THE LIVER’S CIRCADIAN POWER
TEMPORAL CONTROL OF HEPATOCYTES
Abstract-The biological systems of organisms, and in specific humans, rely on the regulated circadian control that covers a 24-hour period. One master controller of this internal clock is located in the brain, however smaller systems are influenced by a local control that is regulated by post-translational and post-transcriptional mechanisms. The liver is one of these systems that has a powerful local controller, and is responsible for most of the metabolism in the body. This local temporal pattern dictates the different metabolic processes.I. Introduction
The physiology of living organisms comprises a vast collection of systems that all function toward the common goal of maintaining life. These systems synchronize with each other in different patterns in which all of them are utilized efficiently. The most dominant regulators of these patterns is the light-dark cycle ; that is a 24-hour period of day and night that is synchronous with the axial rotation of the earth, and the availability of food and its consumption (1). Other inputs to an individual organism such as temperature, noise, and social cues have a significant but substantially weaker effect on cyclic functioning of the entire organism (2). The cues of light and energy intake are processed in the brain; through the eyes, and in the liver; the bodies main metabolic sensor. Therefore these organs in mammals are the central locations that dictate the circadian system for the animal to aptly adapt and respond to its environment. While the entire body depends on the brain for most of this signaling, the cells of the liver, hepatocytes, have rhythms and a temporal system that regulates metabolic processes and protein coding with its own regulating peripheral pattern.
The light-dark oscillation is sensed by retinal photoreceptors in the eye; the only way for light to enter the biological system of the body directly, and is transmitted directly to the paired suprachiasmatic nuclei (SCN) in the ventral hypothalamus (1). The SCN is often considered the central integrator of light via circadian clock genes that secrete protein products used to generate the master circadian rhythm for a mammal body (3). This system involves two negative feedback loop systems that work together in response to the light-dark cycle of the environment. The 24-hour system is maintained by antagonistic control of the proteins produced by the circadian clock genes. This means that while one set of proteins is increasing in number, an opposite set is decreasing at the same time and vice versa (4). The genes, eight have currently been identified, have been discovered participating in this oscillation to control the entire biological clock of the system for optimum performance. The primary feedback loop dependents on PER1, PER2, CRY1, and CRY2 and the PAS-domain basic helix-loop-helix (PAS-bHLH) transcription factors BMAL1, CLOCK, and additionally NPAS2 (4). The repressor genes activated by PAS-bHLH activators are regulated by the secondary feedback loop in which the nuclear receptor REV-ERBa represses Bmal1 promoter (4). REV-ERBa is governed by the primary PAS-bHLH activators and CRY-PER repressors, which link the primary and secondary loops together for a system that functions as a looped regulator (4). The proteins produced by these genes interact in a rather sophisticated way to control the 24-hour rhythm of the body as a whole; because of this the entire complex system the SCN and subsequent genes are nicknamed the master clock.II. The SCN and Local Control
The SCN master clock controls the circadian rhythm of the physiological systems as a whole using the light-dark cycle, but individual organs have peripheral cycle regulators that function on a small scale basis in direct response to the environment (2). One of the most important peripheral tissue cyclic systems is found in the liver. The circadian rhythm of the liver plays a vital role in metabolism for the body, and is actually able to generate diurnal rhythms; making the liver its own biological clock. The liver was found to possess the ability to generate in vitro cycles of gene production in response to feeding pattern; although other factors such as body temperature, insulin, and glucocorticoid levels are also thought to be able to reset the circadian rhythm in the liver (5). Most rhythmic proteins are released during the activity period of an animal, in humans during the day, peaking at about 80% while only 20% of the proteins are released during the rest period in animals, or at night for humans (5). Oscillations of the circadian clock in the liver and in any other rhythmic system in the body are controlled by the protein secretion from multiple genes; just like the SCN, and in the liver this cycle affects the metabolism of urea, sugar, alcohol and bile acid (5).III. Circadian Transciptomes and Proeteomes
A study done in 2006 by Akhilesh B. Reddy and others on his team, clearly show some of these rhythms through their work on certain proteins. The circadian pattern in a local region is expressed through different gene expression of certain ‘circadian transciptomes’; particularly this experiment examined the activity of ‘circadian proteome’, the entire set of proteins expressed by a system, which displayed 20% of the proteins are controlled with this local rhythm of the liver (5). Reddy and his team altered the rhythmic activity of circadian proteins and studied the effect this action had on three particular metabolic enzymes: aldolase, arginase, and catalase (5). It was found that these enzymes are affected and function in time to a circadian clock even without the presence of the master clock gene proteins coming from the SCN (5). This clearly indicates that the metabolic enzymes are in tune with some other local clock of the liver and are in no way completely controlled by the transcription of circadian genes from the master clock (5).
