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.

Impact of Bmal1 Rescue and Time-Restricted Feeding on Liver and Muscle Proteomes During the Active Phase in Mice.

Molecular clocks and daily feeding cycles support metabolism in peripheral tissues. Although the roles of local clocks and feeding are well defined at the transcriptional level, their impact on governing protein abundance in peripheral tissues is unclear. Here, we determine the relative contributions of local molecular clocks and daily feeding cycles on liver and muscle proteomes during the active phase in mice. LC-MS/MS was performed on liver and gastrocnemius muscle harvested 4 h into the dark phase from WT, Bmal1 KO, and dual liver- and muscle-Bmal1-rescued mice under either ad libitum feeding or time-restricted feeding during the dark phase. Feeding-fasting cycles had only minimal effects on levels of liver proteins and few, if any, on the muscle proteome. In contrast, Bmal1 KO altered the abundance of 674 proteins in liver and 80 proteins in muscle. Local rescue of liver and muscle Bmal1 restored ∼50% of proteins in liver and ∼25% in muscle. These included proteins involved in fatty acid oxidation in liver and carbohydrate metabolism in muscle. For liver, proteins involved in de novo lipogenesis were largely dependent on Bmal1 function in other tissues (i.e., the wider clock system). Proteins regulated by BMAL1 in liver and muscle were enriched for secreted proteins. We found that the abundance of fibroblast growth factor 1, a liver secreted protein, requires BMAL1 and that autocrine fibroblast growth factor 1 signaling modulates mitochondrial respiration in hepatocytes. In liver and muscle, BMAL1 is a more potent regulator of dark phase proteomes than daily feeding cycles, highlighting the need to assess protein levels in addition to mRNA when investigating clock mechanisms. The proteome is more extensively regulated by BMAL1 in liver than in muscle, and many metabolic pathways in peripheral tissues are reliant on the function of the clock system as a whole.

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

BACKGROUND AND PURPOSE: Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. METHODS: We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. RESULTS: Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1, accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. CONCLUSIONS: Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders.

Resveratrol Preconditioning Induces a Novel Extended Window of Ischemic Tolerance in the Mouse Brain.

BACKGROUND AND PURPOSE: Prophylactic treatments that afford neuroprotection against stroke may emerge from the field of preconditioning. Resveratrol mimics ischemic preconditioning, reducing ischemic brain injury when administered 2 days before global ischemia in rats. This protection is linked to silent information regulator 2 homologue 1 (Sirt1) and enhanced mitochondrial function possibly through its repression of uncoupling protein 2. Brain-derived neurotrophic factor (BDNF) is another neuroprotective protein associated with Sirt1. In this study, we sought to identify the conditions of resveratrol preconditioning (RPC) that most robustly induce neuroprotection against focal ischemia in mice. METHODS: We tested 4 different RPC paradigms against a middle cerebral artery occlusion model of stroke. Infarct volume and neurological score were calculated 24 hours after middle cerebral artery occlusion. Sirt1-chromatin binding was evaluated by ChIP-qPCR. Percoll gradients were used to isolate synaptic fractions, and changes in protein expression were determined via Western blot analysis. BDNF concentration was measured using a BDNF-specific ELISA assay. RESULTS: Although repetitive RPC induced neuroprotection from middle cerebral artery occlusion, strikingly one application of RPC 14 days before middle cerebral artery occlusion showed the most robust protection, reducing infarct volume by 33% and improving neurological score by 28%. Fourteen days after RPC, Sirt1 protein was increased 1.5-fold and differentially bound to the uncoupling protein 2 and BDNF promoter regions. Accordingly, synaptic uncoupling protein 2 level decreased by 23% and cortical BDNF concentration increased 26%. CONCLUSIONS: RPC induces a novel extended window of ischemic tolerance in the brain that lasts for at least 14 days. Our data suggest that this tolerance may be mediated by Sirt1 through upregulation of BDNF and downregulation of uncoupling protein 2.

Signaling pathways leading to ischemic mitochondrial neuroprotection.

