The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy(definition), is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden. Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden. Calorie restriction (CR) 1The abbreviations used are:AAamino acidAAPPamino acid precursor poolALad libitumBCAbicinchoninic acidBWbody waterCRcalorie restrictionCVcoefficient of variationD0time point at day 0EM0absolute value of change in M0 intensityffractional replacementkturnover rate constantLC-MS/MSliquid chromatography tandem mass spectrometryMIDAmass isotopomer distribution analysisMPEmolar percent excessmtDNAmitochondrial DNAm/zmass-to-charge ratioM0monoisotopic massnnumber of sites within peptide capable of incorporating labelnAAnumber of sites within amino acid capable of incorporating labelNIANational Institute on AgingNIHNational Institutes of Healthpprecursor pool enrichmentPGC-1αperoxisome proliferator-activated receptor gamma coactivator 1-alphaRMSEroot mean square errorROSreactive oxygen speciesSILAMStable Isotope Labeling in Mammals. 1The abbreviations used are:AAamino acidAAPPamino acid precursor poolALad libitumBCAbicinchoninic acidBWbody waterCRcalorie restrictionCVcoefficient of variationD0time point at day 0EM0absolute value of change in M0 intensityffractional replacementkturnover rate constantLC-MS/MSliquid chromatography tandem mass spectrometryMIDAmass isotopomer distribution analysisMPEmolar percent excessmtDNAmitochondrial DNAm/zmass-to-charge ratioM0monoisotopic massnnumber of sites within peptide capable of incorporating labelnAAnumber of sites within amino acid capable of incorporating labelNIANational Institute on AgingNIHNational Institutes of Healthpprecursor pool enrichmentPGC-1αperoxisome proliferator-activated receptor gamma coactivator 1-alphaRMSEroot mean square errorROSreactive oxygen speciesSILAMStable Isotope Labeling in Mammals. is a dietary intervention in which calorie intake is reduced without malnutrition. CR is considered the most robust nongenetic method for increasing life span and has been shown to be effective in yeast (1Guarente L. Calorie restriction and SIR2 genes—towards a mechanism.Mech. Ageing Dev. 2005; 126: 923-928Crossref PubMed Scopus (136) Google Scholar), worms (2Houthoofd K. Vanfleteren J.R. The longevity effect of dietary restriction in Caenorhabditis elegans.Exp. Gerontol. 2006; 41: 1026-1031Crossref PubMed Scopus (45) Google Scholar), flies (3Partridge L. Piper M.D. Mair restriction in Ageing Dev. 2005; 126: PubMed Scopus Google Scholar), The effect of the of life span and the Scholar), and restriction and in PubMed Scopus Google of in on dietary and in a PubMed Scopus Google CR a of including reduced oxygen percent and percent restriction mitochondrial and in PubMed Scopus Google Scholar), reduced L. calorie not in PubMed Scopus Google Scholar), and reduced global rates M.D. The of to calorie restriction on global PubMed Google of and restriction on in and PubMed Scopus Google is to the of and in L. Calorie restricti