Non-alcoholic fatty liver disease: the role of nuclear receptors and circadian rhythmicity
Division of Internal Medicine and Chronobiology Unit, Department of Medical Sciences, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy
Gianluigi Mazzoccoli, MD, Division of Internal Medicine and Chronobiology Unit, Department of Medical Sciences, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza”, Opera di Padre Pio da Pietrelcina, Cappuccini Avenue, San Giovanni Rotondo (FG) 71013, Italy
Division of Internal Medicine and Chronobiology Unit, Department of Medical Sciences, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy
Division of Medicine, Institute for Liver and Digestive Health, University College London, London, UK
EuroMEditerranean Institute of Science and Technology (IEMEST), Palermo, Italy
Division of Internal Medicine and Chronobiology Unit, Department of Medical Sciences, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy
Non-alcoholic fatty liver disease (NAFLD) is the accumulation of triglycerides in the hepatocytes in the absence of excess alcohol intake, and is caused by an imbalance between hepatic synthesis and breakdown of fats, as well as fatty acid storage and disposal. Liver metabolic pathways are driven 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 feeding/activity during wake times, and catabolic processes characterizing fasting/resting during sleep. A number of nuclear receptors in the liver are expressed rhythmically, bind hormones and metabolites, sense energy flux and expenditure, and connect the metabolic pathways to the molecular clockwork throughout the 24-h day. In this review, we describe the role played by the nuclear receptors in the genesis of NAFLD in relationship with the circadian clock circuitry.
Non-alcoholic fatty liver disease (NAFLD) is a form of ectopic lipid amassing, deriving from the accumulation at or above 5% of liver weight of triglycerides (TGs), consisting of a glycerol molecule scaffolding three molecules of fatty acid (FA), in the absence of excess alcohol intake (<20 g/day) [1, 2]. NAFLD is the most common chronic liver disease, with an incidence even superior with respect to that of infection with hepatitis B or C viruses and liver damage from alcohol abuse, and is often associated with metabolic disorders like obesity, metabolic syndrome, type 2 diabetes and cardiovascular disease [3, 4]. The histological classification distinguishes a range varying from hepatic steatosis to non-alcoholic steatohepatitis (NASH), which is characterized by the presence of inflammatory infiltrate and/or fibrotic tissue, and may evolve to cirrhosis, liver failure and hepatocellular carcinoma . The ‘two-hit hypothesis’ proposes that the progression of steatosis to NASH takes place through a predisposing factor represented by hepatic fat accumulation (first hit), and successively through oxidative stress, cytokines, bacterial endotoxin or endoplasmic reticulum (ER) stress representing additional insults (second hit) . In the past 30 years, prevalence numbers for NAFLD have progressively increased worldwide, as a result of the global epidemic of obesity, with resultant insulin resistance (IR), metabolic syndrome and diabetes mellitus, such that up to 30% of the general population in affluent countries have some form of NAFLD, and up to 75% of patients with obesity and diabetes mellitus have NAFLD [6-11].
NAFLD is a consequence of an imbalance between FAs supply, formation, utilization through mitochondrial β-oxidation or production of ketone bodies, and disposal through secretion of TGs in very low-density lipoprotein (VLDL) particles. The liver is a central player in the body's system that manages FA and TG metabolism, and synthesizes, stores, secretes and oxidizes free FA (FFAs). The liver handles FAs deriving from ingested foods, from adipose stores, and from its own de novo production, and precisely diet accounts for about 15%, mobilization of FAs from the adipose tissue for approximately 60%, and de novo lipogenesis for about 25% of the triacylglycerol amount present in the liver . A leading mechanism that provides FAs is the absorption of dietary fats, and in the postprandial phase the pancreas releases insulin, which increases lipogenesis and decreases lipolysis and FA mitochondrial β-oxidation. In the intestinal epithelial cells, FFAs and monoglycerides are absorbed separately, packaged into TGs, and then secreted in lipoproteins with a very high lipid content, called chylomicrons, which upon action of lipoprotein lipase release FFAs to adipose and muscle cells. Chylomicrons depleted of most lipids, defined as remnants, are absorbed by the liver . Another source and the main storage for FAs in humans is adipose tissue. In healthy individuals, fasting induces lipolysis and causes TGs to be released into the plasma pool of non-esterified FAs (NEFAs), whereas during feeding, FAs are got and stored by adipocytes. TGs are incorporated into VLDL particles while being transported out of the liver to peripheral tissues . Adipose tissue, especially visceral adipocytes, functions as a depot for energy that can be released in times of need, and the storage of TGs and FFAs in adipose tissue is mediated by insulin. In healthy individuals, meal eating brings on an augment in plasma insulin concentration and successive suppression of adipocyte lipolysis, with reduction in the plasma NEFA pool. Long-lasting surplus dietary energy intake causes IR, reducing inhibitory effects of insulin on peripheral lipolysis and increasing availability of FFAs, which are released in the bloodstream by visceral adipocytes and flow to the liver . The third source that contributes total supply of FAs to the liver is hepatic de novo lipogenesis, id est the synthesis of FAs from non-fat precursors such as glucose, amino acids and ethanol, which produce acetyl-CoA during their catabolism and can be converted to FAs in the intermediary metabolism. In healthy individuals, this source is a minor contributor while fasting and insulin levels are low . On the other hand, in a fasting state, oxidation of FFAs provides the major energy source for gluconeogenesis. De novo liponeogenesis is an important source of lipids, and the transcription factor sterol regulatory element-binding protein (SREBP)-1c is a key player in the regulation of this process. SREBP-1c is activated by insulin, liver X receptor (LXR)-α, oxysterol-binding protein and suppressor of cytokine signalling (SOCS) 3, whereas leptin and glucagon have opposing effects [15-17]. LXR-α is an oxysterol-activated nuclear receptor (NR) that upon activation induces SREBP-1c transcription through retinoid X receptor (RXR)-α co-activation . On its side, SOCS3 is an adipocyte-excreted cytokine that upregulates hepatic SREBP-1c; tumour necrosis factor (TNF)-α, interleukin (IL)-6 and leptin seem to augment excretion of SOCS3, whereas adiponectin is found to have inhibitory effects .
As regards FA metabolism, in healthy conditions, FFAs are taken up by the mitochondria, which utilize them as a substrate for β-oxidation, and FA oxidation is the main substrate for the production of energy used in gluconeogenesis . In the presence of IR, the quantity of FFAs available for oxidation exceeds the mitochondrial capacity; a bulk of acetyl-CoA enters the citric acid cycle, leading to delivery of electrons to the respiratory chain and production of reactive oxygen species (ROS). Besides, in physiological conditions, transport of TG from hepatocytes occurs through formation of VLDL by the ER in two steps. The microsomal triglyceride transfer protein (MTP), a chaperone indispensable for lipid assembly, catalyses the first step causing lipidation of apolipoprotein (Apo) B and arrangement of so-called pre-VLDL . The pre-VLDL is transported to the smooth ER and additionally lipidated before its migration to the cell membrane once more. The amount of TGs available determines the progression of ApoB to pre-VLDL, and the ApoB protein will degrade if available lipids are lacking. Insulin is a strong promoter of ApoB degradation via the phosphoinositol 3-kinase (PI3K) pathway and can thus influence the number of VLDL particles synthesized . SREBP-1c inhibits the formation of MTTP, thereby reducing the amount of VLDL particles produced. In IR states, signalling of the PI3K pathway is somehow reduced, but the increased insulin levels stimulate the transcription factor SREBP-1c, leading to a decrease in VLDL synthesis, as well as LXR, leading to increased transcription of lipogenic genes. The particle size depends on the amount of TGs stored in the cell, and it has been reported that in fatty livers, VLDL particles are sufficiently larger, most likely as a result of the reduced production, and probably TG export is decreased in IR states .
