplings of the same liver.
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pone-0005242-g002: CR-induced phosphorylation changes in Eif4ebp1.Western blots of unphosphorylated Eif4ebp1 (4EBP1) and Thr69 phosphorylated Eif4ebp1 (p4EBP1) for calorie restricted (CR) and high-calorie-fed (HIGHCAL) mice. Labels at top indicate individual mouse sample codes. CR, Calorie Restriction; FHC, Fixed High Calorie feeding; TAL, True Ad Libitum feeding. TAL1, TAL3, and TAL4 are from livers used to make the RNA pool TAL-P. CR1, CR7, and CR8 are from livers used to make the RNA pool CR-P1. CR4, CR5, and CR6 are from livers used to make the RNA pool CR-P2. FHC3 and FHC3B are separate samplings of the same liver.

Mentions: To test this hypothesis we performed Western blots on Eif4ebp1 and S6K. Two primary isoforms of S6K1, p70 and p90, were detected in our liver samples. We assayed Thr390 of p70 (Thr412 of p90), and for Eif4ebp1 we assayed Thr69, which are recognized targets of phosphorylation by Frap1. Consistent with the current model of the regulation of Eif4ebp1 by Frap1, Thr69 phosphorylation of Eif4ebp1 was increased more than 2-fold by high calorie feeding relative to CR (Figure 2) (P = 6.0×10−4, one-sided Wilcoxon rank sum test). In contrast, the Thr390 phosphorylated p70 isoform of S6k1 was undetectable in our liver samples, including those from TAL-fed mice, while Thr412 of p90 was detected but no significant change was found in HIGHCAL relative to CR mice. These results suggest that CR results in reduced efficiency of initiation of cap-dependent translation through phosphorylation of 4EBP1 but does not significantly alter processes dependent upon phosphorylation of S6K1, such as translation of ribosomal transcripts and ribosome biogenesis [34].


Short-term calorie restriction in male mice feminizes gene expression and alters key regulators of conserved aging regulatory pathways.

Estep PW, Warner JB, Bulyk ML - PLoS ONE (2009)

CR-induced phosphorylation changes in Eif4ebp1.Western blots of unphosphorylated Eif4ebp1 (4EBP1) and Thr69 phosphorylated Eif4ebp1 (p4EBP1) for calorie restricted (CR) and high-calorie-fed (HIGHCAL) mice. Labels at top indicate individual mouse sample codes. CR, Calorie Restriction; FHC, Fixed High Calorie feeding; TAL, True Ad Libitum feeding. TAL1, TAL3, and TAL4 are from livers used to make the RNA pool TAL-P. CR1, CR7, and CR8 are from livers used to make the RNA pool CR-P1. CR4, CR5, and CR6 are from livers used to make the RNA pool CR-P2. FHC3 and FHC3B are separate samplings of the same liver.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667255&req=5

pone-0005242-g002: CR-induced phosphorylation changes in Eif4ebp1.Western blots of unphosphorylated Eif4ebp1 (4EBP1) and Thr69 phosphorylated Eif4ebp1 (p4EBP1) for calorie restricted (CR) and high-calorie-fed (HIGHCAL) mice. Labels at top indicate individual mouse sample codes. CR, Calorie Restriction; FHC, Fixed High Calorie feeding; TAL, True Ad Libitum feeding. TAL1, TAL3, and TAL4 are from livers used to make the RNA pool TAL-P. CR1, CR7, and CR8 are from livers used to make the RNA pool CR-P1. CR4, CR5, and CR6 are from livers used to make the RNA pool CR-P2. FHC3 and FHC3B are separate samplings of the same liver.
Mentions: To test this hypothesis we performed Western blots on Eif4ebp1 and S6K. Two primary isoforms of S6K1, p70 and p90, were detected in our liver samples. We assayed Thr390 of p70 (Thr412 of p90), and for Eif4ebp1 we assayed Thr69, which are recognized targets of phosphorylation by Frap1. Consistent with the current model of the regulation of Eif4ebp1 by Frap1, Thr69 phosphorylation of Eif4ebp1 was increased more than 2-fold by high calorie feeding relative to CR (Figure 2) (P = 6.0×10−4, one-sided Wilcoxon rank sum test). In contrast, the Thr390 phosphorylated p70 isoform of S6k1 was undetectable in our liver samples, including those from TAL-fed mice, while Thr412 of p90 was detected but no significant change was found in HIGHCAL relative to CR mice. These results suggest that CR results in reduced efficiency of initiation of cap-dependent translation through phosphorylation of 4EBP1 but does not significantly alter processes dependent upon phosphorylation of S6K1, such as translation of ribosomal transcripts and ribosome biogenesis [34].

Bottom Line: CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis.Using western analysis we confirmed post-translational inhibition of the TOR pathway.Our data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of females.

View Article: PubMed Central - PubMed

Affiliation: Longenity Inc, Lincoln, MA, USA. pestep@post.harvard.edu

ABSTRACT

Background: Calorie restriction (CR) is the only intervention known to extend lifespan in a wide range of organisms, including mammals. However, the mechanisms by which it regulates mammalian aging remain largely unknown, and the involvement of the TOR and sirtuin pathways (which regulate aging in simpler organisms) remain controversial. Additionally, females of most mammals appear to live longer than males within species; and, although it remains unclear whether this holds true for mice, the relationship between sex-biased and CR-induced gene expression remains largely unexplored.

Methodology/principal findings: We generated microarray gene expression data from livers of male mice fed high calorie or CR diets, and we find that CR significantly changes the expression of over 3,000 genes, many between 10- and 50-fold. We compare our data to the GenAge database of known aging-related genes and to prior microarray expression data of genes expressed differently between male and female mice. CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis. Among the genes showing the largest and most statistically significant CR-induced expression differences are Ddit4, a key regulator of the TOR pathway, and Nnmt, a regulator of lifespan linked to the sirtuin pathway. Using western analysis we confirmed post-translational inhibition of the TOR pathway.

Conclusions: Our data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of females.

Show MeSH