Circadian clock controls rhythms in ketogenesis by interfering with PPARα transcriptional network.

Ketone bodies are energy-rich metabolites and signaling molecules whose production is mainly regulated by diet. Caloric restriction (CR) is a dietary intervention that improves metabolism and extends longevity across the taxa. We found that CR induced high-amplitude daily rhythms in blood ketone bodies (beta-hydroxybutyrate [ßOHB]) that correlated with liver ßOHB level. Time-restricted feeding, another periodic fasting-based diet, also led to rhythmic ßOHB but with reduced amplitude. CR induced strong circadian rhythms in the expression of fatty acid oxidation and ketogenesis genes in the liver. The transcriptional factor peroxisome-proliferator-activated-receptor α (PPARα) and its transcriptional target hepatokine fibroblast growth factor 21 (FGF21) are primary regulators of ketogenesis. Fgf21 expression and the PPARα transcriptional network became highly rhythmic in the CR liver, which implicated the involvement of the circadian clock. Mechanistically, the circadian clock proteins CLOCK, BMAL1, and cryptochromes (CRYs) interfered with PPARα transcriptional activity. Daily rhythms in the blood ßOHB level and in the expression of PPARα target genes were significantly impaired in circadian clock-deficient Cry1,2-/- mice. These data suggest that blood ßOHB level is tightly controlled and that the circadian clock is a regulator of diet-induced ketogenesis.

Two-meal caloric restriction induces 12-hour rhythms and improves glucose homeostasis.

Glucose metabolism is tightly regulated and disrupting glucose homeostasis is a hallmark of many diseases. Caloric restriction (CR), periodic fasting, and circadian rhythms are interlinked with glucose metabolism. Here, we directly investigated if CR depends on periodic fasting and circadian rhythms to improve glucose metabolism. CR was implemented as two-meals per day (2M-CR), provided at 12-hour intervals, and compared with one meal per day CR, mealtime (MT), and ad libitum (AL) feeding. The 2M-CR impacted the circadian rhythms in blood glucose, metabolic signaling, circadian clock, and glucose metabolism gene expression. 2M-CR significantly reduced around the clock blood glucose and improved glucose tolerance. Twenty-four-hour rhythms in mTOR signaling and gene expression observed under AL, MT, and CR, became 12-hour rhythms in 2M-CR. The 12-hour rhythms in behavior, gene expression, and signaling persisted in fasted mice, implicating some internal regulation. The study highlights that the reduction in caloric intake rather than meal frequency and duration of fasting is essential for metabolic reprograming and improvement in glucose metabolism and provides evidence on food-entrained molecular pacemaker, which can be uncoupled from the light-entrained circadian clock and rhythms.

It's about time; divergent circadian clocks in livers of mice and naked mole-rats.

Light is the key regulator of circadian clock, the time-keeping system synchronizing organism physiology and behavior with environmental day and night conditions. In its natural habitat, the strictly subterranean naked mole-rat (Heterocephalus glaber) has lived in a light-free environment for millennia. We questioned if this species retains a circadian clock and if the patterns of this clock and concomitant rhythms differed in liver tissue from mice and naked mole-rats. As expected, in mice, the various circadian clock genes peaked at different times of the day; the Period gene (Per) group peaked in the evening, whereas Brain and Muscle ARNT-like1 (Bmal1) gene peaked in the morning; this phase shift is considered to be fundamental for circadian clock function. In sharp contrast, in the naked mole-rat both Per1 and Per2, as well as Bmal1, peaked at the same time in the morning-around ZT2-suggesting the organization of the molecular circadian oscillator was different. Moreover, gene expression rhythms associated with glucose metabolism and mTOR signaling also differed between the species. Although the activity of mTORC1 was lower, while that of mTORC2 was higher in naked mole-rat livers compared to mice, unlike that of mice where the expression profiles of glucose metabolism genes were not synchronized, these were highly synchronized in naked mole-rats and likely linked to their use of feeding times at zeitgebers.

eIF2A-knockout mice reveal decreased life span and metabolic syndrome.

Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.

Calorie restriction reprograms diurnal rhythms in protein translation to regulate metabolism.

