Reprogramming of the circadian clock by nutritional challenge

Abstract

Circadian rhythms and cellular metabolism are intimately linked. Here, we reveal that a high-fat diet (HFD) generates a profound reorganization of specific metabolic pathways, leading to widespread remodeling of the liver clock. Strikingly, in addition to disrupting the normal circadian cycle, HFD causes an unexpectedly large-scale genesis of de novo oscillating transcripts, resulting in reorganization of the coordinated oscillations between coherent transcripts and metabolites. The mechanisms underlying this reprogramming involve both the impairment of CLOCK:BMAL1 chromatin recruitment and a pronounced cyclic activation of surrogate pathways through the transcriptional regulator PPARγ. Finally, we demonstrate that it is specifically the nutritional challenge, and not the development of obesity, that causes the reprogramming of the clock and that the effects of the diet on the clock are reversible.

Figure 1. A High Fat Diet Alters the Circadian Profile of the Metabolome

(A) Number of hepatic metabolites affected by diet or time. (B) The hepatic circadian metabolome consists of metabolites that oscillate in both groups of animals regardless of diet (“Both”), metabolites that oscillate only in animals fed normal chow (“NC ”) and metabolites that oscillate only in animals fed high fat diet (“HF”). (P<0.05, JTK_cycle, N=5 biological replicates). (C) The number of hepatic metabolites altered by the HF diet at each zeitgeber time (ZT). (D) Percent of metabolites in a metabolic pathway changing at a specific ZT in HF animals. (E) Metabolic landscapes depict the percent of oscillatory metabolites that peak at a specific ZT for each feeding condition compared to the total number of oscillatory metabolites in that metabolic pathway. (F) Proportion of metabolites that oscillate on both diets which are in phase or phase shifted (left) and the direction of the phase shift (right). (G) Phase graph of metabolites that oscillate in Both conditions (left) or only in the NC or HF conditions (right). (H) Heat maps depicting phase delayed or phase advanced metabolites in HF livers. (I) Overlap of metabolites that are both CLOCK-dependent and sensitive to a HF diet.

Figure 2. The Circadian Transcriptome is Reprogrammed by a High Fat Diet

(A) The number of oscillatory transcripts only in NC, only in HF or in both NC and HF groups (P<0.01, JTK_cycle). (B) Heat maps for NC- and HF-only oscillating transcripts (P<0.05). (C) Gene annotation on oscillating genes with a P<0.01 reveals pathways that are oscillatory in both NC and HF livers (unique pathways in bold font). (D) Pathways in which oscillatory expression is lost by the HF diet. (E) KEGG pathways represented by genes oscillatory only in the HF liver. (F) Proportion of the oscillatory transcriptome shared in both liver sets that is phase-shifted (left), and the direction of the phase shift (right). (G) Phase analysis of transcripts that oscillate only in NC or HF. (H) Circadian fluctuations of the metabolome relative to the transcriptome in Both (left), NC-only (middle), or HF-only categories (right). (I) Extent of amplitude changes in transcript abundance (heat map and graph) and metabolites (graph) after HF feeding.

Figure 3. A High Fat Diet Disrupts Circadian Organization between the Transcriptome and Metabolome

(A) Heat map showing the relationships between all pairs of metabolites and enzymes in KEGG. (Note: “flat” is a subset of “not”, where the maximum abundance does not exceed the minimum by 20%.) Circled are the numbers referring to the five most common relationships. (B) Related enzyme transcripts and metabolites (“edges”) that follow a particular temporal profile. (C) Metabolites and related transcripts within the S-adenosylmethionine (SAM) node that gain oscillation in HF. (D) Oscillatory abundance of SAM, SAH, and their related enzymes Ehmt2 and Ahcyl2 only in HF.