The circadian timing system and the hepatic metabolic pathways
In healthy individuals in the fasting state, occurring customarily during the night and characterized by low insulin levels, de novo lipogenesis is a minor supplier of FAs to the hepatocyte, accounting for <5% of the total contribute of FAs. In the feeding state, occurring normally during the day and characterized by high insulin levels, insulin stimulates de novo lipogenesis, supplying in this condition approximately a quarter of the FFAs, as reported above. This nycthemeral rhythm determined by the sleep/wake and fasting/feeding cycle is absent in NAFLD patients, where the role of de novo lipogenesis is constantly relevant . The coordinated regulation of sleep/wake, rest/activity, fasting/feeding and catabolic/anabolic cycles according to circadian rhythmicity (recurring with a frequency characterized by a period of 24 ± 4 h) is crucial for the preservation of optimal health [21-23]. In mammals, the daily timekeeping is driven by the biological clocks of the circadian timing system, composed of molecular oscillators in a master pace-maker within the suprachiasmatic nuclei (SCN) of the hypothalamus as well as in every body organ. The SCN pace the self-sustained and cell-autonomous molecular oscillators in the peripheral tissues through neural outputs, by means of autonomic nervous system fibres reaching target organs, and humoral signals, through the release of circulating systemic factors, such as melatonin and glucocorticoids . The SCN are entrained by the photic inputs deriving from the geophysical light/dark alternation related to Earth's rotation around its axis, perceived by melanopsin containing retinal ganglion cells and conveyed by the fibres of the retino-hypothalamic tract, which use the excitatory amino acid glutamate as major neurotransmitter . The signals transmitted by sympathetic and parasympathetic nerve fibres targeting peripheral tissues are essential for the entrainment of peripheral oscillators to changes of the environmental cues . Besides, melatonin is synthesized by the pineal gland in both diurnal and nocturnal mammals only during the night, and feeds back on the SCN through high-affinity receptors [27, 28]. In diurnal mammals, including humans, cortisol secretion begins in the middle of the night and reaches the zenith in the early morning, whereas in nocturnal animals, such as rodents, cortisol is secreted during the activity phase. Consequently, the secretion of melatonin and cortisol occurs during two opposing halves of the day in diurnal animals, whereas in nocturnal animals, these hormones are secreted during the same phase with respect to the light–dark cycle [29-31].
The SCN are anatomically connected to brain regions involved in the regulation of appetite, energy expenditure and behavioural activity, such as arcuate nucleus, ventromedial, dorsomedial and lateral hypothalamic nuclei [32, 33], and are composed of approximately 15 000–20 000 neurons. These cells in the SCN autonomously show the aptitude to keep going fluctuations of biological processes, and this characteristic is present in more or less each cell in peripheral tissues [34-41]. The oscillatory function is driven by molecular clockworks operated by a set of so-called clock genes and their coded proteins, which generate transcriptional–translational feed-back loops (TTFLs) revolving rhythmically with a roughly 24-h period [42-44] (Fig. 1).
The TTFL is arranged by a positive limb, represented by the Period-Arnt-Single-minded and basic helix-loop-helix (PAS-bHLH) transcription factors CLOCK (circadian locomotor output cycles kaput), and its paralog NPAS2 (neuronal PAS domain protein 2), and by BMAL1-2/ARNTL-2 (brain and muscle aryl-hydrocarbon receptor nuclear translocator-like/aryl-hydrocarbon receptor nuclear translocator-like). These heterodimerize and bind to E-box (5′-CACGTG-3′) cis-regulatory enhancer sequences of their target genes Period (Per1-3) and Cryptochrome (Cry 1-2). The negative limb of the feed-back loop is represented by PER and CRY proteins, which dimerize forming a repressor complex and translocate back to the nucleus where hamper the transcriptional activity of CLOCK/BMAL1-2 [45, 46]. Another component of the molecular clockwork in the Drosophila melanogaster is represented by Timeless (Tim), which is conserved in mammals, and with TIMELESS interacting protein cooperates with the DNA replication system .
The amplitude of oscillation of many clock genes depends on the activity of SIRT1, a type III NAD+-dependent histone/protein deacetylase, which is a core component of the circadian clock and rhythmically deacetylates BMAL1, histone H3 and PER2, decreasing PER2 stability in a circadian way [48, 49]. SIRT1 activity is influenced by the oscillating levels of nicotinamide (NAM) adenine dinucleotide (NAD+), synthesized de novo from tryptophan through nicotinamide phosphoribosyltransferase (NAMPT/visfatin/pre-B-cell colony-enhancing factor), the rate-limiting enzyme driving the NAD+ salvage pathway, which shows circadian rhythmicity of expression [50-52]. NAD+/NADH ratio gauges cellular redox and metabolic status, and in vitro the circadian oscillation of NADH/NADPH directly modulates the DNA-binding activity of CLOCK:BMAL1 heterodimer . Accordingly, the circadian oscillations in cytoplasmic redox parameters (peroxiredoxin oxidation–reduction, haemoglobin tetramerdimer transitions and NADH/NADPH oscillations) link metabolic processes in the cytoplasm and transcriptional processes in the nucleus . In turn, NAD+ drives NAMPT expression in a circadian manner by SIRT1 activation, and through recruitment of CLOCK:BMAL1 heterodimer to the Nampt promoter [55, 56]. Besides, NAD+ and adenosine monophosphate (AMP) are cellular indicators of low energy status, and AMP-activated kinase (AMPK) plays a role in this circuit modulating the balance NAD+/NADH through the activation of NAMPT [57, 58], and works as a nutrient sensor activated by exercise, fasting or hypoxia to restore energy balance [59, 60].
The activity of SIRT1 is inhibited by protein–protein interaction with deleted in breast cancer–1 (DBC1), which regulates SIRT1 activity in metabolically active tissues, and in particular in hepatic cells . When feeding high-calorie diets, DBC1 binds to SIRT1 and inhibits its deacetylase activity, whereas upon starvation or low-calorie diets remains unbound and props up SIRT1 activity .
A process that plays an important role in the function of the clock gene machinery is represented by the post-translational modification, such as phosphorylation, acetylation, sumoylation and ubiquitylation, which influence transcriptional activity and intracellular localization of circadian proteins [63, 64]. In particular, Casein kinases 1-δ and 1-ε (CK1δ and CK1ε) target the PER and CRY proteins and mark them for polyubiquitylation by, respectively, the E3 ubiquitin ligase complex β-TrCP1 (β-transducin repeat containing protein 1) and SCF/Fbxl3 ubiquitin ligase complex (Skp1, Cullin1, F-box and leucine-rich repeat protein 3) [65, 66]. BMAL1 is phosphorilated by the AKT-GSK3β system, and in the absence of GSK3β BMAL1 becomes stabilized, decreasing the dependent circadian gene expression . Besides, AMPK marks the CRY proteins for degradation through the 26S proteasome by means of the SCF/Fbxl3 ubiquitin ligase complex, and also this mechanism could underlay the interplay between metabolic and circadian pathways .