Calorie restriction (CR) delays aging and affects the circadian clocks by reprogramming circadian rhythms in gene expression. To expand on the circadian mechanisms in CR, we assayed rhythms in the protein translation by analyzing polysome-associated mRNAs in the liver of mice fed ad libitum (AL) and CR diets. Global comparison of the diets revealed that <1% of transcripts were differentially abundant in the polysomes. In contrast, the large differential, up to 10%, was detected when CR and AL diets were compared at individual times throughout the day. Most transcripts that were rhythmic under AL lost their rhythms, and many new transcripts gained rhythms under CR. Only a small fraction of transcripts, including the circadian clock genes, were rhythmic under both diets. Thus, CR strongly reprograms translation. CR affected translation of enzymes regulating long-chain acetyl-coenzyme A (Acyl-CoA) metabolism. The expression of the Acyl-CoA thioesterase (ACOT) family was induced upon CR, leading to the increased transcriptional activity of peroxisome proliferator-activated receptor α, the transcriptional factor regulated by the ACOT products. We propose that the differential translation induced by CR leads to a temporal partition and reprogramming of metabolic processes and provides a link between CR, lipid metabolism, and the circadian clock.-Makwana, K., Gosai, N., Poe, A., Kondratov, R. V. Calorie restriction reprograms diurnal rhythms in protein translation to regulate metabolism.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada's syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

The circadian clock and pathology of the ageing brain.

Ageing leads to a functional deterioration of many brain systems, including the circadian clock--an internal time-keeping system that generates ∼24-hour rhythms in physiology and behaviour. Numerous clinical studies have established a direct correlation between abnormal circadian clock functions and the severity of neurodegenerative and sleep disorders. Latest data from experiments in model organisms, gene expression studies and clinical trials imply that dysfunctions of the circadian clock contribute to ageing and age-associated pathologies, thereby suggesting a functional link between the circadian clock and age-associated decline of brain functions. Potential molecular mechanisms underlying this link include the circadian control of physiological processes such as brain metabolism, reactive oxygen species homeostasis, hormone secretion, autophagy and stem cell proliferation.

Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms.

Calorie restriction (CR) increases longevity in many species by unknown mechanisms. The circadian clock was proposed as a potential mediator of CR. Deficiency of the core component of the circadian clock-transcriptional factor BMAL1 (brain and muscle ARNT [aryl hydrocarbon receptor nuclear translocator]-like protein 1)-results in accelerated aging. Here we investigated the role of BMAL1 in mechanisms of CR. The 30% CR diet increased the life span of wild-type (WT) mice by 20% compared to mice on anad libitum(AL) diet but failed to increase life span ofBmal1(-/-)mice. BMAL1 deficiency impaired CR-mediated changes in the plasma levels of IGF-1 and insulin. We detected a statistically significantly reduction of IGF-1 in CRvs.AL by 50 to 70% in WT mice at several daily time points tested, while inBmal1(-/-)the reduction was not significant. Insulin levels in WT were reduced by 5 to 9%, whileBmal1(-/-)induced it by 10 to 35% at all time points tested. CR up-regulated the daily average expression ofBmal1(by 150%) and its downstream target genesPeriods(by 470% forPer1and by 130% forPer2). We propose that BMAL1 is an important mediator of CR, and activation of BMAL1 might link CR mechanisms with biologic clocks.-Patel, S. A., Chaudhari, A., Gupta, R., Velingkaar, N., Kondratov, R. V. Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms.

An extraribosomal function of ribosomal protein L13a in macrophages resolves inflammation.

Inflammation is an obligatory attempt of the immune system to protect the host from infections. However, unregulated synthesis of proinflammatory products can have detrimental effects. Although mechanisms that lead to inflammation are well appreciated, those that restrain it are not adequately understood. Creating macrophage-specific L13a-knockout mice, we report that depletion of ribosomal protein L13a abrogates the endogenous translation control of several chemokines in macrophages. Upon LPS-induced endotoxemia, these animals displayed symptoms of severe inflammation caused by widespread infiltration of macrophages in major organs causing tissue injury and reduced survival rates. Macrophages from these knockout animals show unregulated expression of several chemokines (e.g., CXCL13, CCL22, CCL8, and CCR3). These macrophages failed to show L13a-dependent RNA binding complex formation on target mRNAs. In addition, increased polyribosomal abundance of these mRNAs shows a defect in translation control in the macrophages. Thus, to our knowledge, our studies provide the first evidence of an essential extraribosomal function of ribosomal protein L13a in resolving physiological inflammation in a mammalian host.

