BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis.

Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to "remember" time in the absence of external cues.

Defining the Independence of the Liver Circadian Clock.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

Atlas of Circadian Metabolism Reveals System-wide Coordination and Communication between Clocks.

Metabolic diseases are often characterized by circadian misalignment in different tissues, yet how altered coordination and communication among tissue clocks relate to specific pathogenic mechanisms remains largely unknown. Applying an integrated systems biology approach, we performed 24-hr metabolomics profiling of eight mouse tissues simultaneously. We present a temporal and spatial atlas of circadian metabolism in the context of systemic energy balance and under chronic nutrient stress (high-fat diet [HFD]). Comparative analysis reveals how the repertoires of tissue metabolism are linked and gated to specific temporal windows and how this highly specialized communication and coherence among tissue clocks is rewired by nutrient challenge. Overall, we illustrate how dynamic metabolic relationships can be reconstructed across time and space and how integration of circadian metabolomics data from multiple tissues can improve our understanding of health and disease.

Lung Adenocarcinoma Distally Rewires Hepatic Circadian Homeostasis.

The circadian clock controls metabolic and physiological processes through finely tuned molecular mechanisms. The clock is remarkably plastic and adapts to exogenous "zeitgebers," such as light and nutrition. How a pathological condition in a given tissue influences systemic circadian homeostasis in other tissues remains an unanswered question of conceptual and biomedical importance. Here, we show that lung adenocarcinoma operates as an endogenous reorganizer of circadian metabolism. High-throughput transcriptomics and metabolomics revealed unique signatures of transcripts and metabolites cycling exclusively in livers of tumor-bearing mice. Remarkably, lung cancer has no effect on the core clock but rather reprograms hepatic metabolism through altered pro-inflammatory response via the STAT3-Socs3 pathway. This results in disruption of AKT, AMPK, and SREBP signaling, leading to altered insulin, glucose, and lipid metabolism. Thus, lung adenocarcinoma functions as a potent endogenous circadian organizer (ECO), which rewires the pathophysiological dimension of a distal tissue such as the liver. PAPERCLIP.

Nutrient-sensitive transcription factors TFEB and TFE3 couple autophagy and metabolism to the peripheral clock.

Autophagy and energy metabolism are known to follow a circadian pattern. However, it is unclear whether autophagy and the circadian clock are coordinated by common control mechanisms. Here, we show that the oscillation of autophagy genes is dependent on the nutrient-sensitive activation of TFEB and TFE3, key regulators of autophagy, lysosomal biogenesis, and cell homeostasis. TFEB and TFE3 display a circadian activation over the 24-h cycle and are responsible for the rhythmic induction of genes involved in autophagy during the light phase. Genetic ablation of TFEB and TFE3 in mice results in deregulated autophagy over the diurnal cycle and altered gene expression causing abnormal circadian wheel-running behavior. In addition, TFEB and TFE3 directly regulate the expression of Rev-erbα (Nr1d1), a transcriptional repressor component of the core clock machinery also involved in the regulation of whole-body metabolism and autophagy. Comparative analysis of the cistromes of TFEB/TFE3 and REV-ERBα showed an extensive overlap of their binding sites, particularly in genes involved in autophagy and metabolic functions. These data reveal a direct link between nutrient and clock-dependent regulation of gene expression shedding a new light on the crosstalk between autophagy, metabolism, and circadian cycles.

Adipocyte NMNAT1 expression is essential for nuclear NAD+ biosynthesis but dispensable for regulating thermogenesis and whole-body energy metabolism.

