Gastroenterology 2020;158:1948–1966
Circadian Rhythms in the Pathogenesis and Treatment of Fatty Liver Disease
Anand R. Saran1 Shravan Dave1 Amir Zarrinpar1,2,3,4
From the 1Division of Gastroenterology, University of California, San Diego, La Jolla, California; 2Veterans Affairs Health Sciences San Diego, La Jolla, California; 3Institute of Diabetes and Metabolic Health, University of California, San Diego, La Jolla, California; and 4Center for Microbiome Innovation, University of California, San Diego, La Jolla, California
Circadian clock proteins are endogenous timing mecha- Although the term circadian rhythms usually conjures nisms that control the transcription of hundreds of genes. thoughts of jet lag and sleep deprivation, physiologically, Their integral role in coordinating metabolism has led to they are core cellular processes that affect every organ their scrutiny in a number of diseases, including nonalco- system. The pervasiveness of clock proteins, which are holic fatty liver disease (NAFLD). Discoordination between found in diverse organisms from cyanobacteria to mammals, central and peripheral circadian rhythms is a core feature shows their evolutionary importance to life. Development of of nearly every genetic, dietary, or environmental model of a molecular program to anticipate and prepare for periodic, metabolic syndrome and NAFLD. Restricting feeding to a predictable changes gives a tremendous adaptative advan- fi de ned daily interval (time-restricted feeding) can syn- tage. Organisms have developed specialized sensory cells chronize the central and peripheral circadian rhythms, that allow them to adjust their own rhythmicity to external which in turn can prevent or even treat the metabolic cues (called zeitgebers—German for “time giver”). The daily syndrome and hepatic steatosis. Importantly, a number of light-dark cycle on Earth governs rhythmic changes in the proteins currently under study as drug targets in NAFLD behavior and physiology of most species, including humans. (sterol regulatory element–binding protein [SREBP], Almost all organisms consolidate their biological processes acetyl-CoA carboxylase [ACC], peroxisome proliferator– activator receptors [PPARs], and incretins) are modu- (e.g., sleeping and feeding) to particular times of day. To lated by circadian proteins. Thus, the clock can be used to acknowledge that the periodicity of these oscillations stays maximize the benefits and minimize the adverse effects of pharmaceutical agents for NAFLD. The circadian clock it- Abbreviations used in this paper: ACC, acetyl-CoA carboxylase; AKT (or self has the potential for use as a target for the treatment PKB), v-Akt murine thymoma viral oncogene homolog 1 or (protein kinase of NAFLD. B); AMP, adenosine monophosphate; AMPK, adenosine monophosphate– activated protein kinase; BHLH, basic helix-loop-helix family member; BMAL1, brain and muscle ARNTL-like protein 1; cAMP, cyclic adenosine monphoshate; CK1, casein kinase 1; CLOCK, circadian locomotor output Keywords: Dyssynchrony; Gut Microbiome; Steatohepatitis; cycles kaput; CREB, cAMP response element-binding protein; CREBH, cAMP response element binding protein hepatocyte specific; CRY, cryp- Lipogenesis; Bile Acids. tochrome; CYP7A1, cholesterol 7 alpha-hydroxylase; DBP, D-box binding protein; DEC, (or BHLH), basic helix-loop-helix family member; DIO, diet- induced obesity; ER, endoplasmic reticulum; FBXL3, F-box/LRR-repeat protein 3; FGF, fibroblast growth factor 15; FXR, farnesoid X receptor; he 2017 Nobel Prize in Medicine signaled a recog- GLP-1, glucagon-like peptide; GLUT2, glucose transporter 2; GYS2, glycogen synthase 2; HFD, high-fat diet; IRE1a, inositol-requiring 1a; KO, nition of the fundamental role that circadian clock D T knockout; mTOR, mammalian target of rapamycin; NAD , nicotinamide proteins play in physiology and disease. It was awarded to adenine dinucleotide; NAFLD, nonalcoholic fatty liver disease; NFIL3 Jeffrey C. Hall, Michael Rosbash, and Michael W. Young for (or E4BP4), nuclear factor, interleukin 3 regulated; OR, odds ratio; OSA, obstructive sleep apnea; PER, period; PGC1a, peroxisome proliferator– their discovery of molecular mechanisms controlling circa- activated receptor g coactivator 1a; PPAR, peroxisome proliferator- dian rhythms