Sexual dimorphism of circadian liver transcriptome.

Sexual dimorphism affects various aspects of physiology, metabolism and longevity. Circadian clock is a master regulator of metabolism. Anti-aging dietary interventions reprogram circadian transcriptome in the liver and other tissues, but little is known about sexual dimorphism of circadian transcriptome. We compared circadian transcriptomes in the liver of male and female mice on ad libitum (AL) and 30% caloric restriction (CR) diets. We found that AL female mice had a larger number of oscillating genes than male mice, and the portion of the transcriptome with sex-specific rhythms displayed phase difference. We found that CR increased the number of oscillating genes in both sexes and strongly synchronized the transcriptome without complete elimination of sex dimorphism in rhythms. Sex also had an effect on the response of the rhythms to CR. Gene ontology analysis revealed sex-specific signatures in metabolic pathways, which suggests a complex interaction of sex, circadian rhythms, and diet.

Pharmacological modulators of the circadian clock as potential therapeutic drugs: focus on genotoxic/anticancer therapy.

The circadian clock is an evolutionary conserved intrinsic timekeeping mechanism that controls daily variations in multiple biological processes. One important process that is modulated by the circadian clock is an organism's response to genotoxic stress, such as that induced by anticancer drug and radiation treatments. Numerous observations made in animal models have convincingly demonstrated that drug-induced toxicity displays prominent daily variations; therefore, undesirable side effects could be significantly reduced by administration of drugs at specific times when they are better tolerated. In some cases, these critical times of the day coincide with increased sensitivity of tumor cells allowing for a greater therapeutic index. Despite encouraging results of chronomodulated therapies, our knowledge of molecular mechanisms underlying these observations remains sketchy. Here we review recent progress in deciphering mechanistic links between circadian and stress response pathways with a focus on how these findings could be applied to anticancer clinical practice. We discuss the potential for using high-throughput screens to identify small molecules that can modulate basic parameters of the entire circadian machinery as well as functional activity of its individual components. We also describe the discovery of several small molecules that can pharmacologically modulate clock and that have a potential to be developed into therapeutic drugs. We believe that translational applications of clock-targeting pharmaceuticals are twofold: they may be developed into drugs to treat circadian-related disorders or used in combination with existing therapeutic strategies to improve therapeutic index of a given genotoxic treatment via the intrinsic clock mechanism.

Circadian proteins and genotoxic stress response.

The circadian clock is an evolutionarily conserved time-keeping system that coordinates the physiology of the organism with daily changes in the environment. A growing body of evidence gradually leads to the conception that virtually all aspects of the biochemical, physiological, and behavioral functions of the animal are linked to circadian regulation. Moreover, proper synchronization of various processes through the activity of circadian components is important for the well-being of many organisms, including humans. The focus of this review is the circadian control of an organism's response to genotoxic stress, which is a major contributor to life-threatening human pathologies such as cancer and cardiovascular disease.

Circadian Control of Mitochondria in Reactive Oxygen Species Homeostasis.

Significance: Mitochondria produce most of the cellular ATP through the process of oxidative phosphorylation. Energy metabolism in the mitochondria is associated with the production of reactive oxygen species (ROS). Excessive ROS production leads to oxidative stress and compromises cellular physiology. Energy metabolism in the mitochondria depends on nutrient flux and cellular metabolic needs, which are in turn connected with the feeding/fasting cycle. In animals, the feeding/fasting cycle is controlled by the circadian clock that generates 24-h rhythms in behavior, metabolism, and signaling. Recent Advances: Here, we discuss the role of the circadian clock and rhythms in mitochondria on ROS homeostasis. The circadian clock is involved in mitochondrial ROS production and detoxification through the control of nutrient flux and oxidation, uncoupling, antioxidant defense, and mitochondrial dynamics. Critical Issues: Little is known on the molecular mechanisms of circadian control of mitochondrial functions. The circadian clock regulates the expression and activity of mitochondrial metabolic and antioxidant enzymes. The regulation involves a direct transcriptional control by Circadian Locomotor Output Cycles Kaput/brain and muscle ARNT-like 1(CLOCK/BMAL1), nuclear factor erythroid-2-related factor 2 (NRF2) transcriptional network, and sirtuin-dependent posttranslational protein modifications. Future Perspectives: We hypothesize that the circadian clock orchestrates mitochondrial physiology to synchronize it with the feeding/fasting cycle. Circadian coordination of mitochondrial function couples energy metabolism with diets and contributes to antioxidant defense to prevent metabolic diseases and delay aging. Antioxid. Redox Signal. 37, 647-663.

