Circadian Reprogramming in the Liver Identifies Metabolic Pathways of Aging

Abstract

The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD⁺-related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging.

Keywords: Acetylation; Aging; Circadian Clock; Liver Metabolism; NAD; Reprogramming; SIRT1.

Fig. 1.. Circadian transcriptome analysis in the liver: effect of aging and caloric restriction.

(A) Venn diagram displays the total number (top) and ratio (bottom) of rhythmic genes in the liver from young mice fed a normal diet (YND, green) and old mice fed a normal diet (OND, orange), including common genes. (B) Amplitude plot represents the distribution of oscillatory genes in YND and OND. (C) Heatmaps display rhythmic genes exclusively in YND (left panel) and OND group (right panel). (D) Circadian gene expression of core clock genes, Bmal1, Clock, Rev-erbα, Per2, and Cry1 in YND and OND liver was determined by qPCR. (E) Venn diagram displays hepatic rhythmic genes from YND (green), young mice subjected to CR (YCR, purple), and common genes. (F) Amplitude distribution of rhythmic genes in YND and YCR. (G) Heatmaps display circadian genes found exclusively in YND (left panel) and YCR (right panel). (H) Core clock gene expression in YND and YCR by qPCR. (I) Venn diagram represent circadian genes from OND (orange), old mice subjected to CR (OCR, blue), and common rhythmic genes. (J) Amplitude distribution of circadian genes in OND and OCR. (K) Heatmaps display circadian genes found exclusively in OND (left panel) and OCR (right panel). (L) Determination of core clock gene expression in OND and OCR by qPCR. Data represent mean±SEM and was analyzed by two-way ANOVA using Bonferroni posttest. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively (YND, n=4–5 per time point; OND, n=3–4 per time point; YCR, n=5–6 per time point; OCR, n=3–5 per time point). See also Figure S1, S2, S3, and S4.

Fig. 2.. GO ontology (GO) analysis of rhythmic genes from the circadian hepatic transcriptome.

(A) GO analysis of genes exclusively circadian in YND (left, green) and OND (right, orange). Numbers within the pie charts indicate number of circadian genes identified within each biological process, using a p-value cutoff of 0.01. (B) GO analysis of genes exclusively circadian in YND (left, green) versus YCR (right, purple). (C) GO analysis of genes exclusively circadian in OND (left, orange) versus OCR (right, blue). 3–6 biological replicates per time point were subjected to the hepatic transcriptomic analysis. See also Figure S1, S3, and S4.

Fig. 3.. Distinct phase signature of circadian hepatic genes.

(A) Phase analysis of YND (green), OND (orange), YCR (purple), and OCR (blue) liver-specific oscillating genes selected to be circadian by JTK_cycle, based on a p-value cutoff of 0.01. Phase 1, 2, 3 and 4 indicate ZT 0–6, ZT 12–18, ZT 8–16, and ZT 20–2, respectively. (B) Venn diagrams display the total number (top) and ratio (bottom) of phase-specific rhythmic genes in YND, OND, YCR, and OCR. Phase 1, 2, 3 and 4 correspond to ZT 0–6, ZT 12–18, ZT 8–16, and ZT 20–2, respectively. Yellow squares highlight significant overlap between YND versus YCR, and between OND versus OCR. (C) Tissue-specific phase comparison of gene expression was performed using JTK_cycle, based on p-value cutoff of 0.01. Red lines, green lines, and blue lines indicate the phase of rhythmic genes in the liver, EpSCs, and MsSCs, respectively. 3–6 biological replicates per time point per tissue were subjected to the transcriptomic analysis.

Fig. 4.. Tissue-specific circadian signatures identified by transcriptomic analysis.

(A) Venn diagram displays the number of genes selected to be circadian by JTK_cycle, based on p-value cutoff of 0.01 in each tissue/cell including liver (red), EpSCs (green), and MsSCs (blue). Pie charts display GO terms of genes exclusively circadian in liver (red). Common circadian genes in EpSCs and MsSCs are indicated (light gray) versus rhythmic genes in all three tissues/cells (dark gray). (B) Histogram represents the number of tissue-independent common circadian genes in each group. (C) Histogram represents the significance of each biological process and numbers within the histogram indicate number of circadian genes identified within each biological process. GO terms regarding NAD metabolism/acetylation processes specifically identified by liver-specific circadian genes (left), while GO terms regarding DNA damage responses specifically identified by EpSCs-specific circadian genes (right). Top eight GO terms regarding DNA damage responses identified by EpScs-specific circadian genes were listed. p-value cutoff of 0.01 was used. 3–6 biological replicates per time point per tissue were subjected to the transcriptomic analysis. See also Figure S5.

