Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1

Abstract

Many metabolic and physiological processes display circadian oscillations. We have shown that the core circadian regulator, CLOCK, is a histone acetyltransferase whose activity is counterbalanced by the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase SIRT1. Here we show that intracellular NAD+ levels cycle with a 24-hour rhythm, an oscillation driven by the circadian clock. CLOCK:BMAL1 regulates the circadian expression of NAMPT (nicotinamide phosphoribosyltransferase), an enzyme that provides a rate-limiting step in the NAD+ salvage pathway. SIRT1 is recruited to the Nampt promoter and contributes to the circadian synthesis of its own coenzyme. Using the specific inhibitor FK866, we demonstrated that NAMPT is required to modulate circadian gene expression. Our findings in mouse embryo fibroblasts reveal an interlocked transcriptional-enzymatic feedback loop that governs the molecular interplay between cellular metabolism and circadian rhythms.

Figure 1. Circadian oscillation of NAD⁺ is controlled by the clock

(A and B) Cellular NAD⁺ was extracted from serum-entrained MEFs derived from wild type (wt) (A), clock/clock mutant mice (c/c) and Cry1/Cry2 double KO mice (cry) (B) at indicated time points and analyzed by LC/MSⁿ. Three independent experiments were performed and representative results are shown. All data are means ± SEM of three independent samples. (C) Average NAD⁺ levels in wt, c/c and cry MEFs. Data are shown as means ± SEM of >50 independent samples. (D) Cellular nicotinamide (NAM) was extracted from serum-entrained MEFs derived from wt, c/c and cry mice at indicated time points and analyzed by LC/MSⁿ. (E) Average NAM levels in wt, c/c and cry MEFs. Data are shown as means ± SEM of >50 independent samples.

Figure 2. The circadian clock controls Nampt gene expression

(A) Scheme of the NAD⁺ salvage pathway in mammals. NAM, nicotinamide; NMN, nicotinamide mononucleotide; NAMPT, nicotinamide phosphoribosyltransferase; NMNAT1-3, nicotinamide mononucleotide adenyltransferase. (B) Nampt gene expression in livers from light-entrained wt and c/c mutant mice was quantified by q-PCR. Nampt gene expression at ZT 15 in liver from wt mice was set to 1. (C) Nampt and Dbp gene expressions in serum-entrained MEFs from wt and c/c mutant mice were quantified by q-PCR. Expression at time 0 in wt MEFs was set to 1. All data are means ± SEM of three independent samples.

Figure 3. Regulation of the Nampt promoter by CLOCK:BMAL1 and SIRT1

(A) Schematic diagram of regulatory elements in human Nampt promoter. TSS (Transcription Start Site) primer region for q-PCR is shown as arrowheads. Truncated forms of Nampt promoter used in (C) are shown (arrows). Putative transcription factors binding sites are indicated: HRE, hypoxia-inducible factor-responsible element; SP1, specificity protein 1; CRE, cAMP-response element; AP-1, activator protein 1; GRE, glucocorticoid receptor response element. (B) Conserved E-boxes (capitalized) are shown. Numbers indicate positions from human transcription start site. (C) Schematic diagram of Nampt promoter constructs. Effect of CLOCK:BMAL1 (+CL/BM; black bars) on luciferase activity is shown. Luciferase activity of CLOCK:BMAL1 on the pGL4.10 was set as 1. All data are means ± SEM of three independent samples. (D) Representative results of ChIP assays analyzed by semiquantitative PCR. Dual crosslinked nuclear extracts isolated from MEFs after 16 or 24 hr serum shock and subjected to ChIP assay with anti-SIRT1, anti-CLOCK, anti-BMAL1, or no antibody (ctrl). No antibody and 3’R primers were used as controls for immunoprecipitation and PCR, respectively. (E) Quantification of ChIP by q-PCR. q-PCR was performed on the same samples as described in (D). All data are means ± SEM of three independent samples.

Figure 4. NAMPT modulates the circadian clock

(A) Per2 and Dbp gene expression levels in serum-entrained MEFs treated with 10 nM FK866 or EtOH as control (solvent: ctrl) were analyzed by q-PCR. Highest value for each gene in EtOH treated MEFs was set to 1. All data are means ± SEM of three independent samples. (B) BMAL1 Lys537 acetylation profile in serum-entrained MEFs either treated with 10 nM FK866 or EtOH. Extracts prepared from indicated time points and BMAL1 acetylation was monitored (7). Input samples were probed with anti-BMAL1 and anti-GAPDH antibodies. (C) Scheme of the transcription-enzymatic interplay by which the circadian machinery governs the intracellular levels of NAD⁺. The NAD⁺-dependent deacetylase SIRT1 is thereby controlling the oscillatory synthesis of its own coenzyme.