Metabolic and chemical architecture of the mammalian circadian clock.

Circadian rhythms are endogenous periodic biological processes that occur on a daily timescale. These rhythms are generated by a transcriptional/translational feedback loop that consists of the CLOCK-BMAL1 heterodimeric transcriptional activator complex and the PER1/2-CRY1/2-CK1δ/ε repressive complex. The output pathways of this molecular feedback loop generate circadian rhythmicity in various biological processes. Among these, metabolism is a primary regulatory target of the circadian clock which can also feedback to modulate clock function. This intertwined relationship between circadian rhythms and metabolism makes circadian clock components promising therapeutic targets. Despite this, pharmacological therapeutics that target the circadian clock are relatively rare. In this review, we hope to stimulate interest in chemical chronobiology by providing a comprehensive background on the molecular mechanism of mammalian circadian rhythms and their connection to metabolism, highlighting important studies in the chemical approach to circadian research, and offering our perspectives on future developments in the field.

Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.

Caloric restriction (CR) prolongs life span, yet the mechanisms by which it does so remain poorly understood. Under CR, mice self-impose chronic cycles of 2-hour feeding and 22-hour fasting, raising the question of if it is calories, fasting, or time of day that is the cause of this increased life span. We show here that 30% CR was sufficient to extend the life span by 10%; however, a daily fasting interval and circadian alignment of feeding acted together to extend life span by 35% in male C57BL/6J mice. These effects were independent of body weight. Aging induced widespread increases in gene expression associated with inflammation and decreases in the expression of genes encoding components of metabolic pathways in liver from ad libitum-fed mice. CR at night ameliorated these aging-related changes. Our results show that circadian interventions promote longevity and provide a perspective to further explore mechanisms of aging.

The Disordered Amino Terminus of the Circadian Enzyme Nocturnin Modulates Its NADP(H) Phosphatase Activity by Changing Protein Dynamics.

Endogenous circadian clocks control the rhythmicity of a broad range of behavioral and physiological processes, and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes, and the NOCT protein exhibits phosphatase activity, catalyzing the removal of the 2'-phosphate from NADP(H). In addition to its daily nighttime peak of expression, it is also induced by acute stimuli. Loss of Nocturnin (Noct-/-) in mice results in resistance to high-fat diet-induced obesity, and loss of Noct in HEK293T cells confers a protective effect to oxidative stress. Modeling of the full-length Nocturnin protein reveals a partially structured amino terminus that is disparate from its CCR4 family members. The high sequence conservation of a leucine zipper-like (LZ-like) motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. In vitro biochemical and biophysical techniques demonstrate that the LZ-like domain within the flexible N-terminus is necessary for preserving the active site cleft in an optimal conformation to promote the efficient turnover of the substrate. This modulation occurs in cis and is pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity to provide fine-tuned control over cellular levels of NADPH.

Time to target the circadian clock for drug discovery.

The circadian clock is an intracellular timekeeping device that drives daily rhythms in diverse and extensive processes throughout the body. The clock mechanism comprises a core transcription/translation negative feedback loop that is modulated by a complex set of additional interlocking feedback loops. Pharmacological manipulation of the clock may be valuable for treating many maladies including jet lag, shift work and related sleep disorders, various metabolic diseases, and cancer. We review recent identification of small-molecule clock modulators and discuss the biochemical features of the core clock that may be amenable to future drug discovery.

Natural antisense transcript of Period2, Per2AS, regulates the amplitude of the mouse circadian clock.

In mammals, a set of core clock genes form transcription-translation feedback loops to generate circadian oscillations. We and others recently identified a novel transcript at the Period2 (Per2) locus that is transcribed from the antisense strand of Per2 This transcript, Per2AS, is expressed rhythmically and antiphasic to Per2 mRNA, leading to our hypothesis that Per2AS and Per2 mutually inhibit each other's expression and form a double negative feedback loop. By perturbing the expression of Per2AS, we found that Per2AS transcription, but not transcript, represses Per2 However, Per2 does not repress Per2AS, as Per2 knockdown led to a decrease in the Per2AS level, indicating that Per2AS forms a single negative feedback loop with Per2 and maintains the level of Per2 within the oscillatory range. Per2AS also regulates the amplitude of the circadian clock, and this function cannot be solely explained through its interaction with Per2, as Per2 knockdown does not recapitulate the phenotypes of Per2AS perturbation. Overall, our data indicate that Per2AS is an important regulatory molecule in the mammalian circadian clock machinery. Our work also supports the idea that antisense transcripts of core clock genes constitute a common feature of circadian clocks, as they are found in other organisms.