Nicotinamide adenine dinucleotide (NAD+) functions as an essential cofactor regulating a variety of biological processes. The purpose of the present study was to determine the role of nuclear NAD+ biosynthesis, mediated by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), in thermogenesis and whole-body energy metabolism. We first evaluated the relationship between NMNAT1 expression and thermogenic activity in brown adipose tissue (BAT), a key organ for non-shivering thermogenesis. We found that reduced BAT NMNAT1expression was associated with inactivation of thermogenic gene program induced by obesity and thermoneutrality. Next, we generated and characterized adiponectin-Cre-driven adipocyte-specific Nmnat1 knockout (ANMT1KO) mice. Loss of NMNAT1 markedly reduced nuclear NAD+ concentration by approximately 70% in BAT. Nonetheless, adipocyte-specific Nmnat1 deletion had no impact on thermogenic (rectal temperature, BAT temperature and whole-body oxygen consumption) responses to ß-adrenergic ligand norepinephrine administration and acute cold exposure, adrenergic-mediated lipolytic activity, and metabolic responses to obesogenic high-fat diet feeding. In addition, loss of NMNAT1 did not affect nuclear lysine acetylation or thermogenic gene program in BAT. These results demonstrate that adipocyte NMNAT1 expression is required for maintaining nuclear NAD+ concentration, but not for regulating BAT thermogenesis or whole-body energy homeostasis.

Reshaping circadian metabolism in the suprachiasmatic nucleus and prefrontal cortex by nutritional challenge.

Food is a powerful entrainment cue for circadian clocks in peripheral tissues, and changes in the composition of nutrients have been demonstrated to metabolically reprogram peripheral clocks. However, how food challenges may influence circadian metabolism of the master clock in the suprachiasmatic nucleus (SCN) or in other brain areas is poorly understood. Using high-throughput metabolomics, we studied the circadian metabolome profiles of the SCN and medial prefrontal cortex (mPFC) in lean mice compared with mice challenged with a high-fat diet (HFD). Both the mPFC and the SCN displayed a robust cyclic metabolism, with a strikingly high sensitivity to HFD perturbation in an area-specific manner. The phase and amplitude of oscillations were drastically different between the SCN and mPFC, and the metabolic pathways impacted by HFD were remarkably region-dependent. Furthermore, HFD induced a significant increase in the number of cycling metabolites exclusively in the SCN, revealing an unsuspected susceptibility of the master clock to food stress.

Distinct metabolic adaptation of liver circadian pathways to acute and chronic patterns of alcohol intake.

Binge drinking and chronic exposure to ethanol contribute to alcoholic liver diseases (ALDs). A potential link between ALDs and circadian disruption has been observed, though how different patterns of alcohol consumption differentially impact hepatic circadian metabolism remains virtually unexplored. Using acute versus chronic ethanol feeding, we reveal differential reprogramming of the circadian transcriptome in the liver. Specifically, rewiring of diurnal SREBP transcriptional pathway leads to distinct hepatic signatures in acetyl-CoA metabolism that are translated into the subcellular patterns of protein acetylation. Thus, distinct drinking patterns of alcohol dictate differential adaptation of hepatic circadian metabolism.

Circadian clocks, epigenetics, and cancer.

PURPOSE OF REVIEW: The interplay between circadian rhythm and cancer has been suggested for more than a decade based on the observations that shift work and cancer incidence are linked. Accumulating evidence implicates the circadian clock in cancer survival and proliferation pathways. At the molecular level, multiple control mechanisms have been proposed to link circadian transcription and cell-cycle control to tumorigenesis. RECENT FINDINGS: The circadian gating of the cell cycle and subsequent control of cell proliferation is an area of active investigation. Moreover, the circadian clock is a transcriptional system that is intricately regulated at the epigenetic level. Interestingly, the epigenetic landscape at the level of histone modifications, DNA methylation, and small regulatory RNAs are differentially controlled in cancer cells. This concept raises the possibility that epigenetic control is a common thread linking the clock with cancer, though little scientific evidence is known to date. SUMMARY: This review focuses on the link between circadian clock and cancer, and speculates on the possible connections at the epigenetic level that could further link the circadian clock to tumor initiation or progression.

Intermittent Fasting Sustainably Improves Glucose Tolerance in Normal Weight Male Mice Through Histone Hyperacetylation.