Reddy and his team found that different genes expressed multiple different proteins that are produced in a rhythmic sequence. Two such protiens; translated from the same gene; aldehyde dehydrogenase (Aldh2) and carbarmoyl phosphate synthase (Cpsl1), have isoforms that are expressed in a different manner from each other but both have a rhythmic cycle (5). Aldh2 expresses isoforms in a regular synchronized pattern, while Cps1, a control enzyme found in the urea cycle, has a rhythmic pattern of isoform release following a different pattern than the temporal pattern of Aldh2 (5). These different isoforms demonstrate that post-transcriptional control contributes to the temporal rhythm of local control in the liver (5).
This finding in conjunction with the fact that proteins can in fact be modified in phosphorylation and post-transcriptional processes indicates that local circadian patterns are post-transcriptional developments. Reddy found that phosphorylated and unphosphorylated forms of peroxiredoxin 6 displayed differential phasing when tested in gene mutant mice (5). The liver was found to generate a self-sustained system, of about six cycles, that was independent of translation and transcription from the core clock mechanism (5).
The SCN is the core circadian regulator of biological systems, and operates through a transcriptional auto-regulatory feedback loop utilizing the proteins Bmal1 and Clock as the activators that it regulates (5). The circadian transcriptome, all messenger RNA (mRNA) and proteome demonstrate a drastic difference which indicates that the circadian commands cannot have only the master clock regulation but also the input from the local systems (5). The rhythmic activity of the transcriptome can be influenced directly with the rhythmic protein locally; however not all rhythmic proteins have this correspondence. Some mRNA do not have a 24-hour rhythm, rather they have a post-transcriptional regulation that expresses the circadian pattern (5). Reddy experimented with circadian promoter elements to relate whether or not mRNA and protein expression could be predictors of these local rhythmic expressions (5). It was discovered that the mechanisms for RNA-binding proteins that determine the RNA processing explains the dissociation but not that they are predictors (5). This helps focus on the fact that the local regulation has to have local control. Transcriptional control dictates proteome circadian expression but most of the liver coordination depends on post-translational and post-transcriptional mechanisms. The degree of temporal regulation over the circadian rhythm mechanism of the body in the transcriptome and proteome control relies on the local controller.IV. Insulin as a Known Predictor
Furthermore a 2012 study by Yamajuku and his team discovered one definite predictor for the local liver circadian pattern. The team created a 3-dimensional cell culture of hepatocytes from rats and tested insulin’s ability to synchronize the liver’s circadian clock. The insulin directly regulated the activation of the Per2 and Dbp protein from the regulatory circadian genes; however this control did not sustain itself for more than a few days (4). This is a clear indication that the liver is susceptible to the feeding pattern of mammals when establishing its local clock system and therefore the metabolism of a mammal is disturbed completely dependent on its environment (2). The local clock however could not be maintained for long periods, and thus the proper expression of these metabolic proteins are dependent on a functioning master clock (4,5)V. Conclusion
Each system in the body has its own oscillating genes, and each gene is expressed in its own circadian manner (4). This control is highly regulated not only by the master clock in the SCN but also by post-translational and post-transcriptional control, which occurs in areas close to the functioning local systems. This control relies on the master clock but has the ability to express individual control on its temporal pattern. Temporally expressed gene and protein regulations are more important in the complex role of metabolism then the SCN clock can cover and thus these local patterns are more specialized for the system and vital for the body to function. Maintenance of circadian rhythm in local pathways is dependent upon extensive transcriptional and posttranscriptional regulation as well as posttranslational alteration of clock proteins (3). For the liver, the clock proteins are expressed in different ways and controlled in different manners than they are in the central SCN system. This guarantees that the systems of the liver are controlled correctly and can respond to any changes in their predictors or environment (3). The exact controllers of the hepatic cells are not specifically identified but research has proven that the liver is a self-driven system that entrains important circadian rhythm to maintain the health of the mammalian organism.
 F.W.Turek, R. Allada. “Liver Has Rhythm,” Hepatology. Vol. 35 Issue. 4. 30 Dec 2003.
 K. Stokkan, S. Yamazaki, H. Tei, Y. Sakaki, M. Menaker. “Entertainment of the circadian clock in the liver by feeding,” Science Vol. 291 no. 5503 pp. 490-493. 19 Jan 2001.
 D. Gatfied, G. Le MArtelot, C.E. Veinar, D. Gerlach, O.Schaad, F. Fleury-Olela, A.L. Ruskeepo”a”, et al. “Liver’Tick Tock’- Integration of microRNA miR-122 in heptic circadian gene expression,” Genes Dev 2009;23;1313-1326. Heptology,Vol.50 Issue4. 29 Sept, 2009.
 B. Kornmann, O. Schaad, H. Bujard, J.S. Takahashi, U. Schibler.”System driven and oscillator dependent circadian transcription in mice with a conditionally active liver clock,” Genome Biology 7:234 , Vol 5. Issue 2. Feb 2007.
 D. Yamajuku. T. Inagaki, T. Haruma, S. Okubo, Y. Kataoka, S. Kobayashi, K. Ikegami,T. Laurent, T. Kojima, K. Noutomi, S. Hashimoto, H. Oda. “Real time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock,” Japan, 2Laboratory of Nutritional Biochemistry, Department of Applied Molecular Biosciences, Nagoya University. 1 June 2012.