Fasting Imparts a Switch to Alternative Daily Pathways in Liver and Muscle

Abstract

The circadian clock operates as intrinsic time-keeping machinery to preserve homeostasis in response to the changing environment. While food is a known zeitgeber for clocks in peripheral tissues, it remains unclear how lack of food influences clock function. We demonstrate that the transcriptional response to fasting operates through molecular mechanisms that are distinct from time-restricted feeding regimens. First, fasting affects core clock genes and proteins, resulting in blunted rhythmicity of BMAL1 and REV-ERBα both in liver and skeletal muscle. Second, fasting induces a switch in temporal gene expression through dedicated fasting-sensitive transcription factors such as GR, CREB, FOXO, TFEB, and PPARs. Third, the rhythmic genomic response to fasting is sustainable by prolonged fasting and reversible by refeeding. Thus, fasting imposes specialized dynamics of transcriptional coordination between the clock and nutrient-sensitive pathways, thereby achieving a switch to fasting-specific temporal gene regulation.

Keywords: RNA-seq; circadian; clock; fasting; liver; metabolism; muscle; rhythm; transcriptome.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Temporal Response to Fasting in Liver and Muscle

(A) Depiction of the experimental design for the 24-hr fasting and tissue collection at ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20. (B and C) RNA-seq generated heatmaps of cycling transcripts only in FED (left) or in FAST (right) in (B) liver and (C) skeletal muscle (JTK_CYCLE p

Figure 2.. Temporal Response to Fasting in Distinct Group of Tissue-Specific Genes

(A)Venn diagrams of hepatic and muscle cycling transcripts in FED. (B) Venn diagrams of hepatic and muscle cycling transcripts in FAST. (C) Gene Ontology analysis of top five biological processes enriched in rhythmic hepatic (blue) and muscle (red) genes only in FED, with the number of genes indicated on the graph. (D) Gene Ontology analysis of top five biological processes enriched in rhythmic hepatic (blue) and muscle (red) genes only in FAST, with the number of genes indicated on the graph. (E) Venn diagram of the number of hepatic cycling genes in fasting mice and in time-restricted fed mice (JTK_CYCLE p ad libitum normal chow for 24 hr were subsequently fasted for 24 hr. Arrow indicates time of food removal. (I) Locomotor activity during light and dark phases under FED and FAST conditions. Data are shown as means + SEMs (n = 16 biological replicates). *p ****p < 0.0001 by two-way ANOVA (interaction/phase/group) with Bonferroni post hoc tests. ns, non-significant.

Figure 3.. The Circadian Clock Fails to Sustain Robust Rhythmicity under Fasting Conditions

(A and B) Expression profiles of core clock genes in (A) liver and (B) muscle. Transcripts were normalized to 18S rRNA and presented as means + SEMs (n = 5 replicates per time point per group). ZT0 is double plotted for visualization. *p **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA (interaction/time/group) with Bonferroni post hoc tests. (C and D) Core clock proteins in total lysates from liver (C) and muscle (D) of FED and FAST mice. (E and F) Quantitation of the blot band density of the hepatic (E) and muscle (F) proteins presented as means + SEMs (n = 3 replicates per time point per group). **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA with Bonferroni post hoc tests.

Figure 4.. Temporal Pattern of Differential Gene Regulation by Clock and Fasting-Induced Transcription Factors

(A) Number of oscillating genes among GR, CREB, FOXO, TFEB, and PPARα targets in liver under FED, FAST, or both FED and FAST conditions. (B) Peak phase distribution of liver fasting-sensitive transcription factor (TF) target genes oscillating only in FED (left) and FAST (right). (C) Number of oscillating genes among GR, CREB, FOXO, TFEB, and PPARδ targets in skeletal muscle under FED, FAST, or both FED and FAST conditions. (D) Peak phase distribution of fasting-sensitive TF target genes in muscle oscillating only in FED (left) and FAST (right). (E) Gene set enrichment analysis (GSEA) for hepatic BMAL1 target genes at ZT8 enriched in FED. (F) Differentially expressed BMAL1 liver target genes at ZT8 illustrating fasting-sensitive TF coordinated regulation. (G) GSEA for muscle BMAL1 target genes at ZT8 enriched in FED. (H) Differentially expressed muscle BMAL1 target genes at ZT8 illustrating fasting-sensitive TF coordinated regulation. (I) GSEA for hepatic REV-ERBα target genes at ZT12 enriched in FAST. (J) Differentially expressed REV-ERBα liver target genes at ZT12 illustrating fasting-sensitive TF coordinated regulation. (K) GSEA for muscle HDAC3 target genes at ZT12 enriched in FAST. (L) Differentially expressed HDAC3 muscle target genes at ZT12 illustrating fasting-sensitive TF coordinated regulation. Gene sets with a false discovery rate (FDR)