To explore the mechanism by which intermittent fasting (IF) exerts prolonged effects after discontinuation, we examined mice that had been subjected to 4 cycles of fasting for 72 hours and ad libitum feeding for 96 hours per week (72hIF), followed by 4 weeks of ad libitum feeding, focusing on expression of genes for lipid metabolism in the skeletal muscle and histone acetylation in the promoter region. The 72hIF regimen resulted in metabolic remodeling, characterized by enhanced lipid utilization and mitochondrial activation in the muscle. This long-term IF (72hIF) caused stronger metabolic effects than alternate day fasting (24hIF) wherein fasting and refeeding are repeated every 24 hours. Upregulation of lipid oxidation genes and an increase in oxygen utilization were sustained even at 4 weeks after discontinuation of 72hIF, associated with histone hyperacetylation of the promoter region of uncoupling protein 3 (Ucp3) and carnitine palmitoyl transferase 1b (Cpt1b) genes. An increase in leucine owing to fasting-induced muscle degradation was suggested to lead to the histone acetylation. These findings support the previously unappreciated notion that sustainable promotion of histone acetylation in lipid oxidation genes of the muscle and adipose tissues during and after IF may contribute to sustained metabolic effects of IF.

Association Between Renal Sinus Fat and Cardiometabolic and Renin-Angiotensin System Parameters in Primary Aldosteronism.

Context: Renal sinus fat (RSF) accumulation is associated with cardiometabolic diseases, such as obesity, diabetes, hypertension, and chronic kidney disease. However, clinical implications of RSF in primary aldosteronism (PA) remain unclear. Objective: We aimed to investigate relationships between RSF volume and key cardiometabolic and renin-angiotensin system (RAS) parameters in PA patients and clarify the differences in these relationships between unilateral and bilateral subtypes. Methods: We analyzed data obtained from well-characterized PA patients that involved 45 unilateral (median age: 52 years; 42.2% men) and 92 bilateral patients (51 years; 42.4% men). Results: RSF volume normalized by renal volume (RSF%) was greater in the unilateral group than in the bilateral group (P < .05). RSF% was greater in men than in women (P < .05). RSF% positively correlated with parameters related to cardiometabolic risk, including age, body mass index, visceral fat volume, creatinine, triglycerides/high-density lipoprotein cholesterol ratio, uric acid, fasting glucose, and C-reactive protein regardless of PA subtypes (all P < .05). Intriguingly, RSF% positively correlated with plasma aldosterone concentration (PAC), aldosterone-to-renin ratio, and intact parathyroid hormone (iPTH) (all P < .05) in bilateral patients but did not correlate with RAS parameters and even showed an opposite trend in unilateral patients. In subgroup analyses by sex, these distinctions became more evident in women. After adjustment for potential confounders, RSF% remained positively correlated with PAC and iPTH in bilateral patients. Conclusion: Our results indicate that RSF accumulation is involved in cardiometabolic dysfunction associated with PA. However, there were distinct correlations between RSF volume and RAS parameters according to sex and PA subtypes.

The circadian clock CRY1 regulates pluripotent stem cell identity and somatic cell reprogramming.

Distinct metabolic conditions rewire circadian-clock-controlled signaling pathways leading to the de novo construction of signal transduction networks. However, it remains unclear whether metabolic hallmarks unique to pluripotent stem cells (PSCs) are connected to clock functions. Reprogramming somatic cells to a pluripotent state, here we highlighted non-canonical functions of the circadian repressor CRY1 specific to PSCs. Metabolic reprogramming, including AMPK inactivation and SREBP1 activation, was coupled with the accumulation of CRY1 in PSCs. Functional assays verified that CRY1 is required for the maintenance of self-renewal capacity, colony organization, and metabolic signatures. Genome-wide occupancy of CRY1 identified CRY1-regulatory genes enriched in development and differentiation in PSCs, albeit not somatic cells. Last, cells lacking CRY1 exhibit differential gene expression profiles during induced PSC (iPSC) reprogramming, resulting in impaired iPSC reprogramming efficiency. Collectively, these results suggest the functional implication of CRY1 in pluripotent reprogramming and ontogenesis, thereby dictating PSC identity.

The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes.

