Interaction between stress responses and circadian metabolism in metabolic disease

Abstract

Circadian rhythms play crucial roles in orchestrating diverse physiological processes that are critical for health and disease. Dysregulated circadian rhythms are closely associated with various human metabolic diseases, including type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Modern lifestyles are frequently associated with an irregular circadian rhythm, which poses a significant risk to public health. While the central clock has a set periodicity, circadian oscillators in peripheral organs, particularly in the liver, can be entrained by metabolic alterations or stress cues. At the molecular level, the signal transduction pathways that mediate stress responses interact with, and are often integrated with, the key determinants of circadian oscillation, to maintain metabolic homeostasis under physiological or pathological conditions. In the liver, a number of nuclear receptors or transcriptional regulators, which are regulated by metabolites, hormones, the circadian clock, or environmental stressors, serve as direct links between stress responses and circadian metabolism. In this review, we summarize recent advances in the understanding of the interactions between stress responses (the endoplasmic reticulum (ER) stress response, the oxidative stress response, and the inflammatory response) and circadian metabolism, and the role of these interactions in the development of metabolic diseases.

1. Introduction

The circadian rhythm is the biological process that orchestrates behavior and physiology in most living organisms over a 24 h period. In mammals, circadian oscillations are generated by a network of clock-controlled genes (CCGs) that form a transcriptional auto-regulatory feedback loop. Several interconnected transcriptional and post-translational negative feedback loops play vital roles in generating and maintaining circadian rhythms. The core circadian transcription factors CLOCK and aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1) exist as a heterodimer and constitute the positive arm of the molecular clock. Genes that are directly regulated by the CLOCK/BMAL1 heterodimer are referred to as first-order CCGs. ^1,2 The core clock regulators also drive circadian expression of many transcription factors, thus extending and enhancing their regulatory functions. Expression of the Bmal1 gene is regulated by the nuclear receptor RORα/γ and PPARγ coactivator-1α (PGC-1α),³ while the major negative regulator of Bmal1 mRNA expression is REV-ERBα (also known as NR1D1).⁴ Other circadian proteins, including period 1 (PER1), PER2, PER3, cryptochrome 1 (CRY1), and CRY2, which are targets of BMAL1, also play roles in the negative regulation of Bmal1 expression.⁴ The components of the core clock are also regulated by post-translational modifications (phosphorylation, acetylation, deacetylation, and ubiquitination) which modulate the stability or activity of clock proteins.⁵

The mammalian circadian clock orchestrates diverse physiological processes by synchronizing with the nervous system, cardiovascular system, immune response, and metabolic homeostasis. The master clock oscillators residing in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus orchestrate the cascade of events that control physiological rhythms and ensure temporal coordination of metabolism and behavior through the synchronization of peripheral oscillators.⁶ The circadian oscillators in peripheral organs, such as the liver and kidney, respond differently to entraining signals and control specific physiological outputs, thus producing the coordinated response required to regulate cellular and physiological function. In particular, circadian rhythm and liver metabolism are intrinsically linked in order to synchronize the storage and utilization of energy with the light-dark cycle.⁷ This integration of circadian rhythm and hepatic energy metabolism is mediated through reciprocal crosstalk between these two regulatory networks.

Dysregulation of the circadian rhythm in humans is closely associated with the development of metabolic diseases, including non-alcoholic fatty liver disease (NAFLD), obesity, and type 2 diabetes. Previous work has demonstrated intimate and reciprocal interaction between the circadian clock system and fundamental metabolic pathways.^1,8,9 It has been shown that Clock gene mutations or BMAL1 deficiency impair lipid and glucose metabolism in animals, demonstrated by the development of hyperlipidemia, hepatic steatosis, and defective gluconeogenesis.^10–13 A survey of nuclear receptor mRNA profiles in metabolic tissues suggested that approximately half of the known nuclear receptors and transcriptional regulators exhibit rhythmic expression.¹⁴ Bioinformatic analysis of genome-wide and phase-specific DNA-binding by the core circadian transcriptional oscillators indicated that BMAL1-binding sites are associated with carbohydrate and lipid metabolism.¹⁵ In the liver, nuclear receptors and other transcription factors can be induced by metabolites or hormones, and therefore, they may serve as direct links between metabolic pathways and the circadian control of gene expression. For example, the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), which binds fatty acid ligands, serves as a sensor of nutrient and energy status and temporally entrains peripheral clocks. In addition, PPARα and BMAL1 are reciprocally regulated, providing a feedback loop that integrates lipid metabolism and circadian oscillations.^16–18