Clock at the Core of Cancer Development.

To synchronize various biological processes with the day and night cycle, most organisms have developed circadian clocks. This evolutionarily conserved system is important in the temporal regulation of behavior, physiology and metabolism. Multiple pathological changes associated with circadian disruption support the importance of the clocks in mammals. Emerging links have revealed interplay between circadian clocks and signaling networks in cancer. Understanding the cross-talk between the circadian clock and tumorigenesis is imperative for its prevention, management and development of effective treatment options. In this review, we summarize the role of the circadian clock in regulation of one important metabolic pathway, insulin/IGF1/PI3K/mTOR signaling, and how dysregulation of this metabolic pathway could lead to uncontrolled cancer cell proliferation and growth. Targeting the circadian clock and rhythms either with recently discovered pharmaceutical agents or through environmental cues is a new direction in cancer chronotherapy. Combining the circadian approach with traditional methods, such as radiation, chemotherapy or the recently developed, immunotherapy, may improve tumor response, while simultaneously minimizing the adverse effects commonly associated with cancer therapies.

Reduced caloric intake and periodic fasting independently contribute to metabolic effects of caloric restriction.

Caloric restriction (CR) has positive effects on health and longevity. CR in mammals implements time-restricted (TR) feeding, a short period of feeding followed by prolonged fasting. Periodic fasting, in the form of TR or mealtime, improves metabolism without reduction in caloric intake. In order to understand the relative contribution of reduced food intake and periodic fasting to the health benefits of CR, we compared physiological and metabolic changes induced by CR and TR (without reduced food intake) in mice. CR significantly reduced blood glucose and insulin around the clock, improved glucose tolerance, and increased insulin sensitivity (IS). TR reduced blood insulin and increased insulin sensitivity, but in contrast to CR, TR did not improve glucose homeostasis. Liver expression of circadian clock genes was affected by both diets while the mRNA expression of glucose metabolism genes was significantly induced by CR, and not by TR, which is in agreement with the minor effect of TR on glucose metabolism. Thus, periodic fasting contributes to some metabolic benefits of CR, but TR is metabolically different from CR. This difference might contribute to differential effects of CR and TR on longevity.

CR reprograms acetyl-CoA metabolism and induces long-chain acyl-CoA dehydrogenase and CrAT expression.

Calorie restriction (CR), an age delaying diet, affects fat oxidation through poorly understood mechanisms. We investigated the effect of CR on fat metabolism gene expression and intermediate metabolites of fatty acid oxidation in the liver. We found that CR changed the liver acylcarnitine profile: acetylcarnitine, short-chain acylcarnitines, and long-chain 3-hydroxy-acylcarnitines increased, and several long-chain acylcarnitines decreased. Acetyl-CoA and short-chain acyl-CoAs were also increased in CR. CR did not affect the expression of CPT1 and upregulated the expression of long-chain and very-long-chain Acyl-CoA dehydrogenases (LCAD and VLCAD, respectively). The expression of downstream enzymes such as mitochondrial trifunctional protein and enzymes in medium- and short-chain acyl-CoAs oxidation was not affected in CR. CR shifted the balance of fatty acid oxidation enzymes and fatty acid metabolites in the liver. Acetyl-CoA generated through beta-oxidation can be used for ketogenesis or energy production. In agreement, blood ketone bodies increased under CR in a time of the day-dependent manner. Carnitine acetyltransferase (CrAT) is a bidirectional enzyme that interconverts short-chain acyl-CoAs and their corresponding acylcarnitines. CrAT expression was induced in CR liver supporting the increased acetylcarnitine and short-chain acylcarnitine production. Acetylcarnitine can freely travel between cellular sub-compartments. Supporting this CR increased protein acetylation in the mitochondria, cytoplasm, and nucleus. We hypothesize that changes in acyl-CoA and acylcarnitine levels help to control energy metabolism and contribute to metabolic flexibility under CR.