Figure 5.. Altered Recruitment of BMAL1 and Fasting-Induced TFs after Fasting

(A) Criteria for gene classification of BMAL1-target genes and differential gene expression after fasting. (B) Number of genes identified for each class in liver and skeletal muscle at ZT8 (left) and ZT12 (right). (C–E) Chromatin immunoprecipitation in liver for BMAL1 or immunoglobulin G (IgG) followed by RT-qPCR specific for class I genes (C) Dbp, (D) Per2, and (E) Nr1d1 (Rev-ERBα). (F–I) Chromatin immunoprecipitation in liver for BMAL1, GR, CREB, PPARα, or IgG followed by RT-qPCR specific for class II genes (F) Fkbp5, (G) Gpt2, (H) Acot4, and (I) Cidec. (J–L) Chromatin immunoprecipitation in liver for GR, CREB, PPARα, or IgG followed by RT-qPCR specific for class III genes (J) Got1, (K) G6pc, and (L) Acot2. Data are presented as means + SEMs (n = 3–4 replicates per time point per group). Diagram above shows regions of amplification by primers designed for analysis (arrows) and TSS (transcription start site). Gene expression profiles are shown next to ChIP-qPCR data for visualization. Transcripts were normalized to 18S rRNA and presented as means + SEMs (n = 5 replicates per time point per group). ZT0 is double plotted for visualization. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA (interaction/time/group) with Bonferroni post hoc tests.

Figure 6.. BMAL1 Modulates Fasting-Sensing Pathways

(A and B) Representative gene expression profiles of (A) hepatic and (B) muscle BMAL1-target genes repressed by fasting (class I) in WT and Bmal1—/— mice at ZT8. (C and D) Gene expression profiles of (C) hepatic and (D) muscle BMAL1-target genes induced by fasting (class II) in WT and Bmal1—/— mice at ZT8. (E and F) Gene expression profiles of (E) hepatic and (F) muscle fasting-induced genes of non-BMAL1 targets (class III) in WT and Bmal1—/— mice at ZT8. (G) BMAL1 in hepatic total lysates from WT and Bmal1—/— mice at ZT8 under ad libitum fed (FED) and 24-hr fasting (FAST) conditions. (H) Quantitation of hepatic BMAL1 presented as means + SEMs (n = 3 replicates per group). (I) Fasting-responsive proteins in muscle total lysates from WT and Bmal1—/— mice at ZT8 under FED and FAST conditions. (J) Quantitation of muscle BMAL1 presented as means + SEMs (n = 3 biological replicates per group). (K) Fasting-responsive proteins in hepatic nuclear extracts from WT and Bmal1—/— mice at ZT8 under FED and FAST conditions. Transcript levels were normalized to 18S rRNA and presented as means + SEMs (n = 4–5 replicates per genotype per group). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA with Bonferroni post hoc tests.

Figure 7.. Response to Fasting Is Reversible by Refeeding

(A) Experimental design of 48-hr fasting and 24-hr fasting with 24-hr refeeding. Tissues were collected at ZT8 and ZT20. (B) Body weight before and after ad libitum fed (FED), 48-hr fasting (48h FS), or 24-hr fasting with 24-hr refeeding (REFED). (C) Serum corticosterone levels under FED, 48h-FS, and REFED conditions. Data are shown as means + SEMs (n = 3 replicates per time point per group). (D and E) Gene expression profiles of (D) hepatic and (E) muscle core clock genes normalized to 18S rRNA and presented as means + SEMs (n = 3 replicates per time point per group). (F and G) Core clock proteins in total lysates from liver (F) and muscle (G) of FED, 48h-FS, and REFED mice. (H and I) Gene expression profiles of (H) hepatic and (I) muscle genes displaying rhythmic response to 24-hr fasting (24h-FS) and 48h-FS. Transcript levels were normalized to 18S rRNA and presented as means + SEMs (n = 3 replicates per time point per group). *p **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA with Bonferroni post hoc tests.