There is extensive evidence that ischemic/reperfusion mediated mitochondrial dysfunction is a major contributor to ischemic damage. However data also indicates that mild ischemic stress induces mitochondrial dependent activation of ischemic preconditioning. Ischemic preconditioning is a neuroprotective mechanism which is activated upon a brief sub-injurious ischemic exposure and is sufficient to provide protection against a subsequent lethal ischemic insult. Current research demonstrates that mitochondria are not only the inducers of but are also an important target of ischemic preconditioning mediated protection. Numerous proteins and signaling pathways are activated by ischemic preconditioning which protect the mitochondria against ischemic damage. In this review we examine some of the proteins activated by ischemic precondition which counteracts the deleterious effects of ischemia/reperfusion thereby maintaining normal mitochondrial activity and lead to ischemic tolerance.

Brain-muscle communication prevents muscle aging by maintaining daily physiology.

A molecular clock network is crucial for daily physiology and maintaining organismal health. We examined the interactions and importance of intratissue clock networks in muscle tissue maintenance. In arrhythmic mice showing premature aging, we created a basic clock module involving a central and a peripheral (muscle) clock. Reconstituting the brain-muscle clock network is sufficient to preserve fundamental daily homeostatic functions and prevent premature muscle aging. However, achieving whole muscle physiology requires contributions from other peripheral clocks. Mechanistically, the muscle peripheral clock acts as a gatekeeper, selectively suppressing detrimental signals from the central clock while integrating important muscle homeostatic functions. Our research reveals the interplay between the central and peripheral clocks in daily muscle function and underscores the impact of eating patterns on these interactions.

The epidermal circadian clock integrates and subverts brain signals to guarantee skin homeostasis.

In mammals, the circadian clock network drives daily rhythms of tissue-specific homeostasis. To dissect daily inter-tissue communication, we constructed a mouse minimal clock network comprising only two nodes: the peripheral epidermal clock and the central brain clock. By transcriptomic and functional characterization of this isolated connection, we identified a gatekeeping function of the peripheral tissue clock with respect to systemic inputs. The epidermal clock concurrently integrates and subverts brain signals to ensure timely execution of epidermal daily physiology. Timely cell-cycle termination in the epidermal stem cell compartment depends upon incorporation of clock-driven signals originating from the brain. In contrast, the epidermal clock corrects or outcompetes potentially disruptive feeding-related signals to ensure the optimal timing of DNA replication. Together, we present an approach for cataloging the systemic dependencies of daily temporal organization in a tissue and identify an essential gate-keeping function of peripheral circadian clocks that guarantees tissue homeostasis.

Liver and muscle circadian clocks cooperate to support glucose tolerance in mice.

Physiology is regulated by interconnected cell and tissue circadian clocks. Disruption of the rhythms generated by the concerted activity of these clocks is associated with metabolic disease. Here we tested the interactions between clocks in two critical components of organismal metabolism, liver and skeletal muscle, by rescuing clock function either in each organ separately or in both organs simultaneously in otherwise clock-less mice. Experiments showed that individual clocks are partially sufficient for tissue glucose metabolism, yet the connections between both tissue clocks coupled to daily feeding rhythms support systemic glucose tolerance. This synergy relies in part on local transcriptional control of the glucose machinery, feeding-responsive signals such as insulin, and metabolic cycles that connect the muscle and liver. We posit that spatiotemporal mechanisms of muscle and liver play an essential role in the maintenance of systemic glucose homeostasis and that disrupting this diurnal coordination can contribute to metabolic disease.

Real-Time Measurement of Energy Metabolism Over Circadian Time Using Indirect Calorimetry-Enabled Metabolic Cages.

Indirect calorimetry probes the relationship between fuel consumed and energy produced, and in doing so provides an estimation of whole-body energy expenditure and fuel preference. When assayed continuously in real-time, rhythms appear and illuminate the temporal regulation of energy metabolism by the circadian clock. Here we describe a method for recording circadian energy metabolism in mice using indirect calorimetry-enabled metabolic cages, encompassing mouse entrainment, experimental design, data acquisition and analysis, troubleshooting of common problems, and important considerations. This method is adaptable to the end user's equipment and serves as an effective tool to study, for example, mutant mice, dietary interventions, drug treatments, or circadian disruption.