Importance of circadian timing for aging and longevity.

Dietary restriction (DR) decreases body weight, improves health, and extends lifespan. DR can be achieved by controlling how much and/or when food is provided, as well as by adjusting nutritional composition. Because these factors are often combined during DR, it is unclear which are necessary for beneficial effects. Several drugs have been utilized that target nutrient-sensing gene pathways, many of which change expression throughout the day, suggesting that the timing of drug administration is critical. Here, we discuss how dietary and pharmacological interventions promote a healthy lifespan by influencing energy intake and circadian rhythms.

Periodicity, repression, and the molecular architecture of the mammalian circadian clock.

Large molecular machines regulate daily cycles of transcriptional activity and help generate rhythmic behavior. In recent years, structural and biochemical analyses have elucidated a number of principles guiding the interactions of proteins that form the basis of circadian timing. In its simplest form, the circadian clock is composed of a transcription/translation feedback loop. However, this description elides a complicated process of activator recruitment, chromatin decompaction, recruitment of coactivators, expression of repressors, formation of a repressive complex, repression of the activators, and ultimately degradation of the repressors and reinitiation of the cycle. Understanding the core principles underlying the clock requires careful examination of molecular and even atomic level details of these processes. Here, we review major structural and biochemical findings in circadian biology and make the argument that shared protein interfaces within the clockwork are critical for both the generation of rhythmicity and timing of the clock.

Spatiotemporal regulation of NADP(H) phosphatase Nocturnin and its role in oxidative stress response.

An intimate link exists between circadian clocks and metabolism with nearly every metabolic pathway in the mammalian liver under circadian control. Circadian regulation of metabolism is largely driven by rhythmic transcriptional activation of clock-controlled genes. Among these output genes, Nocturnin (Noct) has one of the highest amplitude rhythms at the mRNA level. The Noct gene encodes a protein (NOC) that is highly conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, but highly purified NOC possesses little or no ribonuclease activity. Here, we show that NOC utilizes the dinucleotide NADP(H) as a substrate, removing the 2' phosphate to generate NAD(H), and is a direct regulator of oxidative stress response through its NADPH 2' phosphatase activity. Furthermore, we describe two isoforms of NOC in the mouse liver. The cytoplasmic form of NOC is constitutively expressed and associates externally with membranes of other organelles, including the endoplasmic reticulum, via N-terminal glycine myristoylation. In contrast, the mitochondrial form of NOC possesses high-amplitude circadian rhythmicity with peak expression level during the early dark phase. These findings suggest that NOC regulates local intracellular concentrations of NADP(H) in a manner that changes over the course of the day.

Chemical and structural analysis of a photoactive vertebrate cryptochrome from pigeon.

Computational and biochemical studies implicate the blue-light sensor cryptochrome (CRY) as an endogenous light-dependent magnetosensor enabling migratory birds to navigate using the Earth's magnetic field. Validation of such a mechanism has been hampered by the absence of structures of vertebrate CRYs that have functional photochemistry. Here we present crystal structures of Columba livia (pigeon) CRY4 that reveal evolutionarily conserved modifications to a sequence of Trp residues (Trp-triad) required for CRY photoreduction. In ClCRY4, the Trp-triad chain is extended to include a fourth Trp (W369) and a Tyr (Y319) residue at the protein surface that imparts an unusually high quantum yield of photoreduction. These results are consistent with observations of night migratory behavior in animals at low light levels and could have implications for photochemical pathways allowing magnetosensing.

Medicine in the Fourth Dimension.