A further regulatory mechanism in circadian systems is represented by the epigenetic regulation. CLOCK has histone acetyltransferase (HAT) activity and plays a role in protein acetylation and chromatin modification, binding to E-boxes with cyclic adenosine monophosphate-(cAMP) response element (CRE)-binding (CREB) protein (CREBBP or CBP)/p300, and acetylating histones H3 and H4 and BMAL1 [69, 70]. Histone methylation catalysed by methyltransferase MLL3 contributes to circadian transcription through whole-genome cycling of activating (H3K4me3) and inhibitory (H3K9me3) chromatin marks , and a role in the function of the circadian molecular oscillator is played also by histone lysine demethylation as well . Transcriptional activation in the clock gene machinery is controlled by a sequential process working through transcription factors association, coactivator complexes recruitment and gene promoters targeting to induce histone acetylation and chromatin remodelling. A decisive role in the rapid assembly of functional pre-initiation complexes at promoters of target genes is played by the direct association of CBP with RNA polymerase II. Histone H3 is preferably acetylated by CBP/p300-associated factor (PCAF), whereas histone H4 and histone H3 are substrates for CBP/p300 . The transcriptional coactivators and HATs CBP/p300, PCAF and ACTR associate with CLOCK and NPAS2 to regulate positively clock gene expression. In particular, NPAS2 recruits the chromatin remodelling machinery in a time-qualified manner and leads to marked acetylation of N-terminal lysine residues on histone H3 surrounding the proximal E-box on the Per1 promoter, a phenomenon that takes place before peak expression of target genes, whereas interruption of HAT-associated complexes on core clock heterodimers induces CRY2-mediated repression .
CLOCK/BMAL1 heterodimer activates the transcription of the NRs REV-ERB (reverse transcript of erythroblastosis gene) α/β and retinoic acid-related (RAR) orphan receptor (ROR) α, β/δ, γ, which operate an additional regulatory loop controlling BMAL1 expression [75, 76]. REV-ERBα and REV-ERBβ are unable to recruit coactivators and activate target gene transcription, bind ROR-specific response elements (RORE) in Bmal1, Clock and Cry1 promoters, and obstruct binding of the positive transcription regulator RORα [77-79]. RORα physically interacts with the transcriptional coactivator peroxisome proliferator-activated receptor (PPAR)γ coactivator-1α (PGC-1α), which recruits chromatin-remodelling complexes to the proximal Bmal1 promoter and activates Bmal1 transcription. On the other hand, REV-ERBα interacts with the complex formed by histone deacetylase 3 (HDAC3) and nuclear receptor corepressor 1 (NCOR1), which is a bulky, multidomain protein recruited by NRs to mediate transcriptional repression, and functions as an activating subunit for HDAC3 . Consequently, Bmal1 expression is rhythmically driven by the dichotomous recruitment of the RORα/PGC-1α activator complex and the REV-ERBα/NCOR1-HDAC3 repressor complex . HDAC3 binds to the mouse liver genome in a circadian fashion and regulates the expression of genes controlling hepatic lipid homeostasis, whose deregulation causes hepatic fat accumulation in vivo . Besides, in mice, interruption of the interplay between HDAC3 and NCOR1 provokes the alteration of clock gene expression and changes in the circadian behaviour, with increased energy expenditure and increased insulin sensitivity . A physiological ligand of REV-ERBα/β is represented by haeme, which binds to the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry augmenting the thermal stability of the proteins, and is synthesized by a pathway regulated by the molecular clockwork through the rate-limiting enzyme delta-aminolevulinate synthase 1 (ALAS1). Haeme binding to REV-ERB causes the recruitment of the co-repressor NCOR1, leading to repression of target genes including Bmal1, whereas haeme dissociation has been shown to derepress the expression of target genes in response to changes in intracellular redox conditions [83-85]. Addition of nitric oxide (NO) reverses transcription repression mediated by haeme-bound REV-ERBs, rendering REV-ERBs highly dynamic receptors responsive also to redox state and gases . Besides, the interaction between haeme and the ligand-binding domain of REV-ERBβ is controlled by a thiol-disulfide redox switch, so that the reduced dithiol state of REV-ERBβ binds haeme five-fold more tightly than the oxidized disulfide state, whereas no effect on haeme binding to the ligand-binding domain of the protein is induced by the changes in the redox state of iron . Furthermore, NPAS2 has a haeme-binding motif, and haeme controls activity of the BMAL1-NPAS2 transcription complex in vitro by inhibiting DNA binding in response to carbon monoxide (CO) .
The clock gene machinery drives the expression of thousands of clock controlled and tissue-specific output genes, which steer cell processes and tissue functions . Above all, CLOCK:BMAL1 heterodimer binds E-boxes in the promoters of proline- and acidic amino acid-rich domain basic leucine zipper (PAR-bZIP) transcription factors, including DBP (albumin gene D-site-binding protein), TEF (thyrotroph embryonic factor), HLF (hepatic leukaemia factor), which in turn direct the expression of downstream genes . In particular, DBP feeds back on the molecular clockwork activating Per1 transcription . On the other hand, REV-ERBs binding to RORE activate the expression of the Nuclear factor interleukin-3 regulated protein (NFIL-3, also known as adenoviral E4 protein-binding protein, E4BP4), strictly related to DBP, but oscillating in an opposite phase with respect to DBP, so that these transcription factors drive the expression of genes covering different time spans in the 24-h day [92-94].
Transcriptional repression/regulation of multiple genes, including downstream circadian output genes, is operated by the bHLH transcription factors differentially expressed in chondrocytes protein 1 (DEC1) and 2 (DEC2) . In the liver, DEC1, whose transcription is activated by CLOCK:BMAL1, in turn represses CLOCK:BMAL1 activity, so that the peak of CLOCK:BMAL1 transcriptional activity corresponds to maximal levels of Dec1 mRNA .
The NRs link the biological clock to the metabolic pathways in the liver
A crucial mechanism to preserve the synchronization of organismal functions with environmental cues is represented by the daily entrainment to light/darkness cycle [97, 98], but the liver plays a key role in the coordination of metabolic processes to daily feeding/fasting cycles . The molecular clockworks in the liver can be entrained by feeding, whereas brain oscillators are not influenced by food-related signals and are entrained primarily by day/night alternation, which in turn acts through the brain clocks to control feeding behaviour and mealtimes .
The circadian systems are endangered by changes in energy supply and metabolic status, and desynchronization of food intake in respect of customary nycthemeral cycles of rest/activity, sleep/wake and fasting/feeding hampers the coupling of metabolic processes with the light-driven neural and humoral outputs from the SCN, which work as ‘light entrainable oscillators’. The organism has the capability to anticipate food availability and manage behavioural rhythms when regular meal schedules become the dominant entraining stimulus through hypothalamic, corticolimbic and brainstem structures, as well as adrenals. This system is defined ‘food-entrainable oscillators’, comprises metabolic hormones such as ghrelin and leptin, and drives the ‘food anticipatory activity’ before a timed meal, consisting in arousal augment, food-seeking behaviours activation and synthesis/secretion of digestive enzymes [101-103].
