Carbon dioxide regulates cholesterol levels through SREBP2.

In mammals, O2 and CO2 levels are tightly regulated and are altered under various pathological conditions. While the molecular mechanisms that participate in O2 sensing are well characterized, little is known regarding the signaling pathways that participate in CO2 signaling and adaptation. Here, we show that CO2 levels control a distinct cellular transcriptional response that differs from mere pH changes. Unexpectedly, we discovered that CO2 regulates the expression of cholesterogenic genes in a SREBP2-dependent manner and modulates cellular cholesterol accumulation. Molecular dissection of the underlying mechanism suggests that CO2 triggers SREBP2 activation through changes in endoplasmic reticulum (ER) membrane cholesterol levels. Collectively, we propose that SREBP2 participates in CO2 signaling and that cellular cholesterol levels can be modulated by CO2 through SREBP2.

Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.

A wide variety of liver functions are regulated daily by the liver circadian clock and via systemic circadian control by other organs and cells within the gastrointestinal tract as well as the microbiome and immune cells. Disruption of the circadian system, as occurs during jetlag, shift work or an unhealthy lifestyle, is implicated in several liver-related pathologies, ranging from metabolic diseases such as obesity, type 2 diabetes mellitus and nonalcoholic fatty liver disease to liver malignancies such as hepatocellular carcinoma. In this Review, we cover the molecular, cellular and organismal aspects of various liver pathologies from a circadian viewpoint, and in particular how circadian dysregulation has a role in the development and progression of these diseases. Finally, we discuss therapeutic and lifestyle interventions that carry health benefits through support of a functional circadian clock that acts in synchrony with the environment.

Beyond circadian rhythms: emerging roles of ultradian rhythms in control of liver functions.

The mammalian liver must cope with various metabolic and physiological changes that normally recur every day and primarily stem from daily cycles of rest-activity and fasting-feeding. Although a large body of evidence supports the reciprocal regulation of circadian rhythms and liver function, the research on the hepatic ultradian rhythms have largely been lagging behind. However, with the advent of more cost-effective high-throughput omics technologies, high-resolution time-lapse imaging, and more robust and powerful mathematical tools, several recent studies have shed new light on the presence and functions of hepatic ultradian rhythms. In this review, we will first very briefly discuss the basic principles of circadian rhythms, and then cover in greater details the recent literature related to ultradian rhythms. Specifically, we will highlight the prevalence and mechanisms of hepatic 12-h rhythms, and 8-h rhythms, which cycle at the second and third harmonics of circadian frequency. Finally, we also refer to ultradian rhythms with other frequencies and examine the limitations of the current approaches as well as the challenges related to identifying ultradian rhythm and addressing their molecular underpinnings.

Input integration by the circadian clock exhibits nonadditivity and fold-change detection.

Circadian clocks are synchronized by external timing cues to align with one another and the environment. Various signaling pathways have been shown to independently reset the phase of the clock. However, in the body, circadian clocks are exposed to a multitude of potential timing cues with complex temporal dynamics, raising the question of how clocks integrate information in response to multiple signals. To investigate different modes of signal integration by the circadian clock, we used Circa-SCOPE, a method we recently developed for high-throughput phase resetting analysis. We found that simultaneous exposure to different combinations of known pharmacological resetting agents elicits a diverse range of responses. Often, the response was nonadditive and could not be readily predicted by the response to the individual signals. For instance, we observed that dexamethasone is dominant over other tested inputs. In the case of signals administered sequentially, the background levels of a signal attenuated subsequent resetting by the same signal, but not by signals acting through a different pathway. This led us to examine whether the circadian clock is sensitive to relative rather than absolute levels of the signal. Importantly, our analysis revealed the involvement of a signal-specific fold-change detection mechanism in the clock response. This mechanism likely stems from properties of the signaling pathway that are upstream to the clock. Overall, our findings elucidate modes of input integration by the circadian clock, with potential relevance to clock resetting under both physiological and pathological conditions.

The human blood transcriptome exhibits time-of-day-dependent response to hypoxia: Lessons from the highest city in the world.

High altitude exposes humans to hypobaric hypoxia, which induces various physiological and molecular changes. Recent studies point toward interaction between circadian rhythms and the hypoxic response, yet their human relevance is lacking. Here, we examine the effect of different high altitudes in conjunction with time of day on human whole-blood transcriptome upon an expedition to the highest city in the world, La Rinconada, Peru, which is 5,100 m above sea level. We find that high altitude vastly affects the blood transcriptome and, unexpectedly, does not necessarily follow a monotonic response to altitude elevation. Importantly, we observe daily variance in gene expression, especially immune-related genes, which is largely altitude dependent. Moreover, using a digital cytometry approach, we estimate relative changes in abundance of different cell types and find that the response of several immune cell types is time- and altitude dependent. Taken together, our data provide evidence for interaction between the transcriptional response to hypoxia and the time of day in humans.