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.

The central clock suffices to drive the majority of circulatory metabolic rhythms.

Life on Earth anticipates recurring 24-hour environmental cycles via genetically encoded molecular clocks active in all mammalian organs. Communication between these clocks controls circadian homeostasis. Intertissue communication is mediated, in part, by temporal coordination of metabolism. Here, we characterize the extent to which clocks in different organs control systemic metabolic rhythms, an area that remains largely unexplored. We analyzed the metabolome of serum from mice with tissue-specific expression of the clock gene Bmal1. Having functional hepatic and muscle clocks can only drive a minority (13%) of systemic metabolic rhythms. Conversely, limiting Bmal1 expression to the central pacemaker in the brain restores rhythms to 57% of circulatory metabolites. Rhythmic feeding imposed on clockless mice resulted in a similar rescue, indicating that the central clock mainly regulates metabolic rhythms via behavior. These findings explicate the circadian communication between tissues and highlight the importance of the central clock in governing those signals.

Tryptophan metabolism is a physiological integrator regulating circadian rhythms.

OBJECTIVE: The circadian clock aligns physiology with the 24-hour rotation of Earth. Light and food are the main environmental cues (zeitgebers) regulating circadian rhythms in mammals. Yet, little is known about the interaction between specific dietary components and light in coordinating circadian homeostasis. Herein, we focused on the role of essential amino acids. METHODS: Mice were fed diets depleted of specific essential amino acids and their behavioral rhythms were monitored and tryptophan was selected for downstream analyses. The role of tryptophan metabolism in modulating circadian homeostasis was studied using isotope tracing as well as transcriptomic- and metabolomic- analyses. RESULTS: Dietary tryptophan depletion alters behavioral rhythms in mice. Furthermore, tryptophan metabolism was shown to be regulated in a time- and light- dependent manner. A multi-omics approach and combinatory diet/light interventions demonstrated that tryptophan metabolism modulates temporal regulation of metabolism and transcription programs by buffering photic cues. Specifically, tryptophan metabolites regulate central circadian functions of the suprachiasmatic nucleus and the core clock machinery in the liver. CONCLUSIONS: Tryptophan metabolism is a modulator of circadian homeostasis by integrating environmental cues. Our findings propose tryptophan metabolism as a potential point for pharmacologic intervention to modulate phenotypes associated with disrupted circadian rhythms.

Communicating clocks shape circadian homeostasis.

Circadian clocks temporally coordinate physiology and align it with geophysical time, which enables diverse life-forms to anticipate daily environmental cycles. In complex organisms, clock function originates from the molecular oscillator within each cell and builds upward anatomically into an organism-wide system. Recent advances have transformed our understanding of how clocks are connected to achieve coherence across tissues. Circadian misalignment, often imposed in modern society, disrupts coordination among clocks and has been linked to diseases ranging from metabolic syndrome to cancer. Thus, uncovering the physiological circuits whereby biological clocks achieve coherence will inform on both challenges and opportunities in human health.

Collecting mouse livers for transcriptome analysis of daily rhythms.

Molecular daily rhythms can be captured by precisely timed tissue harvests from groups of animals. This protocol will allow the investigator to identify transcriptional rhythms in the mouse liver while also providing a template for similar analyses in other whole metabolic organs. We describe steps for mouse entrainment, liver dissection, and rhythmicity analysis from total RNA sequencing data. The resulting rhythmic transcriptome will provide the user with a starting point for defining specific biological processes that undergo daily rhythms. For complete details on the use and execution of this protocol, please refer to Koronowski et al. (2019). A similar protocol for interfollicular epidermal cells is demonstrated in Welz et al. (2019).

Ketogenesis impact on liver metabolism revealed by proteomics of lysine ß-hydroxybutyrylation.

