The endogenous circadian clock is a key regulator of daily metabolic processes. On the other hand, circadian clocks in a broad range of tissues can be tuned by extrinsic and intrinsic metabolic cues. The bidirectional interaction between circadian clocks and metabolism involves both transcriptional and post-translational mechanisms. Nuclear receptors exemplify the transcriptional programs that couple molecular clocks to metabolism. The post-translational modifications of the core clock machinery are known to play a key role in metabolic entrainment of circadian clocks. O-linked N-acetylglucosamine modification (O-GlcNAcylation) of intracellular proteins is a key mediator of metabolic response to nutrient availability. This review highlights our current understanding of the role of protein O-GlcNAcylation in mediating metabolic input and output of the circadian clock.

The rhythm of life

The daily rotation of the earth creates a magnificent rhythm for life. From cycles of eating and fasting to sleep and wake, this consistent cycle facilitated by the sun entails a metabolic cycling within living creatures of energy input and output. With millions of years of adaptation to this constantly cycling environment, it is only natural that life has evolved a mechanism to co-ordinate our inner biology with the external environment. Termed circadian rhythms from the Latin roots of circa meaning ‘around’ and diem meaning ‘day’, these endogenous rhythms recur naturally on a 24-h cycle and are driven by the biological circadian clock—a mechanism that allows for the co-ordination of physiological and behavioral adaptations to environmental cues. Capable of entraining an organism's biological rhythm, these environment cues, including light, temperature, and food availability, are termed ‘Zeitgebers’, German for time-giver or synchronizer [1].

The most important of these Zeitgebers for the mammalian clock is light that entrains the body's central clock in the suprachiasmatic nucleus (SCN), a tiny region in the hypothalamus of the brain [2]. Termed the master pacemaker, this central clock co-ordinates and maintains the synchronicity of all peripheral clocks found in the other tissues of the body while ensuring the alignment of the intrinsic clock with the external environment. Peripheral clocks, on the other hand, are predominantly responsible for maintaining the local rhythm of the tissue and are brought into synchronicity by circadian cues from the SCN [36]. Though almost every cell in the body contains an intrinsic clock [6,7], key organs including the heart and the liver have been extensively studied and found to be highly sensitive to peripheral clocks [8,9]. Peripheral clocks are found in other organs also, including skeletal muscle, pancreas, intestine, and adipose tissue [10]. Taken together, by facilitating co-ordination between the organism's metabolic processes and the environment, the circadian clock system is capable of maximizing energy intake and increasing energy usage efficiency.

The biological circadian clock mechanism

The body does not only function in a reactive manner to external influences, but is also capable of anticipating daily environmental changes through the presence of a robust, self-generating internal clock. The mammalian clock mechanism in both SCN and peripheral clocks operates upon the oscillations of the same core clock genes that are genetically encoded within every cell (Figure 1) [11]. The key circadian clock mechanism features the oscillation of the heterodimer composed of circadian locomotor output cycles kaput (CLOCK) and brain and muscle arnt-like protein-1 (BMAL1). This CLOCK : BMAL1 activity is facilitated by negative feedback of its downstream targets. Specifically, the CLOCK : BMAL1 heterodimer activates the transcription of Period and Cryptochrome genes (PER1, 2, 3 and CRY1, 2), which form a PER : CRY complex that in turn blocks CLOCK : BMAL1 activity. This negative feedback loop entails the inhibition of core clock protein transcription and the generation of a self-sustained molecular oscillation [12]. This primary loop is further reinforced through a second feedback loop involving retinoic acid receptor-related orphan receptor (ROR) and REV-ERB proteins. ROR and REV-ERB, respectively, activate and repress Bmal1 transcription by alternately binding to the same consensus sequences on the Bmal1 promoter, thus creating another level of regulation that results in a more robust and tunable clock mechanism [13,14]. These two interlocked feedback loops serve as the mechanism underlying the biological clock.

Underpinnings of the self-sustained biological clock.

Figure 1.
Underpinnings of the self-sustained biological clock.

BMAL1 and CLOCK proteins form a heterodimer that drives transcription of PER and CRY whose heterodimer in turn suppresses the BMAL1/CLOCK complex. As a second layer of regulation, ROR and REV-ERB promote and suppress, respectively, the transcription of BMAL1.