Time-related changes in gene expression are crucial in tissues implicated in glucose and lipid metabolism, for instance the liver . The nutritional status is constantly gauged by the transcriptional networks that control glucose and lipid metabolism, capable to react to varied physiological signals, so that an almost continuous fine-tuning of metabolic gene expression assures energy and nutrient homeostasis at both the cellular and organismal level . In mammals, meal timing drives metabolic activity and clock phase in peripheral tissues [106, 107], and restricted feeding resets the phase of clock gene expression in oscillators located outside the brain [108, 109], but clock gene entrainment by feeding in the various organs shows different grade and speed of reaction to nutritional signals [110-112]. In particular, microarray studies evaluating gene expression profiles throughout the circadian cycle have evidenced that in the liver, approximately 14% of cyclic transcripts depend on systemic signals and 86% depend on local oscillators. Among the liver genes that are systemically regulated, an important role is played by immediate early genes (IEGs), which communicate rhythmic signals to the hepatic molecular clockworks and are involved in the synchronization of liver clocks . Systemic circadian signals drive the IEG class, including several heat shock protein genes regulated by heat shock transcription factor 1 (HSF1), and target genes of serum response factor 1. On the other hand, in the liver Per2 transcription may be activated by the postprandial rise in body temperature through binding of HSF1 to the cis-acting element in the promoter of the circadian gene [113-115]. Interestingly, the expression of numerous hepatic transcripts that are targets of metabolic and stress regulators [CREB, SREBP, ATF6 and forkhead box protein O1(FOXO1)] is driven by feeding/fasting cycles even in the absence of a functional clock .
A crucial role in the connection between the metabolic pathways and the circadian clock circuitry is played by the NRs, which bind hormones, such as cortisol, melatonin, 3,5,3′-triiodothyronine and metabolites, such as lipids, oxysterols, haeme, bile acids, and gauge the nutritional status and redox balance. The NRs are ligand-dependent transcription factors sharing comparable domain organizations, and in particular, the DNA-binding domain and the ligand-binding domain play a decisive role in magnifying hormone and metabolite signalling via receptor target genes. Ligand binding to the specific receptor induces ligand-induced conformational changes in the receptor, receptor translocation to the nucleus, receptor dimerization, interaction with target gene promoter elements, release/recruitment of coactivators or corepressors, chromatin remodelling, and ultimately interaction with the polymerase II complex to initiate transcription .
PTMs, such as phosphorylation, ubiquitination, SUMOylation, O-GlcNAcylation and acetylation, modulate the functions of NRs . In turn, PER2 is capable of binding rhythmically to several NRs influencing their transcriptional activity . A number of NRs show circadian variations in metabolically active tissues , and ligand binding leads to the recruitment of co-activators and co-repressors, which modulate the control exerted by NRs on gene transcription, and in due course on glucose, lipid and mitochondrial oxidative metabolism .
Important nutrient sensors linking circadian and metabolic pathways are represented by the NRs PPAR α, β/δ and γ, whose expression is controlled by the clock gene machinery.
PPARα binds FA, reduces circulating TGs by up-regulation of FA catabolism in the liver and the ligand-related activation of PPARα induces the transcription of genes-encoding proteins involved in peroxisomal and mitochondrial FA β-oxidation and in the uptake and/or metabolism of lipids, cholesterol and glucose [120, 121]. Heterodimeric association with RXR-α is necessary for the transcriptional activity of PPARα on genes controlling FA β-oxidation. RXRs mediate the biological effects of retinoids in gene activation joining all-trans or 9-cis retinoic acid, homodimerizing as RXR/RXR or heterodimerizing as RXR/RAR and binding to retinoic acid response elements in the promoters of target genes. PPARα cross-talks with LXR in the regulation of lipogenesis, and even if PPARα is essential for FA catabolism in nutrient deprivation, whereas upon pharmacological activation, LXR leads to the overexpression of genes involved in de novo lipogenesis in the liver; there is evidence of an interplay between these NRs controlling hepatic lipid metabolism in response to oxysterol and FAs .
PPARδ/β controls energy substrate homeostasis through harmonized regulation of glucose and FA metabolism in the liver, regulates hepatic metabolic programmes through both direct and indirect transcriptional mechanisms partly mediated by its co-activator PPARγ co-activator-1β, and improves glucose management and insulin sensitivity upon pharmacological activation . Adenovirus-mediated liver-restricted PPARδ/β activation in chow- and high fat-fed mice lessens fasting glucose levels, along with hepatic glycogen and lipid deposition, in addition to up-regulation of glucose utilization and de novo lipogenesis pathways . PPARδ/β increases the production of the PPAR activators monounsaturated FAs, and conversely reduces saturated FAs synthesis. This PPARδ/β-regulated lipogenic programme may protect against lipotoxicity, considering that notwithstanding the increased lipid accumulation, adeno-PPARδ/β-infected livers show less damage and less jun-kinase (JNK) stress signalling. The glucose-lowering activity of PPARδ/β may also be related to a secondary effect on AMPK activation deriving from modified substrate utilization . The activities of hepatic de novo lipogenic gene products peak during feeding, converting carbohydrates into fats that are utilized by peripheral tissues as energy sources, and hepatic PPARδ/β controls the daily expression of lipogenic genes in line with nutrition and behaviour cycles. Lipid synthesis and utilization is coordinated in the liver–muscle axis by a regulatory mechanism relaying on the PPARδ-regulated lipogenic gene acetyl-CoA carboxylase 1 (ACC1), which catalyses the production of the serum lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and liver-specific deletion of either PPARδ/β or ACC1 reduces muscle FA uptake .
PPARγ heterodimerizes with the RXR and binds to PPAR-responsive elements in the regulatory region of target genes involved in various aspects of metabolism. This NR is abundantly expressed in the adipose tissue, which stimulates the differentiation of pre-adipocytes in mature fat cells, and enhances the activation of genes involved in lipogenesis and storage of TGs, controlling adipogenesis and fat storage. Besides, PPARγ is an important regulator of glucose metabolism through its insulin sensitizing activity. Animal experiments using systemic PPARγ null mice have evidenced severe alteration of circadian phenotype and metabolic regulation, highlighting the key role played by PPARγ in the cross-talk between circadian and metabolic pathways [125, 126].
An important interplay between PPARS and the molecular clockwork has been highlighted: PER2 negatively regulates Pparg expression and recruitment to target promoters ; PPARγ positively regulates Bmal1 transcription ; a mutual positive modulation of expression links PPARα and BMAL1 ; PER3 forms a complex with PPARγ and inhibits PPARγ-mediated transcriptional activation ; and SIRT1 binds to and represses PPARγ activity, promoting fat mobilization during fasting . DBP, HLF and TEF drive the rhythmic expression and activity of PPARα , and in the liver, these PAR-bZIP transcription factors contribute to the circadian transcription of genes coding for acyl-CoA thioesterases, leading to a cyclic release of FAs from thioesters. Moreover, Pparg is rhythmically expressed and its circadian oscillation is driven by binding of DBP and E4BP4 to first exon D-sites with functional promoter activity .
A key role in the cross-talk between the circadian clock circuitry and the metabolic pathways is played by the transcription coactivators of PGC-1 family, which control gene expression recruiting specific NRs, transcription factors and chromatin-remodelling enzymatic complexes [105, 134]. PGC-1α modulates hepatic gluconeogenesis, FA β-oxidation, mitochondrial biogenesis and haeme biosynthesis, and sensing nutritional and circadian signals communicates extracellular inputs to the biological clock [105, 135].