The clock-controlled nuclear receptors REV-ERBs are key regulators of circadian lipid biosynthesis in the liver, and their ablation causes hepatic steatosis through de-repression of lipogenesis.^19,20 PGC-1α also provides a link between the clock and energy metabolism, because PGC-1α expression varies rhythmically and is able to stimulate expression of Bmal1 and nuclear receptors of the ROR family.³ Recent studies have shown that the nuclear receptor small heterodimer partner (SHP) is also critical for the control of REV- and ROR-mediated neuronal PAS domain protein 2 (NPAS2) expression in NAFLD, and of FOXA1 in alcoholic fatty liver disease (AFLD).^21,22 Additionally, the clock component CRY1 functions as a circadian regulator of hepatic gluconeogenesis.²³ By interacting with the Gsα subunit of G proteins, CRY1 can temporally regulate glucagon signaling, thereby activating hepatic gluconeogenesis. Furthermore, recent evidence implicates distinct signaling pathways synchronized by the circadian clock in circadian metabolism at the translational or post-transcriptional level.^24,25 For example, the circadian clock synchronizes the rhythmic activation of the primary endoplasmic reticulum (ER) stress sensor IRE1α (inositol requiring enzyme 1).²⁴ Lack of a functional circadian clock disrupts the rhythmic activation of the IRE1α pathway, leading to impaired lipid metabolism through aberrant activation of the sterol-regulated SREBP transcription factors.

2. ER Stress Response and Circadian Metabolism

In mammalian cells, the ER is an essential organelle that is responsible for protein folding and assembly, synthesis of sterols and lipids, and calcium storage and homeostasis.^26–28 As a protein folding compartment, the ER plays a crucial role in cellular protein quality control by ensuring only correctly folded proteins are transported to their final destinations, and by assembling incorrectly folded proteins into native complexes and degrading them. When cellular or environmental stressors disrupt ER function, unfolded or misfolded proteins accumulate in the ER lumen, a condition referred to as ER stress. To deal with ER stress, cells activate a sophisticated and coordinated series of signal transduction pathways known as the unfolded protein response (UPR). The UPR is a survival response which modifies transcriptional and translational programs to restore ER homeostasis. The UPR pathways in mammals are primarily initiated by three major cell stress sensors: IRE1α, double-stranded RNA activated protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Under ER stress conditions, PERK phosphorylates the serine51 residue on the alpha subunit of eIF2α, which attenuates the translation, thus reducing the workload of the ER. In addition, IRE1α, a bi-functional transmembrane protein, functions as a ribonuclease (RNase), splicing the mRNA encoding X-box binding protein (XBP1), leading to the transcriptional reprogramming of stressed cells. Also during ER stress, ATF6 translocates into the Golgi apparatus, where it is processed into its active form by site 1 protease (S1P) and site 2 protease (S2P). Upon proteolytic activation, ATF6 travels to the nucleus, where it induces the expression of UPR target genes by binding to ER stress response elements (ERSEs) located within their promoter regions. It is important to note that the UPR pathway can also be triggered by inflammatory stimuli, chemical toxicity, infection by pathogens, and even by the differentiation of specialized cell types, such as the differentiation of B-lymphocytes into antibody-secreting plasmocytes.^29,30

A number of enzymes involved in liver metabolism are located in the ER membranes. Lipid metabolism is associated with the physiological UPR, and circadian clock circuitry influences hepatic lipid metabolism and ER enzymes. The UPR plays a key role in maintaining hepatic lipid homeostasis, and disruption of the UPR leads to hepatic steatosis and non-alcoholic steatohepatitis (NASH) under conditions of pathophysiological stress. Deletion or suppression of the UPR transducer IRE1α or ATF6 in mouse liver results in hepatic steatosis under acute ER stress or after consumption of a high fat diet.^31–34 Animals with a defect in the liver-enriched, ER-tethered stress sensor cAMP response element binding protein, hepatocyte-specific (CREBH) develop severe NASH when fed an atherogenic high fat diet.^35–40 Additionally, deficiency of the ER stress-induced transcriptional activator XBP1 in the liver also leads to hepatic lipid accumulation.^41–43 Thus, a wealth of evidence indicates that UPR pathways are required to prevent hepatic lipid accumulation and the development of NAFLD under acute ER stress or chronic pathophysiological stress conditions.