Figure 1.
Underpinnings of the self-sustained biological clock.

BMAL1 and CLOCK proteins form a heterodimer that drives transcription of PER and CRY whose heterodimer in turn suppresses the BMAL1/CLOCK complex. As a second layer of regulation, ROR and REV-ERB promote and suppress, respectively, the transcription of BMAL1.

The circadian clock and metabolism

The self-sustained ability of the circadian clock allows an organism to anticipate environmental changes. However, the circadian clock must also be capable of being tuned and reset to reflect the metabolic state of the tissue, time of day, and synchronization with the rest of the body's tissues to maintain metabolic homeostasis. The circadian clock oscillation can be characterized by three core properties: period, amplitude, and phase. These three characteristics have been shown to be independently changed by environmental cues in studies to be discussed further in this review. As metabolism is in constant flux, it is crucial that the clock is readily responsive to deviations. The endogenous 24-h oscillation of clock proteins is involved in complex interactions that allow for external signals to be interpreted and responded to so as to co-ordinate metabolic processes. This co-ordination is made possible through signaling pathways that transduce nutritional and hormonal signals to the core clock machinery, which in turn controls metabolic responses primarily by transcriptional regulation.

Since the incidence of obesity and type 2 diabetes mellitus is linked to disruption of biological rhythms [1518], recent studies have confirmed the close relationship between glucose homeostasis, circadian rhythmicity, and overall health outcomes [19]. Ablation of the SCN results in disruption of feeding and fasting rhythms [20], while minor changes in light cues result in significant increased weight gain in rodents [21]. Muscle-specific Bmal1 knockout shows impaired glucose uptake [22] and disruption of other clock genes also perturbs glucose homeostasis [2325]. Additionally, changes in feeding pattern can lead to disruption and realignment of circadian rhythms in the liver [26,27], implying a potential influence of glucose metabolism on the circadian clock.

Clock control of metabolism

Many genes are known to oscillate transcriptionally in a circadian rhythm including nearly half of all genes in the mouse genome [28,29]. Recent high-throughput studies further suggest that the majority of their oscillatory expression is responsive to genetic and environmental perturbations [30]. Moreover, a significant number of circadian oscillations are not at the transcriptional level but at the translational and post-translational levels [3134]. The central circadian clock has been shown to play a global role in mediating circadian oscillations [3538].

The nuclear receptor superfamily in humans consists of 48 members that regulate diverse aspects of organ physiology and metabolism [39]. Known ligands for nuclear receptors include steroid and thyroid hormones, vitamins, dietary lipids, xenobiotics, and heme [39,40]. The circadian clock regulates expression of nuclear receptors and/or production of cognate ligands in order to maintain daily oscillations of carbohydrate and lipid metabolism, basal temperature, appetite, and xenobiotic metabolism, to name a few [25,39,41]. Circadian transcriptional expression of peroxisome proliferator-activated receptor (PPARα and PPARγ), a key player in lipid metabolism, is CLOCK-dependent [42,43]. On the other hand, clock-controlled NAD+ cycles correspond with circadian oscillations of lipid oxidation and mitochondrial protein acetylation, while Bmal1 knockout results in overall reduced function of oxidative enzymes [44]. Constitutive androstane receptor that regulates xenobiotic metabolism has circadian mRNA expression in phase with clock gene Bmal1 [4547]. Furthermore, functional status of CLOCK : BMAL1 mediates sensitivity to toxic metabolites [48], demonstrating dependence of xenobiotic metabolism on the circadian clock. Key proteins involved in metabolite biosynthesis, including delta-aminolevulinate synthase 1 involved in biosynthesis of heme known as a REV-ERB ligand [49,50], hormonal ligands like glucocorticoids and aldosterone involved in steroid biosynthesis [51,52], and HMG-CoA reductase and cholesterol 7 alpha-hydroxylase involved in bile acid biosynthesis and cholesterol homeostasis [53,54], are also regulated by the circadian clock. Other key processes demonstrating circadian clock dependency include REV-ERBα involved in autophagy [55] and RANKl involved in bone resorption [56,57]. The wide range of circadian clock control demonstrates its importance in global metabolic homeostasis.