Figure 4. A High Fat Diet Disrupts Uridine and NAD⁺ Metabolism by Impairing CLOCK:BMAL1 Recruitment

(A) Validation of microarray data by qPCR of clock and metabolic genes in NC and HF livers (N=10 biological replicates per ZT per feeding condition). (B) BMAL1 protein in nuclear lysates of NC and HF livers (Figure S4). (C) BMAL1 and CLOCK occupancy at the E1 and I1 positions of the Dbp promoter as well as its 3′UTR (CLOCK at Dbpi1- ****-time; **- interaction). (D) Oscillation of NAD⁺, uridine, and uracil in NC (blue) and HF (green) (N=5). (E) Oscillation of Nampt and Upp2 mRNA in NC (black squares) and HF (orange triangles) livers (N=10, Upp2: ***-time, ***-diet, ***-interaction; Nampt ****-time, ****-diet, ****- interaction; two-way ANOVA). (F) BMAL1 occupancy at the promoter of Nampt. (G) Relative binding of BMAL1 and CLOCK at the promoter of Upp2. (BMAL1 binding- **=diet, *-time, *= interaction; CLOCK binding-*=time, *- interaction; two-way ANOVA, N=4). (H) H3K4me3 at the promoter of Upp2 in NC and HF-fed animals (*-diet; two-way ANOVA, N=2 biological replicates). (*-P<0.05, **-P<0.01, ***-P<0.001, ****P<0.0001, Bonferroni posttests. Error bars=SEM.)

Figure 5. High Fat Diet Induced Transcriptional Reprogramming of the Hepatic Clock

(A). Number of newly oscillating genes in HF livers containing PPARγ, SREBP1, CREB1, SRF, STAT1, or AHR sites. (B) Pparγ mRNA in NC (black squares) and HF (orange triangles) livers (N=5). (C) PPARγ abundance in whole cell lysates (WCL), nuclear extracts (NE) and chromatin extracts (Chromatin) in animals fed NC or HF diet for ten weeks (each lane consists of PPARγ protein from 3 pooled livers). (D) Quantification of nuclear PPARγ protein normalized to the nuclear protein p84. Units are expressed as relative optical density. (E) Circadian abundance of Cidec mRNA. (F) Circadian change in H3K4me3 at the Cidec promoter. (G) PPARγ chromatin recruitment and Cidec expression relative to PPARγ in livers of animals on HF treated with PPARγ antagonist, GW9662 or vehicle. (H) Rhythmic PPARγ recruitment to the promoter of Cidec but not its 3′UTR. (I) Pcx mRNA in NC- and HF-fed animals. (I–J) Binding of PPARγ to the promoter PPARγ response elements (PPRE) and 3′UTR at the Pcx gene.

Figure 6. Acute Administration of a High Fat Diet, and not the Presence of Obesity is Sufficient to Reorganize the Hepatic Circadian Clock

(A) The number of hepatic metabolites affected by acute (three-day) high fat diet feeding. (B) Relative abundance of metabolites uracil, uridine, and NAD⁺ after acute HF diet. (0NC=ZT0, normal chow; 0HF=ZT0, high fat). (C) Bmal1 mRNA after three days on NC or HF diets (***-time, N=5). (D) Western blot of nuclear BMAL1 and p84 proteins. (E) BMAL1 targets (Nampt, Upp2 and Dbp), and PPARγ target (Cidec) after acute HF feeding. (Nampt, ***-time; *-diet; Upp2, ***-time; ***-diet, **-time/diet interaction; Dbp, ***-time; Cidec, *-time, N=5.) (F) CLOCK and BMAL1 occupancy at the Upp2 and Dbp promoter after 3-day feeding. (Clock occupancy, Upp2: **-time; **-diet *- interaction; CLOCK occupancy, Dbpi1: *-time; **- interaction; BMAL1 occupancy Dbpi1: *- interaction). (G) H3K4me3 marks at the Upp2 and Dbp promoters after 3-day feeding. (H3K4me3 at Dbpi1: *-time; **- interaction, N=4.) (H) Hepatic expression of Upp2, Dbp, and Pcx in animals removed from the HFD for two weeks (Upp2: ***-time, Dbp; ***-time, N=5). (I) BMAL1 occupancy at ZT12 at the promoters of Upp2 and Dbp in NC mice and mice withdrawn from the HF diet for two weeks. Error bars=SEM; ***-P<0.001, **-P<0.01, *-P<0.05.

Figure 7. Nutrient Insult Restructures the Hepatic Circadian Clock

A high fat diet both blocks oscillations within the previously existing clock system as well as triggering new oscillations where they previously did not exist.