PGC-1α interacts with PPARα and PPARβ/δ for FA oxidation, PPARγ for induction of uncoupling protein-1 (also known as thermogenin), farnesoid X receptor (FXR) for TG metabolism, LXR for lipoprotein secretion, hepatic nuclear factor-4α (HNF-4α), glucocorticoid receptor (GR or GCR) and FOXO1 for gluconeogenic gene expression, nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), PPARα, PPARβ/δ and oestrogen-related receptor (ERR)-α for mitochondrial biogenesis, thyroid hormone receptor (TR) for induction of carnitine palmitoyltransferase-I [105, 136-138]. Upon binding to transcription factors, PGC-1α induces conformational change and increases the affinity of the transcription complex to additional coactivators with HAT activity, such as steroid receptor coactivator-1 (also called nuclear receptor coactivator 1, NCOA1) and CBP/p300, leading to acetylation of histone proteins, conformation alterations and increased accessibility of DNA to the transcription complex . Furthermore, PGC-1α interacts with chromatin-remodelling complexes, such as Mediator coactivator complex (TR-associated protein, vitamin D receptor interacting protein, SRB/MED-containing cofactor, negative regulator of activated transcription, NAT, activator-recruited cofactor, ARC, cofactor required for SP1 activation, CRSP), and SWItch/sucrose nonfermentable (SWI/SNF) chromatin-remodelling complex . PGC1α is deacetylated and activated by SIRT1 [140, 141], and plays an essential role in the induction of the liver in response to starvation , considering that severe impairment of metabolic adaptation in response to stresses is observed in Pgc1a null mice . PGC-1β is in turn regulated by FAs and cytokines, is involved in adipocyte differentiation, and stimulates TG synthesis and lipoprotein secretion in response to dietary fats in the liver .
In the liver, PGC-1α and PGC-1β phases are driven by the circadian clock circuitry through unknown signals, and PGC-1α peaks approximately 4 h after PGC-1β, suggesting control by circuits such as calcineurin A and calcium/calmodulin-dependent protein kinase, AMPK, CBP and NO [134, 143]. In turn, PGC-1α and PGC-1β are necessary for the functioning of the molecular clockwork, and in particular, PGC-1α coactivates RORα, is recruited to the RORE present on the proximal Bmal1 promoter, activates Bmal1 transcription and induces REV-ERBα expression, and in its side, PGC-1β may play compensatory functions [105, 134, 144].
An important role in the control of energy metabolism is played by ERRα (NR3B1), which modulates insulin sensitivity and nycthemeral glucose oscillation, and directs circadian and metabolic networks in the liver, where is expressed rhyhtmically. ERRα interacts with PGC-1α and the homeobox protein PROX1, which represses the transcriptional activity of the ERRα/PGC-1α complex on the promoters of metabolic genes, and hinders ERRα effects on the respiratory capacity of cultured hepatocytes .
Lipid metabolism is highly influenced by the level of thyroid hormones, and in particular of 3,5,3′-triiodothyronine, the natural ligand of TR α and β. A circadian pattern of variation characterizes the serum levels of free 3,5,3′-triiodothyronine [146, 147], TRα oscillates with circadian rhythmicity in the liver as well as in white and brown adipose tissue, whereas TRβ is expressed with 24-h periodicity in white adipose tissue . A large proportion of thyroid hormone-regulated genes are also regulated by LXR, and different but somehow overlapping metabolic pathways important for overall lipid homeostasis have been evidenced for TRs and LXRs, suggesting a physiological convergence of thyroid hormone and oxysterol signalling pathways .
In mouse liver, metabolic and circadian rhythms are coordinated by an NAD(+)-dependent ADP-ribosyltransferase, poly(ADP-ribose) polymerase 1 (PARP-1), which oscillates in a circadian manner and is regulated by feeding/fasting cycles with highest activity levels during fasting. In response to feeding, PARP-1 modifies components of the clock gene machinery, binding to CLOCK-BMAL1 heterodimers and poly(ADP-ribosyl)ating CLOCK, and modulates DNA-binding activity of CLOCK-BMAL1 and its interactions with PER and CRY proteins altering clock gene expression .
The vast range of circadian factors involved in the control of metabolism underlines the complexity and the temporal qualification of the regulatory mechanisms, and suggests that the interaction between circadian and metabolic pathways is fine-tuned to anticipate rhythmic modifications and match unespected changes in cellular and tissue milieu. The maintenance of body homeostasis and metabolic balance is accomplished through a timely harmonization among the oscillations of circadian transcripts, biological mediators, kinases, nutrient fluxes, transcriptional activators and repressors, cofactors, coactivators and corepressors, which keep going epigenetic modifications and transcriptional events ultimately impinging on the signalling pathways involved in the control of essential physiological phenomena, cellular functions, and in particular of metabolic processes especially in the liver (Fig. 2).
Metabolic and genetic circadian players in NAFLD pathogenesis
A critical mechanism in the genesis of hepatic steatosis is represented by the discrepancy between the processes of FAs storing/production and FAs removal through clearance or use, and a central regulatory role in the genesis of NAFLD is played by disruption of insulin signalling pathway, causing IR and altered response to this major anabolic hormone [150-153]. Upon binding to insulin, the insulin receptor is tyrosine phosphorylated, with subsequent tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins 1 and 2. IRS-1 is the initiator in the pathway of glucose metabolism, and upon phosphorylation induces stimulation of the PI3K/AKT/protein kinase B pathway, leading to recruitment of glucose transporters (GLUTs). On the contrary, alteration of IRS-1 by serine phosphorylation at Ser307/612/632 decreases IRS-1 tyrosine phosphorylation, thereby interrupting the pathway for the transport of glucose via the GLUT transporters to the membrane and contributing to onset of IR [154, 155]. IRS-2 activates lipid metabolism in the cell and is a main regulator of de novo lipogenesis via SREBP-1c. SREBP-1c is a member of the SREBP family, a group of transcription factors that play a fundamental role in cellular lipid metabolism and sets in motion the complete programme of cholesterol, TGs and FA synthesis in the liver [156, 157]. In particular, SREBP-1c takes part in lipid synthesis in the liver by stimulating the formation of enzymes essential for TGs synthesis, principally AAC and FA synthase (FAS), which are also regulated by carbohydrate response element-binding protein (ChREBP) [158, 159] (Fig. 3).