Clock entrainment

Many of these aforementioned proteins are also involved in a feedback loop with core clock proteins and mediate clock entrainment. Although circadian rhythms persist in the absence of external cues, clock entrainment ensures proper alignment of circadian clock rhythm to metabolic fluctuations. It has been known that food cues, body temperature, and physical activity entrain the peripheral circadian clocks through physiological mechanisms. These physiological mechanisms manifest as both transcriptional regulation of clock genes and post-translational modifications (PTMs) of clock proteins. Clock entrainment fine-tunes clock control of metabolism.

Clock entrainment through nuclear receptors

The levels of various lipophilic metabolites, such as steroid hormones and other molecules, released from various tissues in the body signal the metabolic state and reflect the levels of important nutrients [58]. They pass freely through the cell membrane to bind to nuclear receptors to regulate gene transcription. A multitude of nuclear receptors have been demonstrated to exhibit circadian rhythm [40]. It has been well established that nuclear receptors mediate hormonal entrainment of the circadian clock [39].

Estrogen has been shown to affect circadian clocks in different tissues. In the uterus, estrogen-bound estrogen receptor induces biphasic rhythms in Per1 and Per2 expression through estrogen response elements, which is important for the timing of female reproductive physiology [59]. Estradiol-mediated alteration of phase and amplitude of Per1 expression has been seen in liver and kidney [60,61]. Increased estrogen–estrogen receptor signaling leading to up-regulation of Clock and deregulation of Per2 is implicated in breast cancer cell morphogenesis [6264]. On the other hand, glucocorticoids are a class of steroid hormones involved in metabolic responses to fasting and stress especially involved in phase resetting in peripheral tissues [65]. It has been shown that restricted eating and physical stress can phase-shift peripheral clocks through glucocorticoid-mediated regulation of Per1 and Per2 promoter activity [6668]. Another example is the PPAR family [69]. It is activated by free fatty acids and eicosanoids, and is involved in metabolism and tumorigenesis. PPARα mediates mitochondrial dynamics in the liver through amplitude regulation of BMAL1 expression [70]. PPARγ mediates diurnal variations in blood pressure and heart rate through regulation of BMAL1 expression in the aorta [71]. Additionally, orphan nuclear receptors, such as some in the estrogen-related receptor (ERR) family, play a key role in energy metabolism and mitochondrial biogenesis while directly regulating the circadian clock. ERRα binds the promoter region of core clock proteins and mediates their expression amplitude and diurnal rhythm [72,73]. In mouse liver, ERRα is responsible for the strong repression of the Bmal1 gene important for maintaining diurnal rhythms of the blood glucose level and other circulating metabolites [72]. Apart from these brief highlights, there are still many other nuclear receptors with varied roles in different tissues that entrain the circadian clock.

Clock entrainment through other transcriptional factors

Transcriptional control is not limited to nuclear receptors. Recently, transcription factor EB (TFEB) has been linked to the circadian clock mechanism. It is previously known to be a regulator of the autophagy–lysosome pathway and is involved in starvation signaling [74,75]. TFEB was found to interact with CLOCK/BMAL1 to enhance transcription of PER3 in a glucose-mediated manner [76]. In addition to TFEB, cyclic AMP (cAMP)-responsive element-binding protein (CREB) and CREB-regulated transcription co-activator 2 (CRTC2) form a transcriptional complex that induces Bmal1 expression by binding its promoter. Activated by glucagon and inhibited by insulin, CREB/CRTC2 relays these temporal signals of fasting and refeeding so as to entrain the hepatic circadian clock [77]. Additionally, insulin entrains the circadian clock by activating Forkhead box class O 3 (FOXO3), a transcription factor that increases Clock transcription in the liver, and has also been shown to regulate the circadian clock in fat [78,79]. Other transcription factors are also involved in mediating core clock complexes and the circadian clock [2,80].

Clock entrainment through PTMs

Another key mechanism by which temporal and nutritional cues entrain the circadian clock is through altering the PTMs of clock proteins. In light-stimulated SCN, G-protein-coupled receptor kinase 2 (GRK2) phosphorylates PER1 and PER2 to increase the amplitude and decrease the period of the central clock [81]. Indeed, the circadian timing itself has been linked to phosphorylation of clock proteins and shown in both Drosophila and cyanobacteria to be controlled by a timed cascade of phosphorylation events [8285].

