Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug;14(4):547-57.
doi: 10.1111/acel.12317. Epub 2015 Mar 23.

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects

Pabalu P Karunadharma  1 , Nathan Basisty  1 , Dao-Fu Dai  1 , Ying A Chiao  1 , Ellen K Quarles  1 , Edward J Hsieh  2 , David Crispin  1 , Jason H Bielas  1   3   4 , Nolan G Ericson  4 , Richard P Beyer  5 , Vivian L MacKay  6 , Michael J MacCoss  2 , Peter S Rabinovitch  1
Affiliations

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects

Pabalu P Karunadharma et al. Aging Cell. 2015 Aug.

Abstract

Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR. Protein abundance and turnover were measured in vivo using (2) H3 -leucine heavy isotope labeling followed by LC-MS/MS, and translation was assessed by polysome profiling. We observed 35-60% increased protein half-lives after CR and 15% increased half-lives after RP compared to age-matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions.

Keywords: aging; calorie restriction; liver; mammalian target of rapamycin; protein turnover; rapamycin.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Subacute treatment of CR and RP prolong liver protein half-lives. (A) Summary of experimental design showing young (3 months at the start of treatment) and old (25 mo at the start of treatment) C57BL/6 female mice started on an ad libitum, encapsulated RP (14 ppm)-containing or CR (40% restricted) diet for 10 weeks. Livers were harvested and frozen for polysome analyses from five mice per cohort at the conclusion of 10 weeks, and the rest were switched to the same diets except that leucine was replaced with 3H2–leucine. Mice were euthanized 3, 7, 12, and 17 days post-3H2–leucine diet feeding. Liver tissue was harvested and LC-MS/MS performed on tryptic peptides of total and mt proteins. Topograph software enabled the calculation of protein half-lives. (B) Protein half-life density plot illustrates the distribution of half-lives of young (dashed) and old (solid) control (CL), calorie restricted (CR), and rapamycin (RP) cohorts. Median half-life in days is indicated on the right of the legend. Protein half-life ratio histograms for two group comparisons: (C,D) total protein half-lives as ratio to those same proteins in YCL (C) or OCL (D). (E,F) Mt protein half-lives as a ratio to YCL (E) and OCL (F). All comparison mean ratios were significantly different from 1.0 with a P < 0.01 by z-test for proportions. OCL/YCL < 1.0, P < 0.001, and all others > 1.0 P < 0.01.
Figure 2
Figure 2
Box plots of liver protein half-lives of CR and RP by pathway and location. (A) Three representative IPA canonical pathways that are among the top fastest turnover (shortest half-lives). Boxes represent the interquartile range for protein HLs, and whiskers extend from 5 to 95% of the data. (B) Three representative canonical pathways with the longest living proteins. Most pathways were metabolic indicating less turnover and higher stability of the metabolic proteins. BCAA, branched-chain amino acid degradation. Old CR extended median HLs of all shortest and longest living pathways except the eIF2 signaling pathway that contains a majority of ribosomal proteins (ribosomal protein HLs shown in panel 2C). (C) Protein HLs by cellular compartment. Peroxisomal proteins were shortest lived, while ribosomal proteins were longest lived. Statistical significance tested with one-way anova, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Numerical detail and HLs for these pathways are in Table S1 (Supporting infomation).
Figure 3
Figure 3
Heatmap of protein half-life differences and scatter plots of top pathway HL ratio comparisons. (A) Total proteins that were significantly altered (q < 0.05) were categorized into the top 19 canonical pathways using IPA. Red indicates longer half-life in the numerator, and blue indicates longer half-life in the denominator. Ratio values ranging from log2 -3 to +4 are depicted in a color gradient from blue to red. BCAA, branched-chain amino acid degradation; FAO, fatty acid beta oxidation; Keto, ketogenesis; and acetone (to MG), acetone degradation to methylglyoxal. (B–E) Scatter plots of log2 ratio comparisons of top metabolic pathways for (B) YRP/YCL vs. YCR/YCL, (C) ORP/OCL vs. OCR/OCL, (D) OCR/OCL vs. YCL/OCL, and (E) ORP/OCL vs. YCL/OCL. (D,E) If changes with aging are reversed, those proteins are located in the top right or bottom left quadrants of the scatter plots. The number of significantly changed proteins that mapped to each pathway is listed in parenthesis in panel (B). All heatmap proteins and their half-life log2 ratios are listed in Table S2 (Supporting information). Values and summary statistics for all other proteins, in addition to these, are in Table S4 (Supporting information).