ER stress and UPR signaling have significant impacts on circadian activity. A recent study has demonstrated that activation of the IRE1α-mediated UPR pathway is circadian in mouse liver ²⁴. Disruption of the circadian clock perturbs the circadian rhythmicity of IRE1α-mediated UPR activation and provokes deregulation of ER enzymes (Fig. 1). This disruption causes aberrant activation of sterol regulatory element-binding protein (SREBP) transcription factors and impaired hepatic lipid metabolism, which can lead to the development of metabolic disorders.

Fig. 1. Integration of circadian rhythm and stress signaling in the endoplasmic reticulum regulates lipid metabolism.

Activation of the ER-tethered stress sensors CREBH and IRE1α is regulated by the circadian clock. ER-to-Golgi transport and proteolytic cleavage of CREBH protein is regulated by the circadian clock through the BMAL1-AKT-GSK3β regulatory axis. Activated CREBH translocates to the nucleus, where it interacts with circadian-regulated nuclear receptors (PPARα and LXRα) or transcriptional modulators (DBP and E4BP4) to regulate expression of genes involved in lipid utilization (FA oxidation, TG lipolysis, and ketogenesis) or storage (de novo lipogenesis) pathways. The circadian clock also regulates activation of the IRE1α-XBP1 UPR pathway, which regulates the UPR and potentially metabolic pathways. Abbreviations: ER, endoplasmic reticulum; DBP, D-site-binding protein; E4BP4, E4-binding protein 4; GSK3β, glycogen synthase kinase 3 β; AKT, protein kinase B; LXRα, liver X receptor α.

Another example of an interaction between ER stress and circadian metabolism is the role of the liver-enriched ER stress sensor CREBH in integrating circadian regulation with hepatic energy metabolism (Fig. 1).^39,40 The proteolytic activation of CREBH protein, but not expression of CrebH mRNA, exhibits typical circadian rhythmicity in mouse liver. This process is controlled by the core clock oscillator BMAL1 and the AKT/glycogen synthase kinase 3β (GSK3β) signaling pathway.⁴⁰ Importantly, GSK3β-mediated phosphorylation of CREBH within its b-ZIP domain modulates the association between CREBH and coat protein complex II (COPII), present in the vesicles responsible for protein transport from the ER to the Golgi, and thus controls ER-to-Golgi transport and subsequent proteolytic cleavage of CREBH in a circadian fashion (Fig. 1).

In addition to proteolytic cleavage, the post-translational modification (lysine acetylation) of CREBH protein also exhibits a circadian rhythm in the liver.³⁹ Functionally, CREBH regulates circadian expression of key genes involved in triglyceride (TG) and fatty acid (FA) metabolism, and is required to maintain circadian variation in blood TG and FA.^37,40 In mice during the light period, CREBH rhythmically regulates and interacts with the hepatic nuclear receptor PPARα to regulate expression of the genes that mediate FA oxidation, TG lipolysis, and ketogenesis for energy utilization (Fig. 1), while during the dark period, CREBH interacts with liver X receptor α (LXRα) to regulate expression of genes involved in de novo lipogenesis for energy storage. The coordination between CREBH and the circadian clock is also evidenced by the rhythmic interactions between CREBH and the circadian oscillation activator DBP and the repressor E4BP4 in their modulation of CREBH transcriptional activity (Fig. 1). Genetic defects in CREBH or impairment in CREBH activity can dysregulate lipid homeostasis, leading to NAFLD and/or hyperlipidemia.^35–37,40 This has been confirmed in human patients with hypertriglyceridemia, who exhibit a high rate of nonsense mutations or accumulation of rare genetic variants in their CREBH genes.^44–46

CREBH is also required to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels.³⁹ It achieves this by regulating rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH deficiency leads to lower blood glucose levels and higher hepatic glycogen levels during the light period.