Phosphorylation and destabilization of CRY and PER by AMP-activated protein kinase (AMPK) mediates circadian clock oscillation timing and affects circadian clock speed [86,87]. AMPK responds to the AMP : ATP ratio and cAMP to mediate glucose and lipid metabolism [88]. Additionally, glucagon, insulin, and epinephrine are upstream regulators of the AMPK pathway [8993]. As described above, glucagon and insulin have been shown to affect the circadian clock through CREB/CRTC2 and FOXO3 [7779]. Insulin has also been shown to suppress the transcriptional activity of hepatic Bmal1 through Akt-mediated phosphorylation [94]. However, there is currently no evidence indicating that AMPK is also involved in the transduction of steroid signals to the circadian clock. Further research in this direction will clarify the extent of AMPK's role in transducing metabolic cues to mediate the circadian clock.

Another notable PTM is deacetylation by NAD+-dependent deacetylase sirtuin-1 (SIRT1). In the presence of NAD+, SIRT1 deacetylases and subsequently destabilizes PER2 leading to its degradation [95]. Additionally, SIRT1 deacetylation of BMAL1 inhibits the recruitment of CRY1 to the CLOCK/BMAL1 complex [96,97]. Both of these roles prolong the activity level of CLOCK/BMAL1 and the transcription of clock-controlled genes. Furthermore, SIRT1 is a key player in the negative feedback loop with NAD+ biosynthesis, moderates mitochondrial oxidative metabolism, and is capable of ADP-ribosylation [98100]. However, it is still unknown how its function in metabolic control interacts with the circadian clock and if SIRT1 partakes in ADP-ribosylation of clock proteins. On the other hand, poly(ADP-ribose) polymerase 1 (PARP1), another NAD+-dependent enzyme, has been shown to poly(ADP-ribosyl)ate CLOCK. Asher et al. [101] show that poly(ADP-ribosyl)ation of PARP1 reduces the DNA-binding activity of CLOCK/BMAL1 and is important in food entrainment of the peripheral circadian clock. Through NAD+ sensing, both SIRT1 and PARP1 entrain the circadian clock to cellular metabolic cues.

The PTMs often function in concert to regulate the clockwork. Two interacting enzymes involved in different PTMs will be discussed in more detail in this review. One is the kinase, glycogen synthase kinase-3 β (GSK3-β), found to interact with most core clock components [98,102]. In addition to interacting with PER2 to facilitate its nuclear translocation [103,104], GSK3-β has been shown to phosphorylate and thus destabilize BMAL1, CLOCK, and mCRY2 [105108], thereby affecting circadian locomotor activity period, period length, and phase delay. GSK3-β is deactivated upon insulin-mediated signaling and acts as a negative signal for nutrient availability [109,110]. The second protein modification is O-GlcNAcylation that has received increased attention in recent years and is now known to be a key player in nutrient metabolism and clock function [111]. A summary of notable PTMs is highlighted in Figure 2. From these discoveries, one can gain a deeper appreciation for the importance of the circadian clock on metabolic physiology as well as the influence of the metabolic system on circadian rhythms within the body.

PTMs of clock components.
Figure 2.
PTMs of clock components.

Key enzymes responsible for PTMs of clock components relay important external and metabolic signals. The effect of the different PTMs varies depending on the mediating enzyme and target protein. Hexosamine biosynthesis pathway (HBP) flux leads to increased UDP-GlcNAc, the substrate for O-GlcNAcylation by OGT. OGT has been shown to add the O-GlcNAc modification on BMAL1, CLOCK, and PER. AMPK responds to an increase in the AMP-to-ATP ratio, leading to phosphorylation of CRY and PER. SIRT1 activity is dependent on increased NAD+ levels in order to deacetylase BMAL1 and PER2. GRK2 responds to increased light signaling through glutamate levels to phosphorylate PER and entrain the central circadian clock in the SCN. Finally, GSK3-β is reactivated upon decreased insulin signaling inhibition to phosphorylate all four clock components. This figure illustrates the complexity of PTM regulation in clock entrainment.