in Drosophila. Clock proteins are ubiquitous activated receptor; ROR, retinoic acid receptor-related orphan receptors; RORE, retinoic acid receptor-related–responsive element; SCFA, short- endogenous timing mechanisms that help maintain energy chain fatty acid; SCN, suprachiasmatic nucleus; SIRT1, sirtuin 1; SREBP, homeostasis by coordinating cellular processes within and sterol regulatory element-binding proteins; T2D, type 2 diabetes; TLR, toll- like receptor; TRF, time-restricted feeding; ULK1, unc-51 like autophagy between organ systems. They do so by regulating the activating kinase 1; UPR, unfolded protein response. transcription of hundreds of genes, including themselves. Most current article Their integral role in coordinating metabolism has led to closer scrutiny of their contribution to a number of diseases, © 2020 by the AGA Institute 0016-5085/$36.00 including nonalcoholic fatty liver disease (NAFLD). https://doi.org/10.1053/j.gastro.2020.01.050 May 2020 Circadian Rhythms and NAFLD 1949 approximately 24 hours, Franz Halberg, a pioneer in the PER for degradation; FBXL3 promotes the degradation of field of biological rhythms, introduced the term circadian CRY.19,20 However, if Ck1ε phosphorylates PER after it is rhythms (from Latin, circa meaning “about” and diem bound to CRY, the 3-protein complex migrates into the nu- meaning “a day”) in 1959. cleus and inhibits the CLOCK:BMAL1 heterodimer.21 By In mammals, the circadian pacemaker resides in hypo- inhibiting their own activators, PERs and CRYs repress the thalamic structures called the suprachiasmatic nuclei (SCN), transcription of their own genes. Posttranslational modifi- and it is entrained by specialized retinal ganglion cells that cation of PERs and CRYs leads to their degradation, which measure ambient light (Figure 1). However, circadian clock then initiates a new circadian cycle with increased binding proteins are not restricted to the SCN. Explants from lung, of CLOCK:BMAL1 to the now open E-box binding sites. liver, and other organs exhibit sustained oscillations In a second regulatory loop, CLOCK:BMAL1 drives the in vitro.1,2 Thus, endogenous functional clocks are also transcription of the nuclear hormone receptors retinoic acid active in peripheral tissues.3 Temporal signals, such as receptor-related orphan receptor (Ror) a and Rorg and Rev- timing of food intake, can reset the hepatic clock without Erba and Rev-Erbb by binding E-box elements on their affecting the SCN central clock rhythms. Consequently, promoters (Figure 3B).22–24 Both of these receptors can different entrainment signals can activate peripheral and bind the ROR-responsive element (RORE) promoter DNA central circadian clocks separately.4,5 However, this does binding sequence (50-AGGTCA-30), but with opposite effects: not undermine the importance of the SCN in coordinating REV-ERBa/b suppresses expression at the site, whereas physiological processes across multiple tissues. RORa/g promotes expression.25,26 Together, REV-ERBs and In this review, we will describe the molecular organiza- RORs generate large-scale cyclic fluctuations in the tran- tion of the circadian clock proteins, the relationship between scription of hundreds of clock-controlled genes, including circadian clock and metabolic master regulators, and clock regulation of the transcription of Bmal1.27 regulation of metabolic processes important to the patho- Recent studies have revealed an additional autonomous physiology of NAFLD. Environmental cues that entrain pe- feedback loop involving basic helix-loop-helix family member ripheral circadian rhythms will be also discussed in detail. e40 (BHLHE40; more commonly, DEC1) and BHLHE41 The effects of circadian dyssynchrony on host metabolism (more commonly, DEC2), which also suppress CLOCK:BMAL1 will be summarized, along with recent preclinical and clinical activity (Figure 3C).28,29 CLOCK:BMAL1 induces transcription studies showing that maintaining peripheral circadian of Dec1 and Dec2 by binding E-box elements on their pro- rhythms by consolidating feeding within a narrow time frame moters. Unlike PERs and CRYs, which act directly on the (ie, time-restricted feeding [TRF]) can restore metabolic CLOCK:BMAL1 heterodimer, DECs compete with CLOCK:- health. Over the last 5 years, there have