Ketone bodies are bioactive metabolites that function as energy substrates, signaling molecules, and regulators of histone modifications. ß-hydroxybutyrate (ß-OHB) is utilized in lysine ß-hydroxybutyrylation (Kbhb) of histones, and associates with starvation-responsive genes, effectively coupling ketogenic metabolism with gene expression. The emerging diversity of the lysine acylation landscape prompted us to investigate the full proteomic impact of Kbhb. Global protein Kbhb is induced in a tissue-specific manner by a variety of interventions that evoke ß-OHB. Mass spectrometry analysis of the ß-hydroxybutyrylome in mouse liver revealed 891 sites of Kbhb within 267 proteins enriched for fatty acid, amino acid, detoxification, and one-carbon metabolic pathways. Kbhb inhibits S-adenosyl-L-homocysteine hydrolase (AHCY), a rate-limiting enzyme of the methionine cycle, in parallel with altered metabolite levels. Our results illuminate the role of Kbhb in hepatic metabolism under ketogenic conditions and demonstrate a functional consequence of this modification on a central metabolic enzyme.

Integration of feeding behavior by the liver circadian clock reveals network dependency of metabolic rhythms.

The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.

Exposure to recurrent hypoglycemia alters hippocampal metabolism in treated streptozotocin-induced diabetic rats.

AIMS: Exposure to recurrent hypoglycemia (RH) is common in diabetic patients receiving glucose-lowering therapies and is implicated in causing cognitive impairments. Despite the significant effect of RH on hippocampal function, the underlying mechanisms are currently unknown. Our goal was to determine the effect of RH exposure on hippocampal metabolism in treated streptozotocin-diabetic rats. METHODS: Hyperglycemia was corrected by insulin pellet implantation. Insulin-treated diabetic (ITD) rats were exposed to mild/moderate RH once a day for 5 consecutive days. RESULTS: The effect of RH on hippocampal metabolism revealed 65 significantly altered metabolites in the RH group compared with controls. Several significant differences in metabolite levels belonging to major pathways (eg, Krebs cycle, gluconeogenesis, and amino acid metabolism) were discovered in RH-exposed ITD rats when compared to a control group. Key glycolytic enzymes including hexokinase, phosphofructokinase, and pyruvate kinase were affected by RH exposure. CONCLUSION: Our results demonstrate that the exposure to RH leads to metabolomics alterations in the hippocampus of insulin-treated streptozotocin-diabetic rats. Understanding how RH affects hippocampal metabolism may help attenuate the adverse effects of RH on hippocampal functions.

Resveratrol Preconditioning Induces Genomic and Metabolic Adaptations within the Long-Term Window of Cerebral Ischemic Tolerance Leading to Bioenergetic Efficiency.

Neuroprotective agents administered post-cerebral ischemia have failed so far in the clinic to promote significant recovery. Thus, numerous efforts were redirected toward prophylactic approaches such as preconditioning as an alternative therapeutic strategy. Our laboratory has revealed a novel long-term window of cerebral ischemic tolerance mediated by resveratrol preconditioning (RPC) that lasts for 2 weeks in mice. To identify its mediators, we conducted an RNA-seq experiment on the cortex of mice 2 weeks post-RPC, which revealed 136 differentially expressed genes. The majority of genes (116/136) were downregulated upon RPC and clustered into biological processes involved in transcription, synaptic signaling, and neurotransmission. The downregulation in these processes was reminiscent of metabolic depression, an adaptation used by hibernating animals to survive severe ischemic states by downregulating energy-consuming pathways. Thus, to assess metabolism, we used a neuronal-astrocytic co-culture model and measured the cellular respiration rate at the long-term window post-RPC. Remarkably, we observed an increase in glycolysis and mitochondrial respiration efficiency upon RPC. We also observed an increase in the expression of genes involved in pyruvate uptake, TCA cycle, and oxidative phosphorylation, all of which indicated an increased reliance on energy-producing pathways. We then revealed that these nuclear and mitochondrial adaptations, which reduce the reliance on energy-consuming pathways and increase the reliance on energy-producing pathways, are epigenetically coupled through acetyl-CoA metabolism and ultimately increase baseline ATP levels. This increase in ATP would then allow the brain, a highly metabolic organ, to endure prolonged durations of energy deprivation encountered during cerebral ischemia.