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Time-restricted eating (TRE), which limits the daily meal timing to a window of 6-12 h, has been shown to reduce the risks of cardiometabolic diseases through consolidating circadian rhythms of metabolism and physiology. Recent advances indicate that canonical circadian clocks are dispensable for the actions of TRE in the liver, and that meal timing entrains circadian rhythms in peripheral tissues in a tissue-specific manner (e.g., the liver and fat are readily entrainable, whereas the heart and kidneys are resistant). Here, we propose that TRE engages clock-modulated checkpoints (CCPs) to reset circadian rhythms of tissue functions. Elucidation of CCPs would reveal the mechanistic basis of tissue responsiveness to TRE, and facilitate the use of TRE in precision medicine for cardiometabolic diseases.

Summary Inverted feeding is a paradigm to study synchronization of circadian clocks by feeding rhythm in tissues more directly. Here, we provide a protocol for performing inverted feeding in mice and analyzing circadian rhythmicity in mouse tissues. We describe setting up inverted feeding and performing tissue dissection, followed by RNA extraction and gene expression analysis, and lastly R software-based analysis of circadian rhythmicity. This protocol can be combined with the use of CircaMetDB database for mechanistic studies of inverted feeding. For complete details on the use and execution of this protocol, please refer to Xin et al. (2021)., Graphical abstract, Highlights • Set up inverted feeding (IF) to evaluate clock synchronization by meal time • Hands-on tutorial of circadian rhythmicity detection and comparison in R • Protocol combined with CircaMetDB resource can be used for mechanistic study of IF, Inverted feeding is a paradigm to study synchronization of circadian clocks by feeding rhythm in tissues more directly. Here, we provide a protocol for performing inverted feeding in mice and analyzing circadian rhythmicity in mouse tissues. We describe setting up inverted feeding and performing tissue dissection, followed by RNA extraction and gene expression analysis, and lastly R software-based analysis of circadian rhythmicity. This protocol can be combined with the use of CircaMetDB database for mechanistic studies of inverted feeding.

Summary Time of eating synchronizes circadian rhythms of metabolism and physiology. Inverted feeding can uncouple peripheral circadian clocks from the central clock located in the suprachiasmatic nucleus. However, system-wide changes of circadian metabolism and physiology entrained to inverted feeding in peripheral tissues remain largely unexplored. Here, we performed a 24-h global profiling of transcripts and metabolites in mouse peripheral tissues to study the transition kinetics during inverted feeding, and revealed distinct kinetics in phase entrainment of diurnal transcriptomes by inverted feeding, which graded from fat tissue (near-completely entrained), liver, kidney, to heart. Phase kinetics of tissue clocks tracked with those of transcriptomes and were gated by light-related cues. Integrated analysis of transcripts and metabolites demonstrated that fatty acid oxidation entrained completely to inverted feeding in heart despite the slow kinetics/resistance of the heart clock to entrainment by feeding. This multi-omics resource defines circadian signatures of inverted feeding in peripheral tissues (www.CircaMetDB.org.cn)., Graphical abstract, Highlights • A multi-omics analysis of food entrainment in mouse peripheral tissues • Inverted feeding rhythm entrains diurnal transcriptomes with distinct kinetics • Phase kinetics of tissue clocks is conditioned by constant light • Cardiac metabolism entrains to feeding fast with slow kinetics of the heart clock, Animal Physiology ; Systems Biology ; Metabolomics ; Transcriptomics

Time of eating synchronizes circadian rhythms of metabolism and physiology. Inverted feeding reverses peripheral circadian clocks, but privileges the light-responsive central clock located in the suprachiasmatic nucleus. However, system-wide changes of circadian metabolism and physiology entrained to inverted feeding in peripheral tissues remain largely unexplored. Here, we performed a 24-h global profiling of transcripts and metabolites in mouse peripheral tissues to study the transition kinetics during inverted feeding, and revealed distinct kinetics in phase entrainment of diurnal transcriptomes by inverted feeding, which graded from fat tissue (near-completely entrained), liver, kidney, to heart. Phase kinetics of tissue clocks tracked with those of transcriptomes, and was gated by light cue. Integrated analysis of transcripts and metabolites demonstrated that fatty acid oxidation entrained completely to inverted feeding in heart despite the slow kinetics of the heart clock. This multi-omics resource defines circadian signatures of inverted feeding in peripheral tissues (www.CircaMetDB.org.cn).

SummaryCircadian clocks are coupled to metabolic oscillations through nutrient-sensing pathways. Nutrient flux into the hexosamine biosynthesis pathway triggers covalent protein modification by O-linked β-D-N-acetylglucosamine (O-GlcNAc). Here we show that the hexosamine/O-GlcNAc pathway modulates peripheral clock oscillation. O-GlcNAc transferase (OGT) promotes expression of BMAL1/CLOCK target genes and affects circadian oscillation of clock genes in vitro and in vivo. Both BMAL1 and CLOCK are rhythmically O-GlcNAcylated, and this protein modification stabilizes BMAL1 and CLOCK by inhibiting their ubiquitination. In vivo analysis of genetically modified mice with perturbed hepatic OGT expression shows aberrant circadian rhythms of glucose homeostasis. These results establish the counteraction between O-GlcNAcylation and ubiquitination as a key mechanism that regulates the circadian clock and suggest a crucial role for O-GlcNAc signaling in transducing nutritional signals to the core circadian timing machinery.