Figure 2.
PTMs of clock components.

Key enzymes responsible for PTMs of clock components relay important external and metabolic signals. The effect of the different PTMs varies depending on the mediating enzyme and target protein. Hexosamine biosynthesis pathway (HBP) flux leads to increased UDP-GlcNAc, the substrate for O-GlcNAcylation by OGT. OGT has been shown to add the O-GlcNAc modification on BMAL1, CLOCK, and PER. AMPK responds to an increase in the AMP-to-ATP ratio, leading to phosphorylation of CRY and PER. SIRT1 activity is dependent on increased NAD+ levels in order to deacetylase BMAL1 and PER2. GRK2 responds to increased light signaling through glutamate levels to phosphorylate PER and entrain the central circadian clock in the SCN. Finally, GSK3-β is reactivated upon decreased insulin signaling inhibition to phosphorylate all four clock components. This figure illustrates the complexity of PTM regulation in clock entrainment.

Protein O-GlcNAcylation

O-linked N-acetylglucosamine (O-GlcNAc) modification is the covalent modification of intracellular proteins at serine or threonine residues. The enzymes O-GlcNAc transferase (OGT) and O-linked β-N-acetylglucosaminase (OGA) mediate this modification by adding and removing the moiety, respectively. The donor substrate uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is synthesized through the hexosamine biosynthetic pathway (HBP) from fructose-6-phosphate and accounts for 2–5% of glucose flux [112,113]. With increased glucose levels in the cell, studies have shown that increased activation of the HBP pathway results in increased protein glycosylation [114]. However, it should be noted that nutrient availability also affects levels of OGT and OGA expression and activity, leading to increased global O-GlcNAcylation in nutrient-deprived states [115]. Using O-GlcNAcylation as a direct molecular readout of the overall nutrient level within the cell is not suitable for all physiological and pathological states (Figure 3).

Nutrient-sensing pathway for O-GlcNAcylation.

Figure 3.
Nutrient-sensing pathway for O-GlcNAcylation.

Glucose influx into the cell can enter the glycogen biosynthesis pathway (GBP), pentose phosphate pathway (PPP), glycolysis, and HBP, among others. Glutamine fructose-6-phosphate amidotransferase (GFAT) is responsible for controlling the flux of glucose into the HBP. The product of the HBP is UDP-GlcNAc, the substrate for O-GlcNAcylation by OGT.

Figure 3.
Nutrient-sensing pathway for O-GlcNAcylation.

Glucose influx into the cell can enter the glycogen biosynthesis pathway (GBP), pentose phosphate pathway (PPP), glycolysis, and HBP, among others. Glutamine fructose-6-phosphate amidotransferase (GFAT) is responsible for controlling the flux of glucose into the HBP. The product of the HBP is UDP-GlcNAc, the substrate for O-GlcNAcylation by OGT.

Originally thought to be a cell-membrane modification when it was first discovered in the 1980s, it is now known that many cytoplasmic, nuclear, and mitochondrial proteins are modified by O-GlcNAcylation [111]. Modulating serine/threonine residues at both the same and different sites from phosphorylation, O-GlcNAc often occurs reciprocally or sequentially with phosphorylation and functions similarly as an on/off switch for many cellular pathways. Unlike individual effectors, changes in nutritional levels often result in the rapid functional alternations of a multitude of proteins via O-GlcNAcylation. Emerging evidence suggests that O-GlcNAc modification co-ordinates global metabolic responses to fluctuating nutrient levels [111,116].

O-GlcNAcylation and its mediating enzymes are implicated in many diseases including but not limited to cancer, diabetes, cardiovascular disease, and neurodegenerative disease [116,117]. Investigation into the role of O-GlcNAcylation has led to many discoveries of its molecular targets and mediators. Modulations in O-GlcNAc levels are correlated to a broad range of pathogenesis. Excess HBP activation leads to insulin resistance, a key hallmark of type II diabetes [118]. Significant up-regulation of O-GlcNAc and OGT levels in addition to down-regulation of OGA levels implicated in the Warburg effect supports cancer growth [119]. Decreased O-GlcNAc levels in the brain combined with the documented absence of O-GlcNAc on tau aggregates are implicated in Alzheimer's disease [118,120122]. Yet, the physiological and pathological roles of O-GlcNAcylation are still vastly unknown and will continue to be a bountiful area of discovery for years to come.