A great quantity of the enzymes involved in liver metabolism are localized in the membrane of the hepatocyte ER, a complex system of tubules and sacs supporting protein synthesis, folding, assembly, modification and transport. The ER of hepatocytes exhibits nycthemeral rhythmicity of function with circadian dilatation, and augmented work requested to ER leads to an imbalance between biological processes within the cell and its coping capacity determining ER stress . ER stress activates the unfolded protein response (UPR), a conserved adaptative response to manage the accumulation of unfolded or misfolded proteins in this subcellular structure. Lipid metabolism is associated with a low level of physiological UPR, and the circadian clock circuitry influences mouse hepatic lipid metabolism and ER-localized enzymes driving a 12-h period secondary rhythmic activation of the UPR pathway, with ultradian activation of UPR-regulated genes . ER stress is also a response to undue metabolic demand and high ER workload in obesity, and the activation of UPR may sustain inflammatory signalling pathways. Liver tissue is a major site for ectopic fat accumulation in obesity , and in hepatocytes, FFAs cause IR in genetically susceptible subjects through defects in the insulin signalling pathway, in particular of the IRS-1/PI3K/AKT/GLUT pathway. Many inflammatory kinases provoke inhibitory serine phosphorylation, such as IKB-kinase-β (IKK-β), JNK-1 and SOCS3. Above all, JNK-1 is an important mediator in the development of inflammation in the enlarged adipose tissue of obesity, and is triggered by ER stress with activation of the UPR, leading to activation of JNK-1, IKK-β and TNF-α, and converting the metabolic stress into an inflammatory signal [163-165].
Circadian oscillations characterize the transcriptional circuits that control the signalling pathways involved in lipid metabolism, as well as bile acid and Apo biosynthesis, leading to nychthemeral changes in lipid levels in the circulatory system. The molecular clockwork drives the expression of genes encoding the enzymatic and transport proteins managing lipogenesis and lipolysis, such as hepatic cytochrome P450 cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), FAS, lipolytic enzymes, apoA-IV and C-III, low-density lipoprotein receptor, FA transport protein 1 (FATP1), fatty acyl-CoA synthetase 1 and adipocyte differentiation-related protein [20, 166-169]. In turn, lipids, such as FAs, cholesterol, bile acids or their metabolites, feed back on the biological clock as ligands of NRs, bringing on the interplay between metabolic and circadian pathways. LXRs and FXRs bind and are activated, respectively, by oxysterols and bile acids , and drive positively the former and negatively the latter the circadian oscillation of CYP7A1, the rate-limiting enzyme in bile acid synthesis . The rhythmic expression of Cyp7a1 is driven also by REV-ERBα, DBP/E4BP4 and DEC2, which grant accurate regulation of the amplitude, phase and wave form of the circadian profile, operating, respectively, via Rev-RORE, DBP/E4BP4-binding elements and E-boxes . RORα binds cholesterol and its derivatives, such as 7-oxygenated sterol, varying its transcriptional activity , and upregulates the expression of a constituent of VLDLs, apoC-III [174, 175], which is down-regulated by REV-ERBα . Besides, REV-ERBα controls SREBP activity steering the nychtemeral variations of the enzyme insulin-induced gene (INSIG) 2, which holds in ER membranes the SREB cleavage-activating protein (SCAP)-INSIG-SREBP complex, a sensor of cholesterol availability . In this way, REV-ERBα controls the timing of cyclic accumulation of SREBP in the nucleus, which in turn regulates the temporal expression of Hmgcr . REV-ERBα influences also bile acid metabolism through oscillations of oxysterol synthesis and LXR activity, and cross-talks with FXR for the regulation of the small heterodimer partner (SHP), down-regulating hepatic SHP and E4BP4 expression and derepressing Cyp7a1 . Lipid metabolism is modulated by REV-ERBα also by means of epigenetic changes induced recruiting HDAC3 via the NCOR1 to lipid metabolic genes, with subsequent chromatin remodelling and histone modification . Genome-wide occupancy of HDAC3 is highly enriched on lipogenic genes, and low REV-ERBα levels reduce HDAC3 association with the liver genome during the activity/feeding time and permit lipid biosynthesis/amassing, while elevated REV-ERBα levels enhance HDAC3 recruitment to liver metabolic genes in the resting/fasting time, hindering lipid biosynthesis .
The circadian clock circuitry and NAFLD pathogenesis in animal models
The components of the molecular clockwork play a key role in the control of metabolic pathways and biochemical processes, and the genetic determinants related to circadian rhythmicity and involved in the pathogenesis of hepatic steatosis have been thoroughly explored through experimental studies performed using genetic animal models (Table 1).
Table 1. Mutations in circadian genes causing metabolic phenotypes and hepatic steatosis in animal models
REV-ERBα/HDAC3 deletion or misalignment between the rhythms of HDAC3 recruitment to target genes with behavioural patterns alters lipid metabolism causing hepatic steatosis. Adult mice with liver-specific Hdac3 knockout show severe hepatic steatosis associated with enhanced de novo lipogenesis and increased expression of lipogenic genes . Notwithstanding severe liver steatosis, mice with liver-specific depletion of Hdac3 are characterized by higher insulin sensitivity without modifications in insulin signalling or body weight compared with wild-type mice. Hdac3 depletion switches metabolic precursors en route for lipid synthesis and storage within lipid droplets and away from hepatic glucose production, increasing perilipin 2, which coats lipid droplets, and the sequestration of hepatic lipids in perilipin 2–coated droplets contributes to the development of both steatosis and improves IR . On the other hand, animal experiments in obese mice have evidenced that synthetic REV-ERBα/β ligands influence the expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue, augment energy expenditure, improve dyslipidaemia and hyperglycaemia, reduce obesity by decreasing fat mass, and are potentially useful for the treatment of metabolic disorders .
The importance of the role played in lipid metabolism by RORs and REV-ERBs is highlighted by the phenotypic and biohumoral changes that characterize transgenic animal models, such as the RORα mutant mouse (RORαsg/sg, Staggerer mouse), which is less susceptible to hepatic steatosis, has reduced body fat notwithstanding higher food consumption and is characterized by smaller fat cells in brown and white adipose tissue, and lower liver triglyceride levels . The Staggerer mouse is genetically characterized by decreased expression of the reverse cholesterol transporters Abca1 and Abca8/G1 in the liver and intestine, reduced expression of Cyp7b1, Srebp-1c and FASN in the liver, increased expression of PGC1α and β, hypo-α-lipoproteinaemia, lower levels of total plasma cholesterol, the high-density lipoprotein, ApoA-I, the major constituent of high-density lipoprotein, ApoC-III, ApoA-II and TGs, compared with wild-type mice when fed a normal diet . Besides, Staggerer mice are resistant to obesity and hepatic steatosis when fed a high-fat diet; anyway, RORαsg/sg mice develop severe atherosclerosis, suggesting that RORα may play an atheroprotective role [182-184].
On the contrary, Rev-erbα−/− mice show elevated VLDL triglyceride levels correlated with elevated levels of ApoC-III mRNA in the liver [176, 182]. Rev-erbβ knockout mice have not yet been produced, but a role played by this NR in lipid metabolism is suggested by decreased expression of several target genes involved in lipid metabolism, such as FA translocase (Fat/CD36), Fabp3, Fabp4, Ucp3, Srebp-1c and Scd-1, upon expression of a dominant negative form of REV-ERBβ lacking the ligand-binding domain . In addition, experiments with mice deficient for both NR subtypes specifically in the liver (Liver-Double Knockout) evidenced that depletion of both Rev-erbα and Rev-erbβ in the liver synergistically derepresses several metabolic genes and causes marked hepatic steatosis, in contrast to relatively subtle changes upon loss of either subtype alone .