Circadian disruption also leads to dysregulated UPR signaling in pancreatic β cells; Bmal1 or an intact circadian clock is required for β cells to adapt to cellular stress. Disruption of circadian control leads to oxidative stress, ER stress and impaired β cell function. Both oxidative stress and ER stress contribute to impaired mitochondrial function and β cell failure.⁴⁷ Indeed, acute or chronic sleep deprivation in mice leads to ER stress in the pancreas of aged mice, and circadian disruption worsens unresolved ER stress, leading to β cell apoptosis, in diabetes-prone human islet amyloid polypeptide expressing rats.⁴⁸

3. Oxidative stress and circadian metabolism

Oxidative metabolism is a major source of reactive oxygen species (ROS). Cells have evolved many mechanisms to prevent the potential damage caused by the generation of ROS, including upregulation of antioxidant enzymes and free radical non-glutathione systems. During the day-night cycle, levels of lipid peroxidation and protein oxidation, the major components of oxidative stress responses, exhibit circadian rhythmicity in key metabolic organs or tissues, such as liver and muscle.^49–52 Many antioxidative enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), peroxiredoxins (Prxs), and melatonin, display circadian rhythmicity in their levels of expression or activity, providing evidence for a circadian oscillation of oxidative and anti-oxidative stress responses (Fig. 2). ROS scavenging systems are critical for the function and survival of metabolic cells, and there is a growing body of evidence to suggest that impairment in redox regulation and circadian rhythms can lead to metabolic disorders.⁵³ Oxidative stress plays a key role in the initiation and progression of NAFLD: ROS cause lipid peroxidation, followed by inflammatory responses and activation of stellate cells during fibrogenesis in NAFLD patients.⁵⁴

Fig. 2. Interaction between oxidative stress and circadian rhythm.

The circadian clock influences mitochondrial metabolism, leading to ROS production and changes in NADP/NADPH and NAD⁺/NADH ratios through oxidative phosphorylation. ROS-associated oxidative and anti-oxidative stress responses exhibit circadian rhythms because of circadian-regulated expression of anti-oxidative enzymes or oxidation of lipids and proteins. NAD⁺/NADH ratio modulates SIRT1 activity, which controls BMAL1 or PER2 activity through deacetylation. Furthermore, NAD⁺/NADP and NADH/NADPH ratios control the activity of the core circadian oscillator BMAL1/CLOCK heterodimer by modulating its binding to circadian effector genes. Abbreviations: PER2, period circadian clock 2; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADH, nicotinamide adenine dinucleotide reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced).

Several molecular regulators of the interactions between circadian rhythm and the oxidative stress response have been identified. In particular, the stress-induced nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacetylase sirtuin 1 (SIRT1) is linked to both the oxidative stress response and circadian rhythms. SIRT1 is involved in the core circadian mechanism because it deacetylates the circadian oscillation proteins BMAL1 and PER2 (Fig. 2).^55,56 The deacetylation of BMAL1 resets the clock to its original state, while PER2 deacetylation targets it for degradation, thereby preventing formation of PER2-CRY heterodimers as part of the negative feedback regulation of the core circadian mechanism.

The coenzyme NAD⁺ also plays critical roles in regulating the oxidative stress response and circadian metabolism. NAD⁺ and its redox partner NADH are important in redox reactions, in which the phosphorylated forms of NAD⁺/NADH, NADP⁺/NADPH, drive oxidation-reduction reactions.⁵⁷ Circadian oscillation of NADH/NADPH directly regulates DNA binding of the core circadian oscillation heterodimers CLOCK/BMAL1 and NPAS2/BMAL1 to their transcriptional targets, while NAD⁺/NADP⁺ inhibit this activity (Fig. 2).⁵⁸ Therefore, cellular oxidative stress and the general redox state affect the redox state of NAD⁺/NADH and NADP⁺/NADPH and circadian rhythms. Furthermore, protein deacetylation by SIRT1 involves the use of NAD⁺ as a cofactor;⁵⁷ therefore, as a coenzyme of SIRT1, NAD⁺ functions as a key regulator of circadian metabolism.

Circadian redox state has a major influence on the function and survival of pancreatic β cells, because they have a poor tolerance for oxidative stress because of low expression of antioxidant genes.

Many of the key ROS scavenging enzyme systems and their rate-limiting genes are transcriptionally regulated by the anti-oxidative response regulator Nrf2. Nrf2 expression peaks in the late light/early dark phase in β cells and is directly regulated by the circadian clock, ⁵⁹ because Bmal1 can bind to the E-box element in the Nrf2 gene promoter. Indeed, the circadian expression of Nrf2 is lost in Bmal1^−/− β cells.^47,59 Disruption of Bmal1 leads to defects in β cell stimulation-secretion coupling, lower ATP production, uncoupling of oxidative phosphorylation, and impaired glucose-induced insulin secretion. Genetic disruption and environmental stressors can induce oxidative stress, mediated by a disruption of the direct transcriptional control by the core molecular clock and Bmal1 on Nrf2, the master antioxidative transcription factor in β cells.

