Abstract
Dietary restriction (DR) has many beneficial effects, but the detailed metabolic mechanism remains largely unresolved. As diet is essentially related to metabolism, we investigated the metabolite profiles of urines from control and DR animals using NMR and LC/MS metabolomic approaches. Multivariate analysis presented distinctive metabolic profiles and marker signals from glucuronide and glycine conjugation pathways in the DR group. Broad profiling of the urine phase II metabolites with neutral loss scanning showed that levels of glucuronide and glycine conjugation metabolites were generally higher in the DR group. The up-regulation of phase II detoxification in the DR group was confirmed by mRNA and protein expression levels of uridinediphospho-glucuronosyltransferase and glycine-N-acyltransferase in actual liver tissues. Histopathology and serum biochemistry showed that DR was correlated with the beneficial effects of low levels of serum alanine transaminase and glycogen granules in liver. In addition, the Nuclear factor (erythroid-derived 2)-like 2 signaling pathway was shown to be up-regulated, providing a mechanistic clue regarding the enhanced phase II detoxification in liver tissue. Taken together, our metabolomic and biochemical studies provide a possible metabolic perspective for understanding the complex mechanism underlying the beneficial effects of DR.
Highlights
From the ‡College of Pharmacy, Natural Product Research Institute, Seoul National University, Sillim-dong, Gwanak-gu, Seoul, Korea, 151-742; ¶Department of Biochemistry, Inha University Hospital and Center for Advanced Medical Education by BK21 project, College of Medicine, Inha University, Incheon, Korea, 400-712; ʈDepartment of Pathology, Inha University Hospital and College of Medicine, Inha University, Incheon, Korea, 400-712; **Department of Medicine, Inha University Hospital and College of Medicine, Inha University, Incheon, Korea, 400-712; ‡‡Respiratory and Allergy Division, Soonchunhyang University Bucheon Hospital, Bucheon, Kyenggi, Korea, 420-767; §§Department of Biological Sciences, Inha University, Incheon, Korea, 402-751
General Assessment of dietary restriction (DR) Effects—To ensure that our DR procedure led to a difference between the control and DR groups, we measured general parameters such as body weight, serum low density lipoprotein (LDL), and serum TG
NMR and LC/MS Metabolomics Analysis of DR Effects— Dietary intake is intimately related to metabolism, but the effects of DR on specific metabolic pathways, as related to the beneficial results, have been little explored
Summary
Chemicals and Reagents—HPLC-grade acetonitrile and water were purchased from Burdick & Jackson (Morristown, NJ). All onedimensional spectra of the urine samples were measured with an NMR spectrometer (Avance 500, Bruker Biospin, Rheinstetten, Germany) operating at a proton NMR frequency of 500.13 MHz. The NMR experiment was performed at the NMR facility at the Korea Basic Science Institute. The identification of metabolites was established using m/z values and MS/MS fragmentation patterns, which were compared with those in the Human Metabolome Project, METLIN, and MassBank databases These identifications were further confirmed using standard samples for all but two metabolites (phenylalanylhydroxyproline and hydroxymethoxyindole glucuronide). After centrifugation at 13,000 rpm for 30 min, the supernatant containing the total protein released from the liver tissue was quantified using a BCATM Protein Assay Kit (Pierce, Appleton, WI). Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), triglyceride (TG), and low density lipoprotein (LDL) levels were measured using commercial kits at Inha University hospital (Incheon, Korea)
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