The importance of circadian biology has rarely been considered in pre-clinical studies, and even more when translating to the bedside. Circadian biology is becoming a critical factor for improving drug efficacy and diminishing drug toxicity. Indeed, there is emerging evidence showing that some drugs are more effective at nighttime than daytime, whereas for others it is the opposite. This suggests that the biology of the target cell will determine how an organ will respond to a drug at a specific time of the day, thus modulating pharmacodynamics. Thus, it is now time that circadian factors become an integral part of translational research.

Nobiletin fortifies mitochondrial respiration in skeletal muscle to promote healthy aging against metabolic challenge.

Circadian disruption aggravates age-related decline and mortality. However, it remains unclear whether circadian enhancement can retard aging in mammals. We previously reported that the small molecule Nobiletin (NOB) activates ROR (retinoid acid receptor-related orphan receptor) nuclear receptors to potentiate circadian oscillation and protect against metabolic dysfunctions. Here we show that NOB significantly improves metabolic fitness in naturally aged mice fed with a regular diet (RD). Furthermore, NOB enhances healthy aging in mice fed with a high-fat diet (HF). In HF skeletal muscle, the NOB-ROR axis broadly activates genes for mitochondrial respiratory chain complexes (MRCs) and fortifies MRC activity and architecture, including Complex II activation and supercomplex formation. These mechanisms coordinately lead to a dichotomous mitochondrial optimization, namely increased ATP production and reduced ROS levels. Together, our study illustrates a focal mechanism by a clock-targeting pharmacological agent to optimize skeletal muscle mitochondrial respiration and promote healthy aging in metabolically stressed mammals.

The Circadian Protein Nocturnin Regulates Metabolic Adaptation in Brown Adipose Tissue.

Fine-tuning of transcriptional responses can be critical for long-term outcomes in response to an environmental challenge. The circadian protein Nocturnin belongs to a family of proteins that include exonucleases, endonucleases, and phosphatases and is most closely related to the CCR4 family of deadenylases that regulate the cellular transcriptome via control of poly(A) tail length of RNA transcripts. In this study, we investigate the role of Nocturnin in regulating the transcriptional response and downstream metabolic adaptations during cold exposure in brown adipose tissue. We find that Nocturnin exhibits dual localization within the cytosol and mitochondria, and loss of Nocturnin causes changes in expression of networks of mRNAs involved in mitochondrial function. Furthermore, Nocturnin-/- animals display significantly elevated levels of tricarboxylic acid cycle intermediates, indicating that they have distinct metabolic adaptations during a prolonged cold exposure. We conclude that cold-induced stimulation of Nocturnin levels can regulate long-term metabolic adaptations to environmental challenges.

A novel mouse model overexpressing Nocturnin results in decreased fat mass in male mice.

Nocturnin (NOCT) belongs to the Mg2+ dependent Exonucleases, Endonucleases, Phosphatase (EEP) family of enzymes that exhibit various functions in vitro and in vivo. NOCT is known to function as a deadenylase, cleaving poly-A tails from mRNA (messenger RNA) transcripts. Previously, we reported a role for NOCT in regulating bone marrow stromal cell differentiation through its interactions with PPARγ. In this study, we characterized the skeletal and adipose tissue phenotype when we globally overexpressed Noct in vivo. After 12 weeks of Noct overexpression, transgenic male mice had lower fat mass compared to controls, with no significant differences in the skeleton. Based on the presence of a mitochondrial target sequence in NOCT, we determined that mouse NOCT protein localizes to the mitochondria; subsequently, we found that NOCT overexpression led to a significant increase in the preadipocytes ability to utilize oxidative phosphorylation for ATP (adenosine triphosphate) generation. In summary, the effects of NOCT on adipocytes are likely through its novel role as a mediator of mitochondrial function.

An evolutionary hotspot defines functional differences between CRYPTOCHROMES.

Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRYPTOCHROME 1/2 (CRY1/2) and PER1/2). To understand the structural principles of regulation, we used evolutionary sequence analysis to identify co-evolving residues within the CRY/PHL protein family. Here we report the identification of an ancestral secondary cofactor-binding pocket as an interface in repressive CRYs, mediating regulation through direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, suggesting that subtle sequence divergences at this site can modulate clock function. Divergence between CRY1 and CRY2 at this site results in distinct periodic output. Weaker interactions between CRY2 and CLOCK/BMAL1 at this pocket are strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. Overall, this work provides a model for the mechanism and evolutionary variation of clock regulatory mechanisms.

Temporal Control of Metabolic Amplitude by Nocturnin.

The timing of food intake and nutrient utilization is critical to health and regulated partly by the circadian clock. Increased amplitude of circadian oscillations and metabolic output has been found to improve health in diabetic and obesity mouse models. Here, we report a function for the circadian deadenylase Nocturnin as a regulator of metabolic amplitude across the day/night cycle and in response to nutrient challenge. We show that mice lacking Nocturnin (Noct-/-) display significantly increased amplitudes of mRNA expression of hepatic genes encoding key metabolic enzymes regulating lipid and cholesterol synthesis, both over the daily circadian cycle and in response to fasting and refeeding. Noct-/- mice have increased plasma triglyceride throughout the night and increased amplitude of hepatic cholesterol levels. Therefore, posttranscriptional control by Nocturnin regulates the amplitude of these critical metabolic pathways, and loss of this activity results in increased metabolic flux and reduced obesity.

Rhythms of metabolism in adipose tissue and mitochondria.

Circadian clocks synchronize the daily functions of organisms with environmental cues like light-dark cycles and feeding rhythms. The master clock in the suprachiasmatic nucleus in the hypothalamus of the brain and the many clocks in the periphery are organized in a hierarchical manner; the master clock synchronizes the peripheral clocks, and the peripheral clocks provide feedback to the master clock in return. Not surprisingly, it has been shown that circadian rhythms and metabolism are closely linked. Metabolic disorders like obesity have a large cost to the individual and society and they are marked by adipose tissue and mitochondrial dysfunction. Mitochondria are central to energy metabolism and have key functions in processes like ATP production, oxidative phosphorylation, reactive oxygen species production and Ca2+ homeostasis. Mitochondria also play an important role in adipose tissue homeostasis and remodeling. Despite the extensive research investigating the link between circadian clock and metabolism, the circadian regulation of adipose tissue and mitochondria has mostly been unexplored until recently, and the emerging data in this topic are the focus of this review.

Circadian Posttranscriptional Regulatory Mechanisms in Mammals.

The circadian clock drives rhythms in the levels of thousands of proteins in the mammalian cell, arising in part from rhythmic transcriptional regulation of the genes that encode them. However, recent evidence has shown that posttranscriptional processes also play a major role in generating the rhythmic protein makeup and ultimately the rhythmic physiology of the cell. Regulation of steps throughout the life of the messenger RNA (mRNA), ranging from initial mRNA processing and export from the nucleus to extensive control of translation and degradation in the cytosol have been shown to be important for producing the final rhythms in protein levels critical for proper circadian rhythmicity. These findings will be reviewed here.

Period2 3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.

Mice under Caloric Restriction Self-Impose a Temporal Restriction of Food Intake as Revealed by an Automated Feeder System.

Caloric restriction (CR) extends lifespan in mammals, yet the mechanisms underlying its beneficial effects remain unknown. The manner in which CR has been implemented in longevity experiments is variable, with both timing and frequency of meals constrained by work schedules. It is commonplace to find that nocturnal rodents are fed during the daytime and meals are spaced out, introducing prolonged fasting intervals. Since implementation of feeding paradigms over the lifetime is logistically difficult, automation is critical, but existing systems are expensive and not amenable to scale. We have developed a system that controls duration, amount, and timing of food availability and records feeding and voluntary wheel-running activity in mice. Using this system, mice were exposed to temporal or caloric restriction protocols. Mice under CR self-imposed a temporal component by consolidating food intake and unexpectedly increasing wheel-running activity during the rest phase, revealing previously unrecognized relationships among feeding, metabolism, and behavior.