Among the circadian proteins operating the positive limb of the TTFL, BMAL1 plays a key role in the control of fat storage and utilization, and its down-regulation reduces the expression of several key adipogenic/lipogenic factors, such as PPARγ, adipocyte FA-binding protein 2 (aP2), CCAAT/enhancer-binding protein (C/EBP)α, SREBP-1a, FAS, leading to diminished adipogenesis. Accordingly, BMAL1 up-regulation increases lipid synthesis activity in adipocytes , whereas in Bmal1 knockout mice, the capacity of fat storage in adipose tissue is reduced, and the increase in the levels of circulating FAs leads to the formation of ectopic fat in the liver. Anyway, tissue-specific (liver or skeletal muscle) Bmal1−/− mice even fed a high-fat diet did not show ectopic fat formation . In addition, hepatic steatosis, hypertriglyceridaemia and hyperglycaemia characterize ClockΔ19/Δ19 double-mutant mice, which show hyperphagia and increased food intake during the day, hyperleptinaemia, hypoinsulinaemia and obesity with metabolic syndrome .
Plasma lipid homeostasis is maintained through the equilibrium between lipoprotein catabolism and production, the latter influenced by apoB and MTP, encoded by the MTTP gene, a clock-controlled gene coding for a circadian transcript. CLOCK negatively regulates MTP expression , and in addition dietary fats, hormones (insulin, leptin, melanocortin), and transcription factors (HNF-4α, PPARα) control MTP in the intestine and liver, and in turn, daily variations in plasma lipid levels are driven by diurnal variations in MTP levels [20, 190]. PPARα in particular plays a crucial role in lipid management preventing hepatic lipid accumulation through activation of FA β-oxidation and lipid hydrolysis. Accordingly in Ppara-null mice fed a high-fat diet, the absence of the NR increases hepatic steatosis and induces greater predisposition to NASH [120, 121].
It is becoming ever clearer that the clock gene machinery tightly controls and fine-tunes lipid metabolism, and disruption of the biological clock induces lipid accumulation in the liver. Deregulation of the circadian clock circuitry or misalignment with feeding behaviour causes altered expression patterns of enzymes involved in lipid metabolism, provokes derangement of adipogenesis and FA flux, and promotes lipotoxicity [191, 192]. On the other hand, central and peripheral clocks are influenced by the amount and types of nutrients. High-fat diet modifies the daily rhythm of eating behaviour and advances (by 5 h) the phase of rhythmicity in the liver, without affecting the oscillation patterns in other tissues . Feeding mice with a high-fat diet leads to perturbations of clock gene expression in the hypothalamus and peripheral tissues, and obesity induced by high-fat/high-calorie diet induces changes in the molecular clockwork in mouse adipose tissue and liver [194, 195]. In mouse liver, long-term high-fat diet in the evening deregulates the rhythmic pattern and amplitude of expression of Clock, Bmal1, Per2, Cry2 and Ppara, altering the lipogenic genes controlled by the transcriptional activity of this NR, such as Cyp7a1, Hmgcr, low-density lipoprotein receptor, lipoprotein lipase and diacylglycerol acyltransferase. In turn, gene deregulation induced by the timed high-fat diet causes augmented hepatic cholesterol and TG levels and lipid accumulation in the liver .
The clock gene machinery controls the expression of a RNA deadenylase encoded by the clock-controlled gene Nocturnin, expressed with circadian rhythmicity in the liver and in other peripheral tissues such as the kidney, and influencing fat absorption, trafficking, mobilization, lipogenesis and energy homeostasis . Nocturnin regulates triglyceride packaging into chylomicrons at the level of the intestine , promotes adipogenesis and positively modulates PPAR-γ activity through nuclear translocation . Nocturnin expression is induced by fasting and by increasing levels of cAMP , and Nocturnin−/− mice are resistant to hepatic steatosis and diet-induced obesity .
Besides, the biological clock in adipose tissue controls adiponectin secretion, and in turn adiponectin feeds back on the molecular clockwork: the transgenic expression of adiponectin in the liver of KK/Ta mice significantly modifies the nycthemeral pattern of locomotor activity and the length of free-running rhythm, and the expression of Arntl, Dbp, Cry2 and Per2 is phase advanced in the liver and skeletal muscle .
The molecular clockwork drives the circadian variation in expression of Patatin-like phospholipase domain containing 3, adiponutrin (Pnpla3), highly expressed in liver and adipocytes and encoding a transmembrane protein with both lipolytic and lipogenic activity in vitro. SREBP-1c activates Pnpla3 expression and inhibits its degradation through the stimulation of FA synthesis , but Pnpla3 knockout mice did not show body weight, body composition or adipose mass changes, as well as hepatic steatosis or IR .
Obesity and diabetes cause deregulation of the molecular clockwork, and deregulation of the biological clock associates with dysmetabolism. IR has been evidenced to influence the expression of the clock genes, in particular of Bmal1, Per2 and Cry1, which anyway retain circadian rhytmicity of oscillation . The treatment with the thiazolidinedione family drug pioglitazone for 2 weeks completely abolished the dysfunction in metabolic parameters and the changes in expression of the deranged clock genes from early IR conditions . These data suggest that the molecular clockwork is sensitive to IR, changes in the hepatic biological clock features an early event in the metabolic disruption associated with IR and thiazolidinediones treatment can resolve changes in the biological clock, counteracting the adverse physiological consequences in the metabolic syndrome .
The circadian clock circuitry and NAFLD pathogenesis in humans
Genomic variants are associated with hepatic steatosis and impact on clinical practice, and polymorphisms in circadian genes impinging on lipid metabolism, oxidative stress, IR and immune regulation predispose to NAFLD development. The classification of the genetic determinants of hepatic steatosis supports the identification of the mechanisms involved in NAFLD pathogenesis and progression, and may be a useful strategy for cardiometabolic risk stratification (Table 2). A number of humoral and molecular mediators regulate lipid metabolism, influencing lipogenesis, lipolysis, fat storage and disposal, modulate insulin sensitivity, are controlled by the biological oscillators and feed back on the molecular clockwork. A key role in the genesis of NAFLD is played by insulin secretory capacity and insulin sensitivity, which shows nycthemeral changes, driven by the SCN through autonomic outputs to the liver and endocrine pancreas, and influenced by the increase in growth hormone secretion during slow-wave sleep . Furthermore, adipose tissue secretes with circadian patterns bioactive peptides, called adipocytokines and comprising adiponectin, leptin, resistin, visfatin and retinol-binding protein 4 [207-216]. In the peripheral blood, adiponectin and leptin levels show opposite circadian variations: adiponectin levels are high in the morning, whereas the zenith of leptin secretion occurs during the night, and in obese subjects, their nychthemeral profiles are altered when compared with healthy lean subjects . Besides, leptin negatively modulates insulin action by autocrine effects , and melatonin upregulates insulin-stimulated leptin expression by means of signalling coactivation [218, 219].