Figure 4
Figure 4
Protein carbonyl content, autophagic degradation, and translation elongation marker with treatment and aging. (A) Protein carbonyls are decreased with treatments. (B) Autophagic marker p62 show a decrease with aging and an increase with ORP, while beclin-1 is decreased with OCR. LC3 II/I ratios did not change with age or treatments. (C) Representative fluorescent Western blot of phospho-eEF2 and total eEF2. Phosphorylation of this elongation factor disrupts translational elongation. The ratio of phospho-eEF2 over total eEF2 is quantified in (D). Rapamycin treatment significantly increased this ratio in both young and old mice. n = 4–6 per group., *P < 0.05,** P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Heatmap of protein abundance differences and scatter plots of top pathway abundance comparisons. (A) Proteins that were significantly altered at q < 0.05 were categorized into canonical pathways using IPA. The top 19 pathways are reported. Log2 fold-changes ranging from −2 to +2 are gradually colored from blue to red. (B–E) Scatter plots of log2 fold-change comparisons of the top metabolic pathway proteins of (B) YRP/YCL vs. YCR/YCL, (C) ORP/OCL vs. OCR/OCL, (D) OCR/OCL vs. YCL/OCL, and (E) ORP/OCL vs. YCL/OCL are shown. (D,E) If changes with aging are reversed, those proteins are located in the top right or bottom left quadrants of the scatter plots. The number of significantly changed proteins that mapped to each pathway is listed in parenthesis in panel (B). All heatmap proteins and their log2 fold-changes are listed in Table S3 (Supporting information).
Figure 6
Figure 6
Subacute CR decreases global protein translation while RP increases ribosome loading in the liver. (A) Representative polysome profiles of young control, RP, and CR groups. The ‘top’ and ‘bottom’ of the gradient are indicated on the x-axis. The peaks are labeled with the name of the ribosome subunit or number of ribosomes that they represent. (B, C) Area under the curve (AUC) measurements shown relative to the total AUC of the single subunits, monosome, and polysome peaks of control, CR, and RP livers of (B) young and (C) old mice. (D) Quantified relative polysome area of young and old livers. n = 5 per group, P < 0.05 anova. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Figure 7
Figure 7
A summary model of the effects observed in this study. With aging, we observed an overall increase in turnover and translation with a concomitant decrease in mt protein HLs and abundance. In combination, these factors can result in increased ROS and the formation of error-prone polypeptides and oxidized proteins, thereby disrupting protein homeostasis. CR reversed these changes effectively recapitulating a more youthful proteome, while RP changes were more subtle but indicated decreased oxidative damage and changes that improved translational fidelity. Short-term CR and RP can both restore protein homeostasis to promote more healthy aging.
See this image and copyright information in PMC

References

    1. Alvers AL, Wood MS, Hu D, Kaywell AC, Dunn WA, Aris JP. Autophagy is required for extension of yeast chronological life span by rapamycin. Autophagy. 2009;5:847–849. - PMC - PubMed
    1. Beretta L, Gingras AC, Svitkin YV, Hall MN, Sonenberg N. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 1996;15:658–664. - PMC - PubMed
    1. Blackwell BN, Bucci TJ, Hart RW, Turturro A. Longevity, body weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet. Toxicol. Pathol. 1995;23:570–582. - PubMed
    1. Cerqueira FM, Cunha FM, Laurindo FR, Kowaltowski AJ. Calorie restriction increases cerebral mitochondrial respiratory capacity in a NO•-mediated mechanism: impact on neuronal survival. Free Radic. Biol. Med. 2012;52:1236–1241. - PubMed
    1. Chondrogianni N, Gonos ES. Proteasome dysfunction in mammalian aging: steps and factors involved. Exp. Gerontol. 2005;40:931–938. - PubMed

Publication types

MeSH terms