Energy Limitation Rebuilds Energy Homeostasis Through Metabolic Remodeling And Translation Inhibition

Calorie restriction(CR)is a nutritional intervention based on reducing calorie intake without causing malnutrition.CR is believed to be the most consistent non-pharmacological intervention extending lifespan and delaying the onset of age-related phenotypes.It has been proved that CR prolongs lifespan in diverse species,including yeast,worms,flies and rodents.Studies in Rhesus monkeys also showed that CR prevents and delays the onset of age-related functional decline and diseases,such as type-2-diabetes,cancer,and cardiovascular diseases.Even if there are no definitive conclusion has been reached on the effect of CR on human lifespan extension,it has been reported that this intervention protects against age-associated pathologies in particular cardiovascular diseases and reduces risk factors for obesity,insulin resistance,and inflammation.The inverse linear relationship between calorie intake and lifespan suggests that the regulation of energy metabolism is important in the actions of CR.Dysregulation of energy metabolism is a hallmark of aging and disease,in contrast,a coordinated increase in the expression of genes involved in energy metabolism is a prominent feature of CR.The transcriptional patterms of calorie-restricted animals suggest that calorie restriction retards the aging process by causing a metabolic reprogramming.In addition,protein synthesis is one of the most energy-consuming cellular processes.It is specifically meaningful to study the proteome,which is sensitive to the energy state,rather than the transcriptome,to solve the mechanism of CR.Nevertheless,the proteomic study of CR is still very limited.Hence,the present study would strive to elucidate the mechanism of calorie restriction using the high coverage and accuracy quantitative proteomics method based on SILAC labeling mouse technique.First,we constructed a calorie restriction mice model and evaluated the validity of this model.CR significantly decreased the whole body weight and changed the body composition compared with ad libitum(AL)mice.The metabolic cage results showed that O2 comsuption,CO2 production,energy expenditure(EE)and respiratory exchange rate(RER)were all decreased in CR mice.The decreased RER revealed a shift from carbohydrate metabolism to fat metabolism during CR.CR mice also had a better glucose regulatory function in glucose tolerance test(GTT).The serum biochemical results also showed that the levels of cholesterol,triglyceride and fed glucose were all decreased in CR mice.These results demonstrated that CR rebuilt the energy homeostasis in a low calorie state.To investigate the mechanism of CR,we firstly examined the hepatic transcriptome of AL and CR mice by DNA microarray.The gene expression profle revealed that CR induced a metabolic shift toward increased energy metabolism and mitochondrial function.We further performed a proteomics analysis of liver tissue from AL and CR mice using the high eoverage and accuraey quantitative proteomies method.SILAC mouse is used as internal standard to ensure the high precision of the quantitative analysis.Gene ontology(GO)analysis showed that CR reprogrammed the hepatic proteome,and in particular,energy metabolism and translation system were significantly affected during CR.The biological process analysis revealed that CR promoted the oxidation reduction,energy metabolism,cellular respiration,tricarboxylic acid cycle(TCA)and fatty acid metabolic process,at the meanwhile,CR decreased translation,protein localization,protein transport,protein folding and glucose metabolic process compared with AL mice.The cellular component analysis also showed that a large amount of proteins were enrich in mitochondria.Based on the analysis of quantitative proteomics,we found that the translation system,which executes the most energetically expensive process,was heavily affected by CR.We found that CR inhibited the mTOR pathway,as well as the expression of ribosomal proteins.Next,we performed polysome profiling to study the translational activity.This method assesses the mRNA translational state based on the separation of mRNAs bound to varying number of ribosomes via density gradient centriftugation.We found that there was an overall reduction in the number of ribosomes bound to mRNAs upon CR,which reveals a lower translational activity in CR mice compared with AL mice.The analysis of quantitative proteomics revealed a metabolism reprogramming toward enhanced energy efficiency during CR.It indicated that mitochondria may play a critical role in CR.So we analyzed the mitochondrial function of AL and CR mice.Firstly,CR increased the expression of genes associated with mitochondrial function,including PGC-1?,PGC-1?,NRF-1,NRF-2,TFAM.Next,we examined the expression and activities of specific electron transport chain complexes.As expected,CR increased the expression of components of the mitochondrial electron transport chain,in addition,the activities of Complex ? and Complex ? were both elevated in CR mice compared with AL mice.The activity of citrate synthetase,a rate-limiting enzyme in TCA cycle,was also enhanced during CR.It has been reported that CR increased mitochondrial biogenesis,a concept that is challenged by recent studies.Here we showed that CR enhanced mitochondrial oxidative capacity and efficiency without increasing mitochondrial abundance.But the relative area of mitochondria was indeed increased.In conclusion,based on the high coverage and precision SILAC quantitative proteomics,the present study systematically analyzed the fimetion and mechanism of calorie restriction in liver.The proteomics results showed that,on the one hand CR activate energy metabolism to enhance mitochondrial efficiency,on the other hand,CR inhibited the translation system to lower energy expenditure.In brief,we found that calorie restriction rebuilt the energy homeostasis through promoting energy efficiency and reducing energy expenditure.