O-GlcNAcylation and the circadian clock

It has been seen that OGT and OGA transcripts exhibit circadian rhythm in various tissues (http://circadb.hogeneschlab.org/) [29]. While OGT protein levels appear constant, OGA protein levels and protein O-GlcNAcylation levels cycle in a diurnal fashion [123125]. This points to a potential link between the O-GlcNAc pathway and the circadian clock. On the other hand, cellular O-GlcNAcylation levels are responsive to nutrient availability [126,127]. As highlighted above, O-GlcNAcylation functions as a sensor of nutrient levels in the cell and a mediator of many metabolic processes. By the combination of circadian and metabolic processes, O-GlcNAc signaling is known to play a crucial role in helping the internal clock synchronize the external cues with internal metabolism.

Clock control over O-GlcNAcylation

The heart is a high-energy demanding organ. It is therefore not surprising that metabolic loops are integrated in the heart circadian clock. Durgan et al. [123] first described the O-GlcNAc modulation by the circadian clock and its modulation of clock proteins, suggesting the importance of O-GlcNAc signaling in linking the cardiomyocyte circadian clock to both metabolic inputs and outputs. Genetic ablation of the core clock proteins in mouse cardiomyocytes abolishes diurnal Ogt expression and accounts for diurnal changes in global O-GlcNAcylated protein levels. Additional analysis shows the loss of rhythmicity in the expression of proteins involved in glucose transport and HBP function. Indirectly, the resulting loss of diurnal glucose oxidation rate fluctuations due to the ablation of core clock proteins additionally represses protein O-GlcNAcylation levels. This provides initial insights into the modulation of O-GlcNAc signaling by the peripheral circadian clock at the transcriptional level. As of the time of writing this review, central circadian clock modulation of O-GlcNAc signaling has not been documented.

Clock entrainment by O-GlcNAcylation

As a key nutrient sensor, O-GlcNAcylation is well poised to relay metabolic cues to the circadian clock. Indeed, several key papers show circadian clock entrainment by O-GlcNAcylation. Durgan et al. show both the depression of PER2 protein levels and the phase advancement of the SCN clock with the administration of an OGA inhibitor. Their results point to a role for O-GlcNAcylation in central clock function. Additionally, they identified BMAL1 as a direct target of O-GlcNAcylation and showed that levels of cardiac BMAL1 O-GlcNAcylation may vary in a diurnal fashion. Their results suggest a regulatory role of this nutrient-sensing modification in clock entrainment [123].

To gain a better understanding of O-GlcNAcylation in clock function, Li et al. show the rhythmic O-GlcNAcylation of both BMAL1 and CLOCK proteins in mouse liver. Their results link glucose flux through O-GlcNAcylation to the inhibition of BMAL1 and CLOCK protein ubiquitination. The resulting stabilization of BMAL1 and CLOCK promotes the activation of clock target genes [125]. In addition, Kaasik et al. show that increased O-GlcNAcylation levels increase the circadian period length in mouse brain and peripheral tissues in Drosophila. They further show that both mouse PER2 and CLOCK and Drosophila dCLK and dPER are O-GlcNAcylated. O-GlcNAcylation modulates dClk transcriptional activity, which is implicated in proper clock function [124]. Olszewski et al. [128] show the regulation of circadian rhythm by plant OGT (SPINDLY) in Arabidopsis thaliana. Taken together, these results illuminate an evolutionarily conserved role for O-GlcNAcylation in the regulation of circadian clocks.

Additional studies further support the notion that O-GlcNAcylation modulates circadian rhythms by regulating the activity, stability, and translocation of clock proteins. Ma et al. [129] show that O-GlcNAcylation enhances BMAL1 transcriptional activity in NIH3T3 fibroblasts. Kim et al. [130] show that OGT overexpression stabilizes dPER and delays dPER translocation into the nucleus, which has been proposed to set the correct pace of the clock in Drosophila. It is also likely that O-GlcNAcylation affects the circadian clock by modulating upstream regulators. For instance, given the extensive cross-talk between O-GlcNAcylation and phosphorylation on Akt, further examination of the role of O-GlcNAcylation in Akt-mediated Bmal1 phosphorylation may provide insights into indirect regulation of the circadian clock by O-GlcNAcylation [94,131]. Overall, these studies highlight a role of O-GlcNAcylation in central and peripheral clock entrainment across species.