been many excellent BMAL1 for the E-box, thus suppressing all E-box genes, reviews of preclinical data regarding the role of the circadian including transcription of their own genes and those of PERs clock in hepatic metabolic processes specifically6–11 and in and CRYs. metabolic syndrome in general.12–16 Our review builds on Other circadian clock–controlled genes bring additional this strong body of work by focusing on key regulatory refinement to this complex interplay of feedback loops. pathways that play an important role in the treatment of CLOCK:BMAL1 modulates the expression of hundreds of steatohepatitis and fibrosis, thus showing the vital role of the other genes by promoting the transcription of D-box binding circadian machinery in helping our understanding of NAFLD. protein (DBP) by binding E-box elements on its promoter (Figure 3D).11,30 DBP rhythmically activates transcription of genes that have D-box elements in their promoter regions. Molecular Organization of the Clock Nuclear factor interleukin 3 (NFIL3, also E4BP4), which is The circadian clock in mammals, expressed in nearly regulated by a RORE promoter region (and thus is suscep- every cell, is comprised of a series of transcription- tible to activation by RORs and suppression by REV-ERBs), translation feedback loops (Figure 2). These include circa- represses DBP-dependent transactivation by competing for dian locomotor output cycles kaput (CLOCK) and brain and the D-box. Computational genomic analysis of D-box ele- muscle ARNT-like 1 (BMAL1) as transcriptional activators. ments has identified nearly 1500 regions that can be These are opposed by period (PER) and cryptochrome recognized by DBP and NFIL3.31 (CRY) as transcriptional repressors (Figure 3A). CLOCK and The transcription-translation feedback loops generate BMAL1 are two central components that make up the pos- large-scale rhythms in the activities and levels of down- itive (or activation) limb of the molecular clock. The stream clock-controlled genes.32 Because these processes CLOCK:BMAL1 heterodimer then translocates into the nu- are temporally controlled, it is important to regulate the cleus to initiate transcription at the E-box (50-CACGTG-30), a abundance and activity of each component to ensure that specific DNA binding sequence on the promoters of its it is in the right place at the right time. This is achieved target genes.17 The main downstream targets of CLOCK:- by posttranslational modifications of clock components, BMAL1 include their own repressors, period (PER1, PER2, such as phosphorylation, acetylation, SUMOylation (Small and PER3) and cryptochrome (CRY1, CRY2), in addition to Ubiquitin-like Modifier proteins; [SUMO can modify the more than 300 other genes.18 Over time, PERs and CRYs function of other proteins by attaching/detaching from accumulate in the cytoplasm, where they are further regu- them]), O-GlcNAcylation (O-Linked b-N-acetylglucosamine; lated by casein kinase 1 (CK1) ε, CK1d, and F-box/LRR- [O-GlcNAc is an intracellular carbohydrate that can repeat protein 3 (FBXL3). CK1ε and CK1d phosphorylate modify protein activity]), and ubiquitylation. Gene 1950 Saran et al Gastroenterology Vol. 158, No. 7 May 2020 Circadian Rhythms and NAFLD 1951
expression studies show that approximately 10%–40% of protein (CREB), v-Akt murine thymoma viral oncogene ho- the rodent genome exhibits a 24-hour rhythm in a highly molog 1 (AKT/PKB), and sirtuin 1 (SIRT1). All have cyclic tissue-specific manner.33 However, humans are diurnal activity mainly driven by feeding behavior and can regu- animals that feed during the day, whereas most rodents late—and are regulated by—circadian clock proteins. are nocturnal animals that feed primarily at night. The AMPK, a fasting-sensitive protein kinase that regulates recent publication of a transcriptome atlas of a baboon, a cellular energy homeostasis, can modulate, and is modu- diurnal primate, shows that the aforementioned percent- lated by, the circadian clock machinery. AMPK can affect the ages from rodent studies do not convey the full picture.3 length of circadian cycling by phosphorylating CRY and Of the approximately 11,000 genes that are commonly targeting it for degradation.34 Moreover, RORa activation expressed in all tissues in the baboon, 96.6% have 