The circadian machinery is biological time-keeping mechanisms which orchestrate the biological changes that follow a 24-hour cycle. In mammals, the superchiasmatic nuclei (SCN) in the hypothalamus takes the light/dark cycle as the entraining signal and acts as the central pace maker that synchronizes the clock genes in the peripheral organs, such as muscles, liver, heart, and kidneys. The rhythmicity of hormonal and metabolic signals play an important role in immune [1] and metabolic functions [2] by maintaining and controlling the phase relations among the various clocks. The robustness of circadian rhythms is associated with a homeostatic state of the host while disruption in the circadian rhythms is observed in a diseased state of the host [3].
Aside from the light/dark cycle, food intake is a strong zeitgeber, a cue given by the environment on host to reset the internal body clock. Feeding rhythms are a more potent zeitgeber compared to the light/dark cycle eventually synchronizing the peripheral clocks away from the SCN, especially in organs governing the energy homeostasis of the host such as the liver. Therefore, an interconnection between the circadian clocks and metabolism is evident, and it is supported by a multitude of studies. At a clinical level, several studies correlate the disruption of circadian rhythms to increased rate of metabolic syndrome. Shift-work and sleep deprivation dampen the circadian rhythms, and are linked with obesity and high level of triglycerides [4]. At a molecular level, Clock mutant mice showed decreased metabolic rate [5] while liver-specific Bmal1 knockout disrupted glucose homeostasis in mice [6]. These studies suggest that a bidirectional relationship exists between the circadian clocks and metabolism, probably as parts of a network of complex signaling cascades that intricately regulate one another.
Moreover, one can realize from these observations that a broader environmental or behavioral cues (such as feeding patterns) can influence specific, low-level targets (such as liver enzymes). To explore this hypothesis, we would like to demonstrate by a mathematical model how the feeding/fasting cycle influences a specific metabolic function in relation to the circadian rhythms entrained to the light/dark cycle. As a first step toward answering this question, we have previously demonstrated via mathematical modeling how the rhythms of the peripheral clock genes (PCGs) in the liver can be completely entrained to the feeding rhythms, synchronizing away from the light/dark cycle. We further developed this model to explore the effects of light-feeding phase relations on hepatic metabolism. We model the transcription of two genes, G6pc and Pck1, as a surrogate for glucose metabolism in the human liver.
In our model, PPARγ coactivator-1α (PGC1-α) is a key molecule that incorporates the effects of light and feeding to the transcription of the gluconeogenic genes. Transcription of PGC-1α is activated by forkhead box protein O1 (FOXO1) binding upstream of Pck1 and G6pc [7]. When in the nucleus, PGC-1α is deacetylated by SIRT1, which results in an increased activity [8]. The glucocorticoid receptor is also required along with HNF-4α for full activation of Pck1 promoter [9]. PGC-1 has shown to be induced synergistically in primary liver cultures by cAMP and glucocorticoids. Additionally, Pck1 promotor requires co-activation of the glucocorticoid receptor and the liver-enriched transcription factor HNF-4α by PGC-1 for full transcriptional activation. HNF-4α is necessary for the action of PGC1, as hepatocytes from mice lacking HNF-4α loses the ability of PGC-1 to activate key genes of gluconeogenesis such as Pck1 and G6Pc [10]. The role of HNF-4α and PGC-1α were lumped for simplicity in our model. FOXO1 also plays an important role in gluconeogenesis. PGC-1α binds and co-activates FOXO1 in a manner inhibited by Akt-mediated phosphorylation [11]. FOXO1 is known to be required for robust activation of gluconeogenic gene expression in hepatic cells and in mouse liver. Since FOXO1 binds to a DNA motif upstream of the gluconeogenic genes, we used it to model the signal transmission from PGC-1α to transcription of gluconeogenic genes [11]. Finally, PGC-1α also activates the transcription of Bmal1 (Equation 7) [12].
Our model builds upon earlier works [13] of a semi-mechanical model for light entrainment on the peripheral clock genes through the HPA axis. The main driver for oscillations in the HPA axis are the negative feedback between glucocorticoid and corticotropin-releasing hormone (CRH)/adrenocorticotropic hormone (ACTH) using a modified Goodwin oscillator. The glucocorticoid released from the central compartment regulates the peripheral clock genes (Per/Cry, Bmal1, and Clock). The feeding/fasting cycle influences the periphery by modifying the redox reactions between NAD+ and NADH [14]. The feeding-entrained NAD+ activates human sirtuin 1 (SIRT1), a class II histone deacetylase. SIRT1 then acts in coordination with CLOCK/BMAL1 complex, interacting with the PCGs in several ways. It facilitates the transcription of NAMPT, which is the rate-limiting enzyme for the NAD+ salvage cycle, regulated by the CLOCK/BMAL1 complex. NAD+, in turn, activates SIRT1, as well as entraining glucocorticoids through a transit compartment that represents neural input through the ventromedial arcuate nucleus [15]. SIRT1 also facilitates the degradation of PER/CRY proteins in the nucleus, exerting more influence on the peripheral clock genes. Finally, the transcription of G6pc/Pck1 (gluconeogenic gene) is controlled by binding of glucocorticoid-receptor complex to the glucocorticoid response element on the promotor region, as well as FOXO1 binding also to the promotor region.
Current model successfully predicts some observations from experimental time-restricted feeding studies. Some basic predictions include that restricting feeding during the active phase results in phase shift of total gluconeogenic enzyme amount and the increase of amplitude in PEPCK total enzyme amount [16]. Another interesting prediction is that when the system is subjected to constant lighting, the oscillation in G6Pase activity becomes less robust [17]. Furthermore, overexpression of CRY protein results in decreased transcription of gluconeogenic genes [18]. Disruption of circadian clocks by the means of Clock gene knockout resulted in over-expression of G6pc and Pck1, while the nominal expression level could be partially recovered by altering the light-feeding phase relations. To analyze this complex network, sensitivity analysis of the model was performed in three parts: 1) local sensitivity analysis of all parameters; 2) selection of important elementary effects by Morris method; and 3) global uncertainty analysis employing the high dimensional model representations (HDMR). Interpreting and integrating the results from all three methods revealed that transcription and activation of PGC-1α and FOXO1 along with Per/Cry transcription had the greatest influence over the expression level of gluconeogenic genes, demonstrating the close relationship between circadian rhythms and hepatic metabolic activities.
References
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