Table 2. Mutations in circadian genes causing metabolic phenotypes and hepatic steatosis in humans
In certain cases, subjects suffering from diabetes show as expected IR notwithstanding their leanness, and the mechanisms of IR without obesity are not completely understood at present. On the other hand, unbroken insulin signalling upstream of AKT is essential for hepatic lipogenesis, hepatosteatosis and hypertriglyceridaemia upon overfeeding, and clinical studies on human inborn defects and genome-wide epidemiological studies on human polymorphisms have evidenced that the association between NAFLD and IR/diabetes is not ever necessary . In the absence of hyperglycaemia or IR, liver steatosis may be caused by hindering of TG secretion, lack of lipolysis, increase in lipogenesis, faults in gluconeogenesis, impairment of FA β-oxidation and sequestration of lipids at the subcellular level . The development and progression of NAFLD may be related to variants within genes involved in these metabolic processes and encoding circadian transcripts (for a comprehensive list of circadian genes, it is possible to refer to http://circadb.org). In particular, two single-nucleotide polymorphisms (SNPs), rs17222723A/T (V1188E) and rs8187710G/A (C1515Y) of ATP-binding cassette subfamily C member 2 (ABCC2, also known as multidrug resistance protein 2, MRP2) were associated with NAFLD severity, and carriers of the A alleles showed lower risk for NAFLD . On the other hand, the genotypes 45TT and 276GT/TT of the gene encoding adiponectin (ADIPOQ) were more prevalent in subjects with hepatic steatosis with respect to controls , and a SNP (rs767870) in the gene-encoding adiponectin receptor 2 (ADIPOR2) was associated with NAFLD . Likewise, two SNPs in the gene-encoding Apo C3 (APOC3), C482T and T455C, were associated with NAFLD . In humans, obesity and metabolic syndrome are associated with CLOCK polymorphisms. In particular, CLOCK 3111T/C single-nucleotide polymorphism in women under habitual living conditions associates with overweight and circadian abnormalities, represented by lower amplitude and greater fragmentation of the rhythm, a less stable circadian pattern, a significantly weakened circadian function and a delayed acrophase, typical of ‘evening-type’ subjects [225-227]. A correlation between the C677T polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR), homocysteinaemia and NAFLD was evidenced, and the T allele was associated with elevated levels of circulating homocysteine and with a greater prevalence of hepatic steatosis in patients suffering from HCV-related hepatitis . Homozygous or compound heterozygous mutation in the MTP gene causes abetalipoproteinaemia, an autosomal recessive disorder of lipoprotein metabolism characterized by the virtual absence of apoB-containing lipoproteins in blood and hepatic lipid accumulation related to hindered TG export from the liver ; hepatic steatosis was found also in patients with −493G/G compared with G/T polymorphism, as the G allele of the MTP gene promoter, −493G/T, has been associated with lower transcriptional activity than the T allele . Moreover, the rs738409 SNP (I148M) in PNPLA3 is associated with NAFLD susceptibility . The allele frequency of PPARα Val227Ala was lower in subjects with hepatic steatosis than controls , the PPARG C161T allele frequency was higher in subjects with NAFLD with respect to healthy controls, and plasma adiponectin concentrations were lower in NAFLD patients with CT/TT genotypes compared with NAFLD CC homozygotes , and a statistically significant association of the PPAR- γ coactivator 1 α gene (PPARGC1A) rs2290602-T SNP was evidenced with the development of hepatic steatosis . In addition, polymorphisms and haplotypes of the clock-controlled gene encoding the signal transducer and activator of transcription 3 (STAT3) are associated with NAFLD susceptibility and disease severity . Furthermore, the expression of a huge number of genes involved in cellular metabolism is regulated by β-catenin, a circadian protein, and an association with hepatic steatosis was found for a variant (rs7903146C/T) in the TCF7L2 gene , encoding Transcription factor 7-like 2, which functions as receptor for β-catenin and is expressed with a circadian pattern.
In addition to circadian gene polymorphisms, dampened circadian rhythms and biological clock disruption, as well as metabolic disorders, may result from persistent lifestyle disturbances, such as professional jet lag, long-term shift work, intentional sleep restriction and night eating leading to desynchronization of the circadian systems with respect to environmental and social cues. The investigation of the interplay between unfavourable effects of alternating shift work, altered circadian clock circuitry and metabolic disturbances is not easy to carry out in humans; however, remarkable evidences of the relationship between chronodisruption and dysmetabolism originated from studies that retrospectively or prospectively followed shift workers [237, 238].
Epigenetic priming of NAFLD and the molecular clockwork
The complex interplay between the rhythmic components of time-qualified systems confirms the importance of a proper synchronization among the periodic changes of circadian factors and signalling pathways at the cellular level, and between behavioural and metabolic cycles at the level of the whole body for preservation of metabolic health, and conversely it highlights the key role played by desynchronization in triggering dysmetabolism. On the other hand, an ever increasing importance is recognized to the maternofetal transfer of nutrient signals and to the influence of the intrauterine environment on the offspring metabolic phenotype. Maternal obesity and lactation transmit to the offspring a predisposition to a dysmetabolic and NAFLD phenotype through maternal breast milk and leptin derived from neonatal adipose tissue . The early postnatal period is of crucial importance and probably entails changes of signalling in the hypothalamic nuclei controlling appetite. A maternal high-fat diet leading to obesity modifies, through covalent modifications of histones, the epigenomic profile of fetal chromatin structure of the developing offspring in primates, and up-regulates the expression of the core clock gene Npas2 [240, 241]. The epigenetic modifications resulting from nutrition during early development mediate persistent changes in the expression of key metabolic genes and contribute towards an adult metabolic syndrome phenotype, corroborating the role of circadian alteration in metabolic derangement and the fetal origins of adult disease hypothesis [242, 243]. Maternal obesity causes in utero influences that are particularly relevant if followed by an obesogenic diet throughout post-natal life, and leads to liver injury evidenced by increase in alanine aminotransferase, hepatic TGs and hepatic expression of IL-6, TNF-α, transforming growth factor (TGF)-β, α-smooth muscle actin and collagen . A role of the innate immune system in liver injury is highlighted by increased Kupffer cell numbers with weakened phagocytic function and elvated ROS synthesis, in addition to reduced natural killer T cells and raised IL-12 and IL-18 . Interestingly, the circadian clock circuitry drives immune system function [245, 246] and in turn inflammatory mediators influence the functioning of the biological clock in several tissues including the liver [247-250], suggesting that dysmetabolism sets in motion a vicious cycle that auto-maintains and aggravates progressively, leading to poorer function of the molecular clockwork, derangement of cellular processes driven by the biological clock, and ultimately to loss of anatomic integrity.
The circadian clock circuitry controls the metabolic pathways involved in lipid synthesis, transport, amassing, and breakdown, fine-tuning these processes in line with energy flux and expenditure, and matching the timing of anabolic/catabolic processes, with behavioural cycles of sleep/weak, rest/activity and fasting/feeding, entrained by environmental cues, principally light/dark alternation and temperature oscillation. The NRs gauge nutrient levels and cellular redox state, and recruiting co-receptors, co-repressors, HATs and HDACs induce chromatin remodelling and histone modifications, driving time-related changes of epigenetic modification, transcriptional activity and gene expression, synchronizing circadian and metabolic pathways. Mistiming of body circadian rhythmicity with respect to environmental cues and loss of internal synchronization cause deregulation of metabolic processes, with altered balance between fat storage and disposal, leading ultimately to fat accumulation in the hepatocytes and hepatic steatosis, a frontline player in the dangerous concert of metabolic disorders, and a protagonist in the global scenario of obesity, metabolic syndrome and diabetes mellitus epidemic.
Financial support: This study was supported by the ‘5 × 1000’ voluntary contribution and by a grant from the Italian Ministry of Health (RC1302ME31) through Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza,” Opera di Padre Pio da Pietrelcina, San Giovanni Rotondo (FG), Italy.
Conflict of interest: The authors do not have any disclosures to report.