24- can modulate lipid metabolism and prevent hepatic stea- hour rhythmicity in at least 1 tissue. Greater than 80% tosis by activating AMPK, which controls key lipid regula- of the 18,000 protein-coding baboon genes were circa- tors (discussed later in the article).35 Feeding, on the other dian. More importantly, more than 80% of the genes hand, activates CREB, which up-regulates Per1 transcrip- encoding proteins targeted by pharmaceuticals are tran- tion.36 This relationship connects G protein–coupled re- scribed cyclically. This implies that most physiological ceptors, which use cAMP as a second messenger for processes are under circadian control, and thus, the transcription of circadian clock genes, with the circadian disruption of circadian rhythms can result in widespread clock.37 Several metabolic hormones, including glucagon downstream pathology. and epinephrine, both of which activate hepatic gluconeo- genesis, can influence circadian clock machinery through CREB activation.38–40 In addition, CRYs can independently The Circadian Clock Regulates suppress CREB, thereby not only affecting hepatic gluco- Metabolic Processes neogenesis but also modulating Per transcription.39 Finally, Hepatic metabolic processes, including glucose, lipid, AKT, a protein kinase activated by feeding that regulates and cholesterol/bile acid metabolism, are highly dynamic, glucose metabolism, among other cellular processes (eg, cell influenced by feeding/fasting and circadian rhythms. Data proliferation and apoptosis), can modulate the circadian supporting these relationships come from several lines of clock machinery by phosphorylating BMAL1 and CLOCK in the cytosol and preventing their translocation across the research, including metabolic phenotyping data from 41 circadian clock genetically modified mouse lines, molec- nuclear membrane. ular biology studies showing direct interactions between Sensing the nutrient status is a critical regulation step regulators of nutrient homeostasis and circadian clock in the maintenance of homeostasis. Metabolic stressors such as starvation induce autophagy to use glycogen and proteins, and the relationship of feeding/fasting cycles 42 and metabolic gene expression. In this section, we discuss lipid components for generating fuel. Circadian induction the relationship between circadian clock machinery and of autophagy during low nutrient states (eg, inactive state) fi various aspects of metabolism that are dysfunctional in allows ef cient coordination of these cellular processes. patients with NAFLD. AMPK, under glucose starvation conditions, phosphory- lates and activates the autophagy-initiating kinase ULK1 (Unc-51 like autophagy activating kinase 1), thus promot- Circadian Clock and Regulators of Nutrient ing autophagy. However, under nutrient sufficiency, Homeostasis and Autophagy mammalian target of rapamycin (mTOR), another meta- The liver contains several master metabolic regulators bolic master regulator, phosphorylates ULK1 and disrupts that sense feeding/fasting states and control the expression its interaction with AMPK, thus inhibiting autophagy.43 of hundreds of downstream metabolic targets. These include Interestingly, liver-specific Bmal1-null mice display adenosine monophosphate (AMP)–activated protein kinase dampened rhythms of autophagy gene expression, along (AMPK), cyclic AMP (cAMP) response element-binding with increased mTOR activation.44 Thus, autophagy has
= Figure 1. Central and peripheral circadian rhythms. Retinal ganglion cells detect ambient light and entrain the SCN. Their projections travel through the retinohypothalamic tract, although indirect signals are also received from the thalamus. The SCN contains the central circadian clock, which can modulate other oscillatory networks, including sleep/wake, temperature, and feeding/fasting homeostasis, through the hypothalamus. In addition, the SCN regulates the circadian release of hypothalamic- releasing hormones (eg, corticotropin-releasing hormone) and, thus, pituitary hormones (eg, adrenocorticotropic hormone). Hormones released from the pituitary gland are one way that the central clock can regulate the peripheral organs. The SCN also regulates the peripheral clock through poorly understood neurohumoral mechanisms. SCN can drive