Fig. 5.. Hepatic cyclic protein acetylation and protein hyperacetylation under CR.

(A and B) Circadian lysates from livers of YND, OND, YCR, and OCR were probed with a pan acetylated lysine antibody by western blot and quantified using three independent samples. p84 was used as a loading control. (C-F) Abundance of acetylated H3K9/K14 and H3K27 was determined by ChIP analysis using YND (green) versus YCR (purple) liver and OND (orange) versus OCR (blue) liver at Cry1 (C), Per2 (D), Pck1 (E), and Por (F) promoters (YND, n=4–5 per time point; OND, n=3–4 per time point; YCR, n=5–6 per time point; OCR, n=3–5 per time point). IgG was used as a negative control. Corresponding gene expression as determined by qPCR is shown on the left. Data represent mean±SEM and was analyzed by two-way ANOVA using Bonferroni posttest. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively. See also Figure S6 and S7.

Fig. 6.. Elevation of hepatic NAD⁺ and related metabolites by CR.

(A) Heatmap displays the levels of NAD⁺ and related metabolites in YND (n=3 per time point), YCR (n=3 per time point), OND (n=3 per time point), and OCR (n=3 per time point) groups at ZT 0 (left) and ZT 12 (right). (B) Histograms show the concentrations of NAD⁺ metabolites (NAD⁺, NADP⁺, NR, NMN, NAM, ADRP, NAAD, and Me4PY). (C) Schematic summary of the impact of aging (blue arrows) and CR (yellow arrows) on the NAD⁺ salvage pathway. Data represents mean±SEM from three independent samples and was analyzed by two-way ANOVA using Bonferroni posttest. *, **, and *** indicate P<0.05, P<0.01, and P<0.001 versus YND at corresponding time points, respectively. #, ##, and ### indicate P<0.05, P<0.01, and P<0.001 versus OND at corresponding time points, respectively. † indicates P<0.05 between ZT 0 and ZT 12, respectively. Abbreviations: AOX1, aldehyde oxidase1; Me2PY, N-methyl-2-pyridone-5-carboxamide; Me4PY, N-methyl-4-pyridone-5-carboxamide; MeNAM, methylnicotinamide; NAAD, nicotinic acid adenine dinucleotide; NAD⁺/NADH, oxidized/reduced nicotinamide adenine dinucleotide; NADK, nicotinamide adenine dinucleotide kinase; NADP⁺/NADPH, oxidized/reduced nicotinamide adenine dinucleotide phosphate; NADS, nicotinamide adenine dinucleotide synthase; NAM, nicotinamide; NAMPT, nicotinamide phosphoribosyltransferase; NMN, nicotinamide mononucleotide; NMNAT, nicotinamide/nicotinic acid mononucleotide adenylyltransferase; NNMT, NAM N-methyltransferase; NR, nicotinamide riboside; NRK, nicotinamide riboside kinase; NT5, 5-nucleotidase; NUDT, nudix hydrolase; PNP, purine nucleoside phosphorylase; SIRT1, sirtuin1.

Fig. 7.. CR activates SIRT1 and modulates circadian acetyl CoA metabolic pathways.

(A) Venn diagrams display the overlap between hepatic SIRT1-dependent circadian genes (Masri et al., 2014) and rhythmic genes in YND (green), OND (orange), YCR (purple), and OCR (blue). Histogram displays the number of overlapping circadian genes in each group. JTK_cycle was used for the selection of circadian genes, based on p-value cutoff of 0.01, for both transcriptome datasets. (B) Circadian gene expression of Nampt, the rate-limiting enzyme of the NAD⁺ salvage pathway. (C) Acetylated BMAL1 from liver whole cell extracts was determined by western blot. p84 was used as a loading control. (D) Gene expression of glycolytic enzymes, Gck and Pklr, compared between YND (green) and YCR (purple). (E) ACLY protein levels in liver nuclear extracts were determined by western blot, compared to the p84 loading control. (F) Gene expression profiles of SREBP-dependent lipogenic target genes, Fasn, Acaca, and Scd1. (G) Cellular acetate concentrations in liver as measured by colorimetric assay. (H) Circadian acetylation of AceCS1 levels as determined by western blot using liver nuclear extracts from YND, OND, YCR, and OCR. p84 was used as a loading control. (I) Scheme represents the circadian reorganization of CR-dependent metabolism which may rescue age-associated circadian decline in the liver. Western blot images represent a minimum of three independent experiments. Data represent mean±SEM and was analyzed by two-way ANOVA using Bonferroni posttest. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively (YND, n=4–5 per time point; OND, n=3–4 per time point; YCR, n=5–6 per time point; OCR, n=3–5 per time point).