A role of the circadian system and circadian proteins in aging.

Circadian rhythms are genetically determined biological rhythms that are considered an important adaptive mechanism to the cyclical light/dark alterations in the Earth environment. Age-related changes in the circadian time-keeping mechanism are well known, and seemingly contribute to various pathologies of aging. Recent findings demonstrate that the circadian system and circadian proteins play direct roles in many physiological processes, including those associated with aging. The core circadian proteins BMAL1 and PERIODs, in addition to their known functions in the circadian oscillator, play essential non-redundant roles in the control of tissue homeostasis and aging. Although the exact mechanisms are unknown, the involvement of circadian proteins in the regulation of metabolism, genotoxic stress response and reactive oxygen species (ROS) homeostasis can be responsible for the premature aging, observed in some circadian mutants. The understanding of the molecular mechanisms of these non-circadian activities of the circadian proteins will ultimately lead to the improvement in prevention and treatment of age-related pathologies.

Dual role of the CLOCK/BMAL1 circadian complex in transcriptional regulation.

The basic helix-loop-helix (bHLH) -PAS domain containing transcription factors CLOCK and BMAL1 are two major components of the circadian molecular oscillator. It is known that the CLOCK/BMAL1 complex positively regulates the activity of E-box containing promoters. Here we demonstrate that the CLOCK/BMAL1 complex can also suppress the activity of some promoters upon its interaction with CRYPTOCHROME (CRY). Such a dual function of the circadian transcriptional complex provides a mechanistic explanation for the unpredicted pattern of circadian gene expression in the tissues of Bmal1 null mice. We speculate that the switch from transcriptional activation to transcriptional repression may provide a highly efficient mechanism for circadian control of gene expression. We also show that CLOCK/BMAL1 can interfere with promoter regulation by other, non-circadian, transcription factors including N-MYC and ETS, leading to attenuation or abrogation of transcription of CLOCK/BMAL1-controlled stress-induced genes. We propose that, based upon these results, both circadian repression and activation of the transcription of different target genes are required for circadian responses to various external stimuli, including genotoxic stress induced by anticancer treatment.

Calorie restriction effects on circadian rhythms in gene expression are sex dependent.

The rhythms in the expression of circadian clock genes are affected by calorie restriction (CR), a dietary paradigm known to increase lifespan. Many physiological effects of CR differ between males and females; here we investigated if the sex of animals affects the CR induced changes in the circadian rhythms. The liver expression of some circadian clock genes such as Bmal1 and three Periods (Per1, Per2 and Per3) and the effect of CR on the expression of these genes were sex independent, while the expression of Rev-Erb alpha, Ror gamma and both Cryptochome (Cry1 and Cry2) genes was different between males and females. The effect of CR on Rev-Erb alpha, Ror gamma and Cry1 gene expression was sex dependent. The expression and the effects of CR were sex-specific for several genes previously reported to be regulated by CR: Fmo3, Mup4, Serpina12 and Cyp4a12, while the expression of Cyp4a14a was sex independent. IGF signaling plays an important role in aging and CR effects. Igf-1 expression is regulated by CR and by the circadian clock, we found that rhythms in Igf-1 expression have sexual dimorphism. Our data provide molecular evidence that the sex of animals is an important modulator of circadian rhythms in gene expression and their response to CR.

Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13.

Calorie restriction (CR) is a dietary intervention known to delay aging. In order, to understand molecular mechanisms of CR, we analyzed the expression of 983 MicroRNAs (miRNAs) in the liver of female mice after 2 years of 30% CR using micro-array. 16 miRNAs demonstrated significant changes in their expression upon CR in comparison with age-matched control. mmu-miR-125a-5p (miR-125a-5p) was significantly upregulated upon CR, and in agreement with this, the expression of mRNAs for its three predicted target genes: Stat3, Casp2, and Stard13 was significantly downregulated in the liver of CR animals. The expression of precursor miRNA for miR-125a-5p was also upregulated upon CR, which suggests its regulation at the level of transcription. Upon aging miR-125a-5p expression was downregulated while the expression of its target genes was upregulated. Thus, CR prevented age-associated changes in the expression of miR-125a-5p and its targets. We propose that miR-125a-5p dependent downregulation of Stat3, Casp2, and Stard13 contributes to the calorie restriction-mediated delay of aging.