O-GlcNAcylation and clock mechanism

On the basis of the mutually supporting studies above, we propose a working model of the interactions between OGT and clock proteins (Figure 4). As direct targets of OGT, BMAL1 and CLOCK proteins are stabilized by O-GlcNAcylation [124,125], leading to increased transcriptional activity, of which Ogt has been shown to be a target [123]. dPER is also a direct target of OGT. dPER is stabilized and retained by OGT in the cytoplasm, resulting in a delay in nuclear translocation and, subsequently, a delay in dPER's inhibition of BMAL1/CLOCK activity [130]. In this way, increased O-GlcNAcylation as a result of increased glucose levels stabilizes clock proteins and increases clock transcriptional activity. The resulting increased transcription of clock-controlled genes includes a positive feedback loop to increase OGT levels.

Competing roles of OGT and GSK3-β in clock entrainment.

Figure 4.
Competing roles of OGT and GSK3-β in clock entrainment.

Acting in competing manners, OGT promotes the stability of CLOCK and BMAL1, allowing for the transcription of clock-controlled genes (CCG) facilitated by the heterodimer, while GSK3-β primes the two proteins for ubiquitination and subsequent degradation. Additionally, PER protein O-GlcNAcylation delays nuclear translocation, while phosphorylation by GSK3-β promotes nuclear translocation, leading to inhibition of BMAL1/CLOCK function.

Figure 4.
Competing roles of OGT and GSK3-β in clock entrainment.

Acting in competing manners, OGT promotes the stability of CLOCK and BMAL1, allowing for the transcription of clock-controlled genes (CCG) facilitated by the heterodimer, while GSK3-β primes the two proteins for ubiquitination and subsequent degradation. Additionally, PER protein O-GlcNAcylation delays nuclear translocation, while phosphorylation by GSK3-β promotes nuclear translocation, leading to inhibition of BMAL1/CLOCK function.

On the other hand, GSK3-β, a key regulator of the circadian clock mentioned earlier in this review, functions opposite to OGT. GSK3-β promotes the nuclear translocation of PER while phosphorylating and priming BMAL1 and CLOCK for degradation [103108]. Thus, when glucose levels are low, decreased O-GlcNAcylation protection results in phosphorylation by GSK3-β and degradation of BMAL1/CLOCK proteins. Additional inhibition by PER from GSK3-β-mediated nuclear translocation further lowers promotion of clock-controlled genes.

Additionally, OGT and GSK3-β are direct substrates of one another [132]. Kaasik et al. show that OGT is phosphorylated by GSK3-β, resulting in an increase in OGT activity, and Lubas and Hanover show that GSK3-β is a glycosylation target of OGT [124,133]. Although the details on their cross-talk with regard to the circadian rhythm are currently unknown, these results indicate that OGT and GSK3-β may engage in reciprocal regulation [124,132,133]. Thus, the circadian clock and O-GlcNAcylation with its interaction with GSK3-β are likely to create a robust system for effective metabolic activities based on nutrient levels within the cell (Figure 5).

Clock entrainment by nutrient sensing through O-GlcNAc and GSK3-β.

Figure 5.
Clock entrainment by nutrient sensing through O-GlcNAc and GSK3-β.

Nutrition increase leads to up-regulation in the HBP, leading to increased O-GlcNAcylation and OGT activity, while up-regulation of the insulin signaling pathway (ISP) leads to down-regulation of GSK3-β. They work in competition to modulate the clock and, subsequently, clock-controlled genes (CCGs). Additionally, these two proteins are believed to regulate one another, though the effect of their modification on either's function remains to be elucidated.

Figure 5.
Clock entrainment by nutrient sensing through O-GlcNAc and GSK3-β.