The link between oxidative stress and circadian rhythmicity in liver metabolism is evidenced by the rhythmicity of mitochondrial dynamics and energy homeostasis. Recent studies have revealed that mitochondrial dynamics (mitochondrial fission and mitophagy) and biogenesis are transcriptional targets of the circadian regulator BMAL1 in mouse liver, and exhibit circadian rhythmicity in synchronization with diurnal energy demands. ⁶⁰ Liver-specific Bmal1 knockout (LBmal1KO) mice accumulate oxidative damage in the liver and develop hepatic insulin resistance, while restoration of hepatic BMAL1 activity in high fat diet-fed mice improves these metabolic outcomes. These results suggest that clock genes are evolutionarily conserved regulators of cellular energetics.

4. Inflammatory responses and circadian metabolism

Chronic disruption of circadian timing during shift work or chronic jetlag leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation.⁶¹ Impaired circadian rhythm is linked to inflammatory responses that are associated with the development of metabolic diseases. Uncoupling of peripheral and master clock gene rhythms by a reversed feeding cycle leads to exacerbated inflammatory responses in mouse models of polymicrobial sepsis.⁶² In particular, when mice were fed during the light, rather than the dark, period, it was found that serum levels of inflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNFα), IL-1α, IL-9, eotaxin, and granulocyte colony-stimulating factor, were higher in mice fed during the day than in those fed at night, after cecal ligation-puncture to induce sepsis. Consistent with a greater inflammatory response, expression levels of the anti-oxidative regulators SIRT1 and PPARγ were lower in the livers of day-fed mice. This study demonstrates that the uncoupling of circadian clock rhythmicity by an inverted feeding cycle exacerbates inflammatory responses in the liver.

However, inflammation can also cause dysregulation of circadian rhythms. Following a pro-inflammatory response triggered by TNF-α, the enzyme USP2a deubiquitinates and stabilizes the circadian protein CRY1.⁶³ USP2a is regulated by the circadian clock and interacts with CRY1 in the liver to enhance its stability via deubiquitination, which leads to repression of hepatic Per2 gene expression. Interestingly, the pro-inflammatory cytokine TNF-α also increases CRY1 protein expression and inhibits circadian gene expression in an USP2a-dependent fashion, suggesting that USP2a may mediate circadian disruption by suppressing CRY1 degradation during inflammation. In addition, physical and inflammatory stressors, including forced swimming, immobilization, and lipopolysaccharide injection, increased expression of the mouse circadian clock gene Per1 in the hypothalamic paraventricular nucleus (PVN), further demonstrating that inflammatory stressors can modulate circadian regulation through upregulation of Per1 expression.⁶⁴

5. Dysregulated circadian metabolism in NAFLD

NAFLD represents a spectrum of disorders ranging from relatively benign and reversible steatosis to end stage liver disease (cirrhosis). It is characterized by the accumulation of TG in hepatocytes which develops because of an imbalance between hepatic synthesis and breakdown of lipids, and between fatty acid storage and disposal. It causes liver enlargement and manifests in the absence of alcohol ingestion. There are three distinct sources of FAs in the liver: the diet, lipolysis, and de novo lipogenesis. The liver uses or shunts the excess FA by re-esterification to TG and storage as lipid droplets, β-oxidation or export as low-density lipoprotein (VLDL). Thus, hepatic fat deposition can occur as a consequence of lower oxidation or export of lipid and/or greater synthesis.⁶⁵ Liver metabolic pathways are regulated by circadian biological clocks, and hepatic health is maintained by proper timing of circadian patterns of metabolic gene expression, with the alternation of anabolic processes corresponding to the feeding/active state during wakefulness, and catabolic processes characterizing the fasting/resting state during sleep.⁶⁶

