Fig. 2: Daytime NADH elevation regulates genome-wide transcription of fatty acid and amino acid metabolism genes during TRF-CR.

a, RNA-Seq in liver in the morning (ZT4) demonstrating the effect of TRF-CR in null-transduced (left) (n = 6) and LbNOX-transduced (middle) (n = 6 for TRF-Reg, n = 5 for TRF-CR) 4–6-month-old male C57BL/6J mice for genes differentially expressed (DESeq2 FDR-adjusted P < 0.05) by TRF-CR in null-transduced mice (930 genes). Venn diagram (right) displays overlap in differentially expressed genes by TRF-CR in null- and LbNOX-transduced mice. b, Quadrant plot comparing transcriptional responses between TRF-CR (x axis) and LbNOX in TRF-CR (y axis). Each point indicates a gene that is differentially expressed by TRF-CR in null-transduced mice (930 genes). Colouring indicates genes within quadrant 2 (green) and quadrant 4 (red), and the percentages within each quadrant are shown. c,d, For the genes within quadrants 2 and 4 from b, the top 15 (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms enriched (P < 0.05) following gene ontology analysis and (d) JASPAR motifs enriched (P < 0.05) following HOMER DNA motif enrichment analysis are shown. e, Quadrant plots comparing the transcriptional response to TRF-CR in null-transduced mice (x axis) with that of genetic ablation of either Pparα (top) or Bmal1 (bottom) (y axis) in animals fed ad libitum. Each point indicates a gene that is differentially expressed by TRF-CR in null-transduced animals. Genes that have an absolute log₂(fold change) > 0.5 for both comparisons are coloured blue or black, and the percentage of genes was determined by quadrant (n = 3). f, LC–MS metabolomics profiling of amino acids in liver during the daytime (ZT4). The log₂(fold change) from TRF-CR (blue) (n = 5) and LbNOX in TRF-CR (white) (n = 6) is shown for select differential amino acids (two-tailed, unpaired Student’s t test with Benjamini and Hochberg adjustment for multiple measures FDR P < 0.05; see Supplementary Table 2 for full list of amino acids). Box and whisker plots depict the following: line, median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles. g, Model depicting the interrelationship of NADH during TRF-CR to the activity of PPARα and BMAL1 and the transcription of downstream oxidative gene networks.