feeding/fasting rhythms through the hypothalamus. Diet and feeding/fasting rhythms modulate cyclic fluctuations in the gut microbiome, which then create cyclic changes in secondary metabolite production, such as SCFAs and bile acids. Secondary metabolites and/or bacterial products are necessary for the maintenance of peripheral circadian rhythms. Feeding/fasting rhythms are powerful drivers of the hepatic circadian clock. The rhythmic expression of genes in the liver generates rhythmicity in the transcripts and activity of genes involved in critical metabolic processes such as glucose, lipid, and bile acid metabolism, nutrient homeostasis, autophagy, and ER stress. Metabolic syndrome occurs in the setting of central and peripheral circadian rhythm dyssynchrony. 1952 Saran et al Gastroenterology Vol. 158, No. 7
Figure 2. Overview of circadian clock machinery. Humans/primates are diurnal animals, and their feeding/active period oc- curs during the light hours. However, mice (and most rodents) are nocturnal, with the feeding/active period occurring during the dark hours. The cellular molecular clock is a series of transcription-translation feedback loops that include the transcriptional activators (CLOCK and BMAL1; the positive limb) and repressors (PER and CRY; the negative limb). The PER:CRY complex repress their own activa- tors of CLOCK:BMAL1. CLOCK:BMAL1 also pro- motes REV-ERBs and RORs, which add another layer of regulation over the circadian clock and regu- late hundreds of additional genes. multiple layers of regulation from metabolic regulators and NAFLD is well described and makes these pathways a the circadian clock. target for therapeutic interventions.50–52 SIRT1, an anti-aging and nutrient-sensing protein, can up-regulate the expression of a number of genes involved Circadian Clock and Glucose Homeostasis in autophagy and energy metabolism by binding to the E- Glucose metabolism is regulated by the circadian clock box elements in a complex with the CLOCK:BMAL1 heter- machinery. The relationship between circadian changes in odimer.45,46 This complex in particular creates daily os- metabolically important hormones (ie, glucocorticoids, in- cillations in nicotinamide phosphoribosyltransferase, sulin, ghrelin, incretins, adiponectin, and leptin) and which is the rate-limiting enzyme in the nicotinamide ambient light provided the initial clues that the central clock þ adenine dinucleotide (NAD ) salvage pathway, thus plays an important role in glucose and nutrient homeosta- inducing circadian oscillation of intracellular NADþ.47,48 sis.53,54 Moreover, these hormones can directly affect the SIRT1canfurtheractoncircadianclockproteinsby circadian clock in their target cells. Insulin is a regulator of deacetylating BMAL1 and PER2, as well as other key Per2 and Rev-erba, and both glucose and insulin can affect metabolic regulators, through its regulation of NADþ.48 the expression of Dec1 and Dec2.55,56 Low glucose/fasting SIRT1 histone deacetylation leads to a repressive chro- induction of AMPK can repress the cryptochromes, as matin configuration, essentially down-regulating their described in the previous section. Thus, the relationship downstream targets.49 As a result of SIRT1 interaction with between the circadian clock and glucose homeostasis is CLOCK:BMAL1 and deacetylation of key regulator proteins, bidirectional. these genes have day-night rhythmicity to their transcrip- Of particular interest, incretins, which are already the tional activity and temporal regulation of their target target of pharmaceuticals approved by the US Food and genes. Thus, the role of autophagy in the pathogenesis of Drug Administration aimed to treat type 2 diabetes (T2D) May 2020 Circadian Rhythms and NAFLD 1953