RATIONALE: The (pro)renin receptor [(P)RR], encoded in ATP6AP2, plays a key role in the activation of local renin-angiotensin system (RAS). A truncated form of (P)RR, termed M8.9, was also found to be associated with the vacuolar H(+)-ATPase (V-ATPase), implicating a non-RAS-related function of ATP6AP2. OBJECTIVE: We investigated the role of (P)RR/ATP6AP2 in murine cardiomyocytes. METHODS AND RESULTS: Cardiomyocyte-specific ablation of Atp6ap2 resulted in lethal heart failure; the cardiomyocytes contained RAB7- and lysosomal-associated membrane protein 2 (LAMP2)-positive multivesicular vacuoles, especially in the perinuclear regions. The myofibrils and mitochondria remained at the cell periphery. Cardiomyocyte death was accompanied by numerous autophagic vacuoles that contained undigested cellular constituents, as a result of impaired autophagic degradation. Notably, ablation of Atp6ap2 selectively suppressed expression of the V(O) subunits of V-ATPase, resulting in deacidification of the intracellular vesicles. Furthermore, the inhibition of intracellular acidification by treatment with bafilomycin A1 or chloroquine reproduced the phenotype observed for the (P)RR/ATP6AP2-deficient cardiomyocytes. CONCLUSIONS: Genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase. The gene product of ATP6AP2 is considered to act as in 2 ways: (1) as (P)RR, exerting a RAS-related function; and (2) as the V-ATPase-associated protein, exerting a non-RAS-related function that is essential for cell survival.

Prorenin receptor is essential for normal podocyte structure and function.

The prorenin receptor is an accessory subunit of the vacuolar H(+)-ATPase, suggesting that it has fundamental functions beyond activation of the local renin-angiotensin system. Podocytes express the prorenin receptor, but its function in these cells is unknown. Here, podocyte-specific, conditional, prorenin receptor-knockout mice died of kidney failure and severe proteinuria within 4 weeks of birth. The podocytes of these mice exhibited foot process effacement with reduced and altered localization of the slit-diaphragm proteins nephrin and podocin. Furthermore, the podocytes contained numerous autophagic vacuoles, confirmed by enhanced accumulation of microtubule-associated protein 1 light chain 3-positive intracellular vesicles. Ablation of the prorenin receptor selectively suppressed expression of the V(0) c-subunit of the vacuolar H(+)-ATPase in podocytes, resulting in deacidification of intracellular vesicles. In conclusion, the prorenin receptor is important for the maintenance of normal podocyte structure and function.

Chromatin Immunoprecipitation and Circadian Rhythms.

Organisms exhibit daily changes of physiology and behavior to exert homeostatic adaptations to day-night cycles. The cyclic fluctuation also takes place at transcriptional levels, giving rise to rhythmic gene expression. Central to this oscillatory transcription is the core clock machinery which constitutes a circuit of transcriptional-translational feedback and achieves circadian functions accordingly. Chromatin immunoprecipitation provides understanding of such mechanisms that clock and non-clock transcription factors along with co-regulators and chromatin modifications dictate circadian epigenome through cyclic alterations of chromatin structures and molecular functions in a concerted fashion. Besides, innovation of high-throughput sequencing technology has broadened our horizon and renewed perspectives in circadian research. This article summarizes the methodology of a chromatin immunoprecipitation experiment in light of circadian rhythm research.

Activation of the intestinal tissue renin-angiotensin system by transient sodium loading in salt-sensitive rats.

BACKGROUND: The renal tissue renin-angiotensin system is known to be activated by salt loading in salt-sensitive rats; however, the response in other organs remains unclear. METHOD: Spontaneously hypertensive rats were subjected to normal tap water or transient high-salt-concentration water from 6 to 14 weeks of age and were thereafter given normal tap water. From 18 to 20 weeks of age, rats given water with a high salt concentration were treated with an angiotensin II type 1 receptor blocker, valsartan. RESULTS: Sustained blood pressure elevation by transient salt loading coincided with a persistent decrease in the fecal sodium content and sustained excess of the circulating volume in spontaneously hypertensive rats. Administration of valsartan sustainably reduced the blood pressure and normalized the fecal sodium levels. Notably, transient salt loading persistently induced the intestinal tissue renin-angiotensin system and enhanced sodium transporter expression exclusively in the small intestine of salt-sensitive rats, suggesting the potential connection of intestinal sodium absorption to salt sensitivity. CONCLUSION: These results reveal the previously unappreciated contribution of the intestinal tissue renin-angiotensin system to sodium homeostasis and blood pressure regulation in the pathophysiology of salt-sensitive hypertension.