Nutrition increase leads to up-regulation in the HBP, leading to increased O-GlcNAcylation and OGT activity, while up-regulation of the insulin signaling pathway (ISP) leads to down-regulation of GSK3-β. They work in competition to modulate the clock and, subsequently, clock-controlled genes (CCGs). Additionally, these two proteins are believed to regulate one another, though the effect of their modification on either's function remains to be elucidated.

Implications of O-GlcNAcylation and the clock

Misalignment between internal metabolism and the environment has been known to increase risk factors for human diseases [16,134]. Misalignment can result from the dysfunction of clock proteins, OGT, and OGA, leading to perturbations in rhythmic O-GlcNAcylation and the disruption of circadian clock entrainment. As both the circadian clock and O-GlcNAc modification engage in a great range of cellular effects, their individual dysfunction has been implicated in many different disease states [16,116122,134,135]. However, few ailments due to a specific interaction between the circadian clock and O-GlcNAcylation have been documented. The mutation of serine 662 of PER2 results in a familial sleep-phase disorder [136]. This site is known to be competitively phosphorylated and O-GlcNAcylated [124,136], suggesting a role for O-GlcNAcylation in the pathogenesis of circadian rhythm disorder. Though it is possible that other specific mutations causing disturbances in O-GlcNAcylation of clock proteins may result in similar specific defects, they are rare. O-GlcNAcylation of clock proteins resulting in misalignment of metabolic cues and the circadian clock is more likely to cause global systemic effects, leading to long-term metabolic problems and increasing the risk for a plethora of other ailments [17].

Conclusion

The main thrust of research on phosphorylation and its biological functions only began in earnest 25 years after the initial discovery of the modification. Phosphorylation has since become the most well-understood modification. A similar timeline appears to be the case for O-GlcNAcylation. With the recent surge of discoveries in the cellular and physiological functions of O-GlcNAcylation, it will be exciting to see how this growing field will move forward. Although a basic model of the interactions between the molecular clock and O-GlcNAc signaling has been established, it is unclear whether other clock-related proteins are modified and to what extent the role of O-GlcNAc plays. It is also largely unknown how these interactions sense and respond to systemic and tissue-specific metabolic changes. Furthermore, both the long-term and short-term physiological and pathological impacts of these interactions should be examined. It will also be important to examine whether and how other nutrient-sensing mechanisms such as the AMPK, mTOR, and SIRT1 pathways interact with O-GlcNAc signaling in the context of the circadian clock. Thus far, these discoveries have relied on a reductionist approach to elucidate specific relationships between individual components of the circadian clock. A systems biology approach should be embraced to gain a comprehensive understanding of the link between the circadian clock and O-GlcNAcylation in metabolic homeostasis.

Abbreviations

     
  • AMPK

    AMP-activated protein kinase

  •  
  • BMAL

    brain and muscle arnt-like protein

  •  
  • cAMP

    cyclic AMP

  •  
  • CREB

    cAMP-responsive element-binding protein

  •  
  • CRTC2

    CREB-regulated transcription co-activator 2

  •  
  • CLOCK

    circadian locomotor output cycles kaput

  •  
  • CRY

    Cryptochrome

  •  
  • ERR

    estrogen-related receptor

  •  
  • FOXO3

    Forkhead box class O 3

  •  
  • GRK2

    G-protein-coupled receptor kinase 2

  •  
  • GSK3-β

    glycogen synthase kinase-3 β

  •  
  • HBP

    hexosamine biosynthetic pathway

  •  
  • OGA

    O-linked β-N-acetylglucosaminase

  •  
  • O-GlcNAc

    O-linked N-acetylglucosamine

  •  
  • OGT

    O-GlcNAc transferase

  •  
  • PARP1

    poly(ADP-ribose) polymerase 1

  •  
  • PER

    Period

  •  
  • PPAR

    peroxisome proliferator-activated receptor

  •  
  • PTMs

    post-translational modifications

  •  
  • ROR

    retinoic acid receptor-related orphan receptors

  •  
  • SCN

    suprachiasmatic nucleus

  •  
  • SIRT1

    sirtuin-1

  •  
  • TFEB

    transcription factor EB

  •  
  • UDP-GlcNAc

    uridine 5′-diphosphate-N-acetylglucosamine

Funding

This work was supported by the National Institutes of Health, American Cancer Society, and Ellison Medical Foundation to X.Y.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

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