Circadian clocks' interactions with oxygen sensing and signalling.

In mammals, physiology and metabolism are shaped both by immediate and anticipatory responses to environmental changes through the myriad of molecular mechanisms. Whilst the former is mostly mediated through different acute signalling pathways the latter is primarily orchestrated by the circadian clock. Oxygen is vital for life and as such mammals have evolved different mechanisms to cope with changes in oxygen levels. It is widely accepted that oxygen sensing through the HIF-1 signalling pathway is paramount for the acute response to changes in oxygen levels. Circadian clocks are molecular oscillators that control 24 hours rhythms in various aspects of physiology and behaviour. Evidence emerging in recent years points towards pervasive molecular and functional interactions between these two pathways on multiple levels. Daily oscillations in oxygen levels are circadian clock-controlled and can reset the clock through HIF-1. Furthermore, the circadian clock appears to modulate the hypoxic response. We review herein the literature related to the crosstalk between the circadian clockwork and the oxygen-signalling pathway in mammals at the molecular and physiological level both under normal and pathologic conditions.

Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2.

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase-AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2-/- but not of Bmal1-/- mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2-/- mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2-/- genes.

Circa-SCOPE: high-throughput live single-cell imaging method for analysis of circadian clock resetting.

Circadian clocks are self-sustained and cell-autonomous oscillators. They respond to various extracellular cues depending on the time-of-day and the signal intensity. Phase Transition Curves (PTCs) are instrumental in uncovering the full repertoire of responses to a given signal. However, the current methodologies for reconstructing PTCs are low-throughput, laborious, and resource- and time-consuming. We report here the development of an efficient and high throughput assay, dubbed Circadian Single-Cell Oscillators PTC Extraction (Circa-SCOPE) for generating high-resolution PTCs. This methodology relies on continuous monitoring of single-cell oscillations to reconstruct a full PTC from a single culture, upon a one-time intervention. Using Circa-SCOPE, we characterize the effects of various pharmacological and blood-borne resetting cues, at high temporal resolution and a wide concentration range. Thus, Circa-SCOPE is a powerful tool for comprehensive analysis and screening for circadian clocks' resetting cues, and can be valuable for basic as well as translational research.

Circadian Organelles: Rhythms at All Scales.

Circadian clocks have evolved in most light-sensitive organisms, from unicellular organisms to mammals. Consequently, a myriad of biological functions exhibits circadian rhythmicity, from behavior to physiology, through tissue and cellular functions to subcellular processes. Circadian rhythms in intracellular organelles are an emerging and exciting research arena. We summarize herein the current literature for rhythmicity in major intracellular organelles in mammals. These include changes in the morphology, content, and functions of different intracellular organelles. While these data highlight the presence of rhythmicity in these organelles, a gap remains in our knowledge regarding the underlying molecular mechanisms and their functional significance. Finally, we discuss the importance and challenges faced by spatio-temporal studies on these organelles and speculate on the presence of oscillators in organelles and their potential mode of communication. As circadian biology has been and continues to be studied throughout temporal and spatial axes, circadian organelles appear to be the next frontier.

Clock proteins and training modify exercise capacity in a daytime-dependent manner.

Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior.

NQO1 Binds and Supports SIRT1 Function.

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

The liver-clock coordinates rhythmicity of peripheral tissues in response to feeding.

The mammalian circadian system consists of a central clock in the brain that synchronizes clocks in the peripheral tissues. Although the hierarchy between central and peripheral clocks is established, little is known regarding the specificity and functional organization of peripheral clocks. Here, we employ altered feeding paradigms in conjunction with liver-clock mutant mice to map disparities and interactions between peripheral rhythms. We find that peripheral clocks largely differ in their responses to feeding time. Disruption of the liver-clock, despite its prominent role in nutrient processing, does not affect the rhythmicity of clocks in other peripheral tissues. Yet, unexpectedly, liver-clock disruption strongly modulates the transcriptional rhythmicity of peripheral tissues, primarily on daytime feeding. Concomitantly, liver-clock mutant mice exhibit impaired glucose and lipid homeostasis, which are aggravated by daytime feeding. Overall, our findings suggest that, upon nutrient challenge, the liver-clock buffers the effect of feeding-related signals on rhythmicity of peripheral tissues, irrespective of their clocks.

Monitoring daytime differences in moderate intensity exercise capacity using treadmill test and muscle dissection.

There is growing interest in medicine and sports in uncovering exercise modifiers that enhance or limit exercise capacity. Here, we detail a protocol for testing the daytime effect on running capacity in mice using a moderate intensity treadmill effort test. Instructions for dissecting soleus, gastrocnemius plantaris, and quadriceps muscles for further analysis are provided as well. This experimental setup is optimized for addressing questions regarding the involvement of daytime and circadian clocks in regulating exercise capacity. For complete details on the use and execution of this protocol, please refer to Ezagouri et al. (2019).

A Lipidomics View of Circadian Biology.