Antibiotic-induced microbiome depletion remodels daily metabolic cycles in the brain.

The gut microbiome influences cognition and behavior in mammals, yet its metabolic impact on the brain is only starting to be defined. Using metabolite profiling of antibiotics-treated mice, we reveal the microbiome as a key input controlling circadian metabolic cycles in the brain. Intra and inter-region analyses characterise the influence of the microbiome on the suprachiasmatic nucleus, containing the central clockwork, as well as the hippocampus and cortex, regions involved in learning and behavior.

Circadian rhythms in the tissue-specificity from metabolism to immunity: insights from omics studies.

Creatures on earth have the capacity to preserve homeostasis in response to changing environments. The circadian clock enables organisms to adapt to daily predictable rhythms in surrounding conditions. In mammals, circadian clocks constitute hierarchical network, where the central pacemaker in hypothalamic suprachiasmatic nucleus (SCN) serves as a time-keeping machinery and governs peripheral clocks in every other organ through descending neural and humoral factors. The central clock in SCN is reset by light, whilst peripheral clocks are entrained by feeding-fasting rhythms, emphasizing the point that temporal patterns of nutrient availability specifies peripheral clock functions. Indeed, emerging evidence revealed various types of diets or timing of food intake reprogram circadian rhythms in a tissue specific manner. This advancement in understanding of mechanisms underlying tissue specific responsiveness of circadian oscillators to nutrients at the genomic and epigenomic levels is largely owing to employment of state-of-the-art technologies. Specifically, high-throughput transcriptome, proteome, and metabolome have provided insights into how genes, proteins, and metabolites behave over circadian cycles in a given tissue under a certain dietary condition in an unbiased fashion. Additionally, combinations with specialized types of sequencing such as nascent-seq and ribosomal profiling allow us to dissect how circadian rhythms are generated or obliterated at each step of gene regulation. Importantly, chromatin immunoprecipitation followed by deep sequencing methods provide chromatin landscape in terms of regulatory mechanisms of circadian gene expression. In this review, we outline recent discoveries on temporal genomic and epigenomic regulation of circadian rhythms, discussing entrainment of the circadian rhythms by feeding as a fundamental new comprehension of metabolism and immune response, and as a potential therapeutic strategy of metabolic and inflammatory diseases.

Combined Gene Expression and Chromatin Immunoprecipitation From a Single Mouse Hippocampus.

All neuronal cells hold the same genetic information but vary by their structural and functional plasticity depending on the brain area and environmental influences. Such variability involves specific gene regulation, which is driven by transcription factors (TFs). In the field of neuroscience, epigenetics is the main mechanism that has been investigated to understand the dynamic modulation of gene expression by behavioral responses, stress responses, memory processes, etc. Nowadays, gene expression analyzed by real-time quantitative PCR and TF binding estimated by chromatin immunoprecipitation (ChIP) enables one to dissect this regulation. Because of the wide range of transgenic models, as well as cost-effective aspects, mouse models are widely used neuroscience. Thus, we have set up a protocol that allows extraction of both RNA for gene expression analysis and chromatin for ChIP experiment from a single mouse hippocampus. Using such protocols, information regarding gene expression and regulatory molecular mechanisms from the same animal can be integrated and correlated with neurobiological and behavioral outcomes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Hippocampus isolation from mouse brain Basic Protocol 2: RNA extraction and gene expression analysis from a mouse half hippocampus Basic Protocol 3: ChIP from one hemisphere side mouse hippocampus.