Figure 3. Circadian clock machinery. (A) In mammals, the core molecular clock comprises a series of transcription-translation feedback loops that include the transcriptional activators (CLOCK and BMAL1). The CLOCK:BMAL1 heterodimer regulates genes at a specific DNA binding sequence, the E-box, to regulate expression of hundreds of genes, including their own re- pressors (Per1, Per2, Per3, Cry1, and Cry2). Once translated, PERs and CRYs accumulate in the cytoplasm, where they are regulated by CK1ε/d and AMPK, respectively. If these PERs or CRYs are individually phosphorylated, they will undergo ubiquitylation (Ub) and proteasomal degradation. However, if PER and CRY form a heterodimer before phosphorylation by CK1ε/d, the 3-protein complex is transported to the nucleus, where it directly inhibits the CLOCK:BMAL1 heterodimer. (B) CLOCK:BMAL1 also regulates the expression of REV-ERBs and RORs. Once translated, REV-ERBs and RORs bind to the RORE DNA binding sequence, but with opposite effects. RORs promote, whereas REV-ERBs suppress, at this DNA binding sequence. Together, these clock proteins control the regulation of hundreds of genes, including the expression of Bmal1.(C)A third loop of the circadian clock involves DEC1 and DEC2, which are transcriptionally activated by CLOCK:BMAL1. DEC1 and DEC2 proteins migrate back into the nucleus and inhibit Per1 transactivation by competing for E-box binding. (D) CLOCK:- BMAL1 promote the transcription of Dbp, and REV-ERB/ROR regulate the expression of Nfil3. Once translated, DBP and NFIL3 promote or suppress, respectively, gene expression at the D-box DNA binding sequence. The D-box DNA binding sequence controls the expression of hundreds of clock-controlled genes. 1954 Saran et al Gastroenterology Vol. 158, No. 7 and obesity, are being investigated as therapeutic agents in therapeutic agents targeting circadian clock components as NAFLD.57 Glucagon-like peptide 1 (GLP-1), an incretin potential therapeutic targets for T2D and NAFLD. secreted by the intestinal L cells located primarily in the ileum, is an insulin sensitizer.58 GLP-1 release correlates Circadian Clock and Lipid and Bile Acid with the pattern of insulin secretion, showing an increase Homeostasis immediately preceding the feeding period. Thus, GLP-1 In addition to glucose homeostasis, the circadian clock contributes to the decrease in blood sugar levels by regulates lipid and bile acid metabolism. This includes reg- enhancing glucose-dependent insulin secretion.57,59 Patients ulatory transcription factors such as peroxisome with NAFLD display diminished concentrations of active proliferator-activated receptors (PPARs) and sterol regula- incretins.60–62 Release of GLP-1 is circadian in rodents59 tory element-binding proteins (SREBP) 1c (lipid metabolism) and humans,63 and this rhythmicity is lost with the con- or rate-limiting enzymes such as acetyl-CoA carboxylase sumption of a high fat/palmitate diet64,65 and sleep depri- (ACC) (fatty acid biosynthesis) and cholesterol 7 alpha- vation.66 Knockdown of Bmal1 or Clock results in hydroxylase (CYP7A1) (cholesterol and bile acid meta- impairment of insulin release from b-cells in response to bolism). Dysfunction in these regulatory pathways is part of exendin-4.67,68 Thus, circadian clock control of glucose- the pathophysiology of NAFLD. modifying hormones is not limited to the hypothalamic- One of the main reasons for lipid accumulation in the pituitary axis. hepatocytes of patients with NAFLD is dysregulation of de Circadian clock machinery regulates glucose at a cellular novo lipogenesis. SREBP1c is one of several transcription level as well. Glucose homeostasis is altered in nearly all factors that can drive the induction of lipogenic genes. transgenic lines where the circadian clock is perturbed Overexpression of SREBP1c in the liver is associated with a (Table 1).12 The liver-specific Bmal1-knockout (KO) mice 2-fold increase in alanine aminotransferase, aspartate helped elucidate the relationship between circadian clock aminotransferase, hepatic triglyceride levels, and serum – and GLUT2, the main hepatic glucose transporter.69 71 The free fatty acids.78 Transgenic mice that overexpress expression of Glut2 in mice is characterized by the peak and SREBP1c displayed a fatty liver phenotype along with hy- trough of expression corresponding to the inactive/daytime perglycemia and hyperinsulinemia.78 The activity of this and active/nighttime states, respectively. This homeostatic regulator is dependent on the fasting-feeding cycle and stage is disrupted with loss of Bmal1, which results in nutritional status.79 However, more recent studies show constitutively low expression of both transcript and protein that SREBP1c, and its targets, are also regulated by the levels of Glut2. Hepatocyte-specific deletion of Bmal1 in circadian clock