By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.

PERSPECTIVES were cultured in different glucose concentrations. The amplitude of circadian oscillations in gene expression correlated to glucose concentrations only in wild-type cells, but not in the absence of AMPK. In mouse liver, the accumulation and nuclear localization of AMPK, as well as the phosphorylation of known AMPK target proteins, oscillated in a circadian manner. Thus, perturbation of nutrient availability-and consequently, of AMPK activity-alters output of the circadian clock. Although AMPK is an attractive candidate for coupling metabolic and circadian cycles, additional regulators are likely involved. Thus, the ratio of oxidized nicotinamide ade-nine dinucleotide phosphate (NADP +) to its reduced form (NADPH)-which, like the AMP/ATP ratio, constitutes a diagnostic signature of a cell's metabolic state-has been proposed to affect circadian gene expression through diverse mechanisms. At least in vitro, the binding of the heterodimeric core clock transcription factors CLOCK-BMAL1 and NPAS2-BMAL1 to their cognate DNA sequences (so-called E-boxes) is enhanced by NADPH and impaired by NADP + (6). The transcriptional regulatory protein peroxisome proliferator-activated receptor γ (P PA R γ) coactivator 1α (PGC-1α), a well-known mediator of glucose and lipid metabolism, has been proposed to be another important player in connecting metabolism to circadian gene expression. This transcriptional coacti-vator associates with nuclear receptors of the ROR family and thereby modulates the transcription of the clock genes Bmal1 and Rev-erbα. Finally, the NAD +-dependent protein deacetylase sirtuin 1 infl uences the stability and activity of the core clock components PER2 and BMAL1, respectively (7, 8). Why are metabolic processes under tight circadian control? A simple explanation arises from the necessity to separate incompatible enzymatic processes within the same cell. Because complete spatial separation of anabolic and catabolic processes is frequently impossible, these have to be gated to different time windows. This necessity is well illustrated by the temporal sequestration of oxida-tive and reductive phases in yeast by an ultra-dian respiratory clock. For example, DNA is replicated exclusively in the reductive phase, when the concentration of genotoxic reactive oxygen species generated by mitochondrial respiration is minimal (9). In a yeast mutant in which the reductive phase is too short to allow for the completion of DNA synthesis, the mutation rate increases dramatically (10). In mammals, the master pacemaker in the SCN is phase-entrained primarily by light-dark cycles and thus cannot readily adapt to altered feeding rhythms. Hence, when food availability changes, nutrient-dependent synchronization cues must dominate the more direct signals from the SCN to maintain proper homeostasis of metabolism in peripheral tissues (1). This could explain the multitude of metabolic phase entrainment cues that synchronize the circadian core clock machinery in metabolically active peripheral organs. A major challen...