Using metabolomics profiling, we show that CR mice had significantly higher levels of neurotransmitters , neuronal integrity markers , essential fatty acids , and biochemicals associated carnitine metabolism (related to reduced oxidative stress and inflammation) in the cerebral cortex and hippocampus at 2-h. These biochemicals remained at high levels at the 6-h postprandial time-point. The AL mice did not show the similar increases in essential fatty acid and carnitine metabolism until the 6-h time-point, and failed to show increases in neurotransmitters and neuronal integrity markers at any time-point. On the other hand, metabolites related to glucose utilization—glycolysis and pentose phosphate pathway (PPP)—were low in the CR mice throughout the 6-h period and significantly increased at the 6-h time-point in the AL mice. Our findings suggest that CR induces distinct postprandial responses in metabolites that are essential to maintain brain functions. CR mice produced higher levels of essential brain metabolites in a shorter period after a meal and sustained the levels for an extended period, while maintaining a lower level of glucose utilization. These early brain metabolism changes in the CR mice might play a critical role for neuroprotection in aging. Understanding the interplay between dietary intervention and postprandial metabolic responses from an early age may have profound implications for impeding brain aging and reducing risk for neurodegenerative disorders.Caloric restriction (CR) has been shown to extend longevity and protect brain function in aging. However, the effects of CR in young adult mice remain largely unexplored. In addition to the fundamental, long-term changes, recent studies demonstrate that CR has a significant impact on transient, postprandial metabolic flexibility and turnover compared to control groups. The goal of this study was to identify the brain metabolic changes at a transient (2 h) and steady (6 h) postprandial state in young mice (5–6 months of age) fed with CR or  Caloric restriction (CR), without malnutrition, has been demonstrated repeatedly to extend lifespan in various species . A largeThe impacts of CR on brain function at an early age are largely unexplored, however. We were interested to know if CR also made significant effects in young adult mice that might be associated with the neuroprotection seen with aging, including metabolites related to neurotransmitters, WMI, glycolysis, and inflammation. Further, the metabolic changes induced by CR may also be postprandial time-dependent. Recent studies demonstrated that individuals with CR had significant differences in transient, postprandial metabolic flexibility, and turnover compared to the control groups –13. Therad libitum . We used metabolomics profiling to determine the levels of metabolites of interest. We focused on the brain regions that are highly associated with cognitive functions in rodents, including cerebral cortex and hippocampus. We hypothesized that CR may have significant effects on postprandial brain metabolism in young mice.In this study, our goal was to identify the brain metabolic changes at transient (2 h) and steady (6 h) postprandial states between young mice fed with CR or N = 14 for each group). The 40% CR was administrated to the animals by week 16 and the diet was continued over the lifetime. The vitamin-fortified NIH-31 (NIH-31 fortified) diet fed to CR mice provided 60% of the calories and additional vitamins supplement consumed by ad libitum mice. After arriving at our facilities, mice were housed individually (1 mouse per cage) in a specific pathogen-free facility. The CR mice were fed a pellet of the CR diet between 7 a.m. and 9 a.m. everyday. The mice were situated for 3–4 weeks before sending for brain extracts. All experimental procedures were performed according to NIH guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky (UK).We obtained male C57BL/6N mice from the National Institute of Aging (NIA) Caloric Restriction Colony with groups of young adult mice (5–6 months of age)  fed withN = 7) was subjected under 2-h and the other half under 6-h postprandial brain tissue collection. Brain samples were sent to Metabolon Inc. for biochemical profiling and statistical analysis. Metabolon's standard solvent extraction method was used to prepare the samples, which were then equally split for analysis via liquid chromatography/mass spectrometry (LC/MS) or gas chromatography/mass spectrometry (GC/MS) using their standard protocol  source and linear ion-trap (LIT) mass analyzer. Aliquots of the vacuum-dried sample were reconstituted, one each in acidic or basic LC-compatible solvents containing 8 or more injection standards at fixed concentrations (to both ensure injection and chromatographic consistency). Extracts were loaded onto columns  and gradient-eluted with water and 95% methanol containing 0.1% formic acid (acidic extracts) or 6.5 mM ammonium bicarbonate (basic extracts). The instrument was set to scan 99–1,000 m/z and alternated between MS and MS/MS scans.Samples destined for analysis by GC-MS were dried under vacuum desiccation for a minimum of 18 h prior to being derivatized using bis(trimethylsilyl)trifluoroacetamide (BSTFA) as described . DerivatMetabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon . IdentifThe LIMS system encompasses sample accessioning, preparation, instrument analysis and reporting, and advanced data analysis. Additional informatics components include data extraction into a relational database and peak-identification software; proprietary data processing tools for QC and compound identification; and a collection of interpretation and visualization tools for use by data analysts. The hardware and software systems are built on a web-service platform utilizing Microsoft .NET technologies, which run on high-performance application servers and fiber-channel storage arrays in clusters to provide active failover and load balancing.t-test to was used to identify biochemicals that were significantly different between groups. Levels of statistical significance were reached when p < 0.05.Log transformations and imputation of missing values with the minimum observed values for each metabolite was performed. Welch's two-tailed glutamate, N-acetylglutamate, glycine, and serine (Glutamate is an excitatory neurotransmitter and associated with cognitive function (glycine and serine (a precursor of glycine) are inhibitory neurotransmitters  and N-acetyl-aspartyl-glutamate (NAAG) were also found significantly higher in the CR mice at the 2-h time-point. NAA and NAAG have been used as markers for neuronal integrity as they are most abundant in neurons and are also used as an index of neuron quantity  , docosapentaenoate , docosapentaenoate , and docosahexaenoate  at the 2-h time-point. These are omega-3, polyunsaturated fatty acids . DHA helaenoate) . Carnitiaenoate) . As carnaenoate) .glutamate, N-acetylglutamate, NAA, NAAG. The results suggest that AL mice may not be as effective in producing these metabolites after a meal, especially those related to improving neuronal integrity.Interestingly a similar pattern of metabolite increases were not found in the AL mice until the 6-h postprandial time-point (dihomolinolenate (20:3n3 or n6) and docosapentaenoate  , fructose-6-phosphate, and lactate stayed constant in the CR mice, whereas they significantly increased at 6-h in the AL mice; glucose was also higher in AL mice at 6-h compared to 2-h, but did not reach significance. A similar pattern was found with alanine, an amino acid produced from pyruvate (a product of glycolysis), as well as metabolites associated with pentose phosphate pathway (PPP), including arabitol and xyulose-5-phosphate and ribulose-5-phosphate  at the University of Kentucky (UK).LMY contributed to the major analysis and interpretation of data for the work. LEAY contributed to the data analysis. RM, MAK, and EA contributed biostatistical support for the metabolomic profiling. A-LL contributed to the major design, analysis and interpretation of data for the work. LMY, JH, and A-LL drafted and revised the work for important intellectual content. LMY, LEAY, JH, RM, MAK, EA, and A-LL approved of the final version and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.