The pathogenesis of NAFLD is complex and multifactorial; therefore, both genetic and/or environmental disruptors may affect the circadian clock, leading to the development of metabolic diseases or exacerbation of pathological states, and indeed it has been shown that common genetic variations in the Clock gene are associated with NAFLD in humans. Clock gene polymorphisms and haplotypes involving the CLOCK transcription factor are correlated with the susceptibility and severity of NAFLD.⁶⁷ Modern lifestyle demands force large numbers of people into asynchrony between actual time and their circadian clocks, resulting in a constant state of social jetlag. Recent findings have indicated that interactions between altered energy metabolism and disruptions in the circadian clock can lead to the development of metabolic disorders. In human NAFLD patients, disruption of the sleep-wake cycle, manifesting as daytime lethargy, is positively associated with biochemical and histologic surrogates of NAFLD severity and insulin resistance. Recent studies have demonstrated that inadequate, poor quality sleep is linked with NAFLD risk in middle-aged people. Moreover, a pilot study has shown favorable effects of using melatonin, a circadian hormone, as an antioxidant that ameliorates NAFLD. ⁶⁸ Food intake is now more common during the night. NASH is the potential outcome of a relatively benign and reversible condition, hepatic steatosis, and is characterized by hepatocyte injury and tissue inflammation due to oxidative stress and recruitment of inflammatory mediators, such as cytokines. It has been shown that a high fat diet increases the incidence of NASH in adulthood, associated with altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression. ⁶⁹

Disruption of normal circadian rhythmicity represents an independent risk factor for hepatocellular carcinoma (HCC) in experimental animals and has revealed opposing roles for the nuclear receptors FXR and constitutive androstane receptor (CAR) in disease progression from NAFLD to HCC.⁷⁰ Studies have also shown that chronic circadian misalignment, independent of germ line mutation and dietary manipulation, promotes the development of malignant hepatoma; for example, chronic jet-lag induces spontaneous HCC in wild-type mice. ⁷¹ This process commences with the development of simple steatosis, which progresses to steatohepatitis and fibrosis, before the onset of HCC. This pathophysiological pathway is driven by jetlag-induced genome-wide gene dysregulation and metabolic dysfunction in the liver, with nuclear receptor-controlled cholesterol/bile acid and xenobiotic metabolism among the most highly dysregulated pathways. A study performed on jetlagged mice revealed higher expression of genes required for glycogenolysis, amino acid and lipid synthesis and storage, oxidative stress, glycolysis, and hepatocyte proliferation and death, together with suppression of genes responsible for stimulating tumor suppression, DNA damage repair, glycogen synthesis, β-oxidation, and FA transport.⁷¹

The deregulation of vital circadian genes (Per1, Cry1, Clock and Bmal1), together with dysregulation of molecular markers of HCC, highlights the importance of the circadian homeostasis of liver metabolism in the suppression of hepatocarcinogenesis. In addition, ablation of farnesoid X receptor dramatically increases enterohepatic bile acid levels and jetlag-induced HCC, while loss of CAR, a well-known promoter of the development of liver tumors that mediates toxic bile acid signaling, inhibits NAFLD-induced hepatocarcinogenesis. ^71,72 Circadian disruption activates CAR by promoting cholestasis, peripheral clock disruption, and sympathetic dysfunction. The association of dysregulated circadian rhythm with HCC was confirmed by the observation that chronic circadian dysfunction disrupts liver gene expression and metabolism, promoting NAFLD and cancer. ⁷³

6. Summary and future perspectives

Dysregulation of circadian rhythms is closely associated with human cardio-metabolic diseases, such as type 2 diabetes, cardiovascular disease, and NAFLD. Due to modern lifestyles, the problem of irregular circadian rhythm is not limited to shift workers, but has a wide impact on public health. The circadian rhythm aligns molecular signal transduction networks with physiological activities. While the central clock is set by the Clock gene, circadian oscillators in peripheral organs, particularly in the liver, can be entrained by metabolic changes or stress signals, providing the flexibility to adapt to environmental challenges. However, under chronic stress conditions, circadian metabolism is dysregulated, which represents a significant risk factor for the development of metabolic diseases.

For future research, it is still critical to understand the connections between stress responses and circadian rhythms for the maintenance of metabolic homeostasis. Although significant progress has been made, the mechanisms by which environmental or intracellular stressors modulate circadian metabolism, and their roles in the progression of metabolic disorders, remain to be fully elucidated. In particular, recent studies have suggested that the rhythmicity of the human microbiome may affect metabolic homeostasis.^74,75 Feeding time has been shown to shape the rhythmicity and composition of the intestinal microbiota, and disruption of the microbiome is associated with diet, obesity, and metabolic disease. Controlling the circadian rhythmicity of human microbiota, through changes in diet and lifestyle or by the use of drugs, may represent a novel intervention for the treatment of metabolic diseases. As we gain a greater understanding of the molecular basis and the extent to which inflammatory stress responses modulate circadian metabolism, it might be possible to design novel therapies to minimize the deleterious metabolic consequences of circadian disruptions by targeting the activation of stress-associated circadian regulators and mediators.