machinery. SREBP1c transcripts, as well as mice led to fasting hypoglycemia, hypoinsulinemia, and loss protein levels, display daily rhythmicity.80 This rhythmicity of rhythmicity in the expression of hepatic glucose meta- is also reflected in the binding pattern of SREBP1c to some bolic genes.69–71 of its canonical target genes.81 SREBP1c is regulated by The influence of the circadian clock is not limited to BMAL1, REV-ERBa,RORa,RORg,andDEC1.80,82,83 For glucose transport. BMAL1 also regulates the expression of example, REV-ERBa can control hepatic cholesterol and Pgc1a (peroxisome proliferator-activated receptor-gamma lipid metabolism through the controlled expression of coactivator 1a), a coactivator of gluconeogenesis.72 Hepatic Insig2 (insulin-induced gene 2) which is an inhibitor of glycogenesis is controlled by CLOCK through transcriptional SREBP1c.83 activation of glycogen synthase 2 (Gys2), the rate-limiting Cyp7a1, the gene for the rate-limiting enzyme that con- enzyme in glycogen synthesis.70 The increased expression verts cholesterol to bile acids, is also regulated by the of Gys2 is temporally timed with the surge of postprandial circadian clock and expressed cyclically. REV-ERBa84 and glucose. Clock-mutant mice display severely disturbed DBP85 regulate its transcription. Per1/Per2 double-KO mice feeding rhythms and glucose dysregulation (ie, hyperglyce- have abnormal expression of Cyp7a1 and other key bile acid mia, hypoinsulinemia).71,73 In addition, negative mutations enzymes, suggesting that they have at least indirect regu- in Clock and Bmal1 lead to impaired gluconeogenesis.74 lation of bile acid synthesis.86 Moreover, Cyp7a1 transcrip- Per1/Per2 double-KO mice have severe hypoglycemia in tion is regulated by hepatic farnesoid X receptor (FXR) and the fasting/inactive period and impaired glucose toler- small heterodimer partner (SHP), as well as fibroblast ance.69 Cry1/Cry2 double-KO mice also show glucose intol- growth factor (FGF) 15/19, which is released as a result of erance with elevated circulating corticosterone levels.75 FXR activation in the ileum. Fxr and Shp transcription (and CRYs are transcriptional repressors that regulate gluco- thus, also transcription of Fgf15/19) are also clock- neogenic enzymes through the repression of glucocorticoid gated.87,88 FXR agonists, such as obeticholic acid, are receptors75 and CREB.39,76 currently being investigated as potential therapeutic agents Not all circadian clock transgenic mice have unfavorable for NAFLD.89 Finally, it should be noted that BMAL1 can glucose homeostatic responses. Mice with liver-specificKO further influence triglyceride metabolism through PGC1a of Rorg are protected against insulin resistance and hy- regulation of Fxr, further showing the circadian clock’s perglycemia.77 Incidentally, Rorg-KO mice are also pro- multiple layers of regulatory control.90 Altogether, the cyclic tected against diet-induced hepatic steatosis.35 Likewise, activity of CYP7A1 and its regulating enzymes generates mice with hepatic overexpression of Cry1 had normoglyce- circadian fluctuation of serum and fecal bile acids, which has mia and improved insulin sensitivity in response to dietary implications for lipid absorption and microbiome challenge.39 These findings have bolstered the argument for composition.91,92 Table 1.Circadian Clock Genetically Modified Lines and Their Effects on NAFLD 2020 May
D Serum Mouse strain Diet/intervention Liver phenotype D Glucose D Insulin triglycerides D Weight D Adiposity References
Rev-erba KO þ Hepatic NCD Marked hepatic steatosis with deficiency of both Y 177 Reverb b KO Rev-erbs, but only subtle changes with loss of either subtype alone
Rev-erba KO NCD Higher hepatic TG levels (trend) [[ 4[ 178 Normal ITT HFD (53% fat) Higher hepatic TG levels (trend) [[ [[
Per1/2 KO NCD Lower hepatic TG levels, depending on what time 179 of day they were eating (oscillating)
Cry1/2 double KO NCD [4 Y 180 HFD (45% fat) Hepatic steatosis Impaired GTT [ [[ Impaired ITT
Bmal1 KO NCD [Y 181 HFD (60% fat) Hepatic steatosis in global (not tissue specific) [YY Bmal1-KO mice, but not in control mice
ClockD19 KO NCD [4[[[ 73 HFD (45% fat) Lipid engorgement of hepatocytes in Clock-KO [[ mice compared with WT
ClockD19 KO NCD Normal 4 182 HFD Lower hepatic TGs 4