Lipidomics approaches provide quantitative characterization of hundreds of lipid species from biological samples. Recent studies highlight the value of these methods in studying circadian biology, and their potential goes far beyond studying lipid metabolism per se. For example, lipidomics analyses of subcellular compartments can be used to determine daily rhythmicity of different organelles and their intracellular dynamics. In this chapter we describe in detail the procedure for around the clock shotgun lipidomics, from sample preparation to bioinformatics analyses. Sample preparation includes biochemical fractionation of nuclei and mitochondria from mouse liver harvested throughout the day. Lipid content is determined and quantified, in unbiased manner and with wide coverage, using multidimensional mass spectrometry shotgun lipidomics (MDMS-SL). Circadian parameters are then determined with nonparametric statistical tests. Overall, the approach described herein is applicable for various animal models, tissues, and organelles, and is expected to yield new insight on various aspects of circadian biology and lipid metabolism.

Hypoxia induces a time- and tissue-specific response that elicits intertissue circadian clock misalignment.

The occurrence and sequelae of disorders that lead to hypoxic spells such as asthma, chronic obstructive pulmonary disease, and obstructive sleep apnea (OSA) exhibit daily variance. This prompted us to examine the interaction between the hypoxic response and the circadian clock in vivo. We found that the global transcriptional response to acute hypoxia is tissue-specific and time-of-day-dependent. In particular, clock components differentially responded at the transcriptional and posttranscriptional level, and these responses depended on an intact circadian clock. Importantly, exposure to hypoxia phase-shifted clocks in a tissue-dependent manner led to intertissue circadian clock misalignment. This differential response relied on the intrinsic properties of each tissue and could be recapitulated ex vivo. Notably, circadian misalignment was also elicited by intermittent hypoxia, a widely used model for OSA. Given that phase coherence between circadian clocks is considered favorable, we propose that hypoxia leads to circadian misalignment, contributing to the pathophysiology of OSA and potentially other diseases that involve hypoxia.

Physiological and Molecular Dissection of Daily Variance in Exercise Capacity.

Physical performance relies on the concerted action of myriad responses, many of which are under circadian clock control. Little is known, however, regarding the time-dependent effect on exercise performance at the molecular level. We found that both mice and humans exhibit daytime variance in exercise capacity between the early and late part of their active phase. The daytime variance in mice was dependent on exercise intensity and relied on the circadian clock proteins PER1/2. High-throughput gene expression and metabolic profiling of skeletal muscle revealed metabolic pathways that are differently activated upon exercise in a daytime-dependent manner. Remarkably, we discovered that ZMP, an endogenous AMPK activator, is induced by exercise in a time-dependent manner to regulate key steps in glycolytic and fatty acid oxidation pathways and potentially enhance exercise capacity. Overall, we propose that time of day is a major modifier of exercise capacity and associated metabolic pathways.

Oxygen and Carbon Dioxide Rhythms Are Circadian Clock Controlled and Differentially Directed by Behavioral Signals.

Daily rhythms in animal physiology are driven by endogenous circadian clocks in part through rest-activity and feeding-fasting cycles. Here, we examined principles that govern daily respiration. We monitored oxygen consumption and carbon dioxide release, as well as tissue oxygenation in freely moving animals to specifically dissect the role of circadian clocks and feeding time on daily respiration. We found that daily rhythms in oxygen and carbon dioxide are clock controlled and that time-restricted feeding restores their rhythmicity in clock-deficient mice. Remarkably, day-time feeding dissociated oxygen rhythms from carbon dioxide oscillations, whereby oxygen followed activity, and carbon dioxide was shifted and aligned with food intake. In addition, changes in carbon dioxide levels altered clock gene expression and phase shifted the clock. Collectively, our findings indicate that oxygen and carbon dioxide rhythms are clock controlled and feeding regulated and support a potential role for carbon dioxide in phase resetting peripheral clocks upon feeding.

Crosstalk between metabolism and circadian clocks.

Humans, like all mammals, partition their daily behaviour into activity (wakefulness) and rest (sleep) phases that differ largely in their metabolic requirements. The circadian clock evolved as an autonomous timekeeping system that aligns behavioural patterns with the solar day and supports the body functions by anticipating and coordinating the required metabolic programmes. The key component of this synchronization is a master clock in the brain, which responds to light-darkness cues from the environment. However, to achieve circadian control of the entire organism, each cell of the body is equipped with its own circadian oscillator that is controlled by the master clock and confers rhythmicity to individual cells and organs through the control of rate-limiting steps of metabolic programmes. Importantly, metabolic regulation is not a mere output function of the circadian system, but nutrient, energy and redox levels signal back to cellular clocks in order to reinforce circadian rhythmicity and to adapt physiology to temporal tissue-specific needs. Thus, multiple systemic and molecular mechanisms exist that connect the circadian clock with metabolism at all levels, from cellular organelles to the whole organism, and deregulation of this circadian-metabolic crosstalk can lead to various pathologies.