A Matter of Time. Circadian Rhythms and Therapeutic Treatments

We tend to think of time as something external, measured by clocks or calendars, but our body also has an internal time tracking system: the circadian clock that regulates multiple biological functions, from sleep and wake cycles, energy levels, concentration, coordination, and learning, to physiological processes such as bowel movements, thermoregulation, blood pressure, muscle strength, and metabolism. The circadian system acts as an internal clock, synchronizing our body primarily with the daily cycles of light and dark, and helps us anticipate and prepare for changes in the environment.

OUR INTERNAL CLOCK
From the point of view of anatomy, the circadian system is hierarchically organized, with distinct clocks that oscillate subordinated to a master pacemaker located in a region of the hypothalamus in the brain, called the suprachiasmatic nucleus. In turn, this brain nucleus integrates and transmits time information to the rest of the clocks located in every organ and cell of the body. To correctly recognize, maintain, and inform the time of day, the suprachiasmatic nucleus must synchronize daily with the external environment through specific signals called “zeitgebers” (from the German words zeit, time, and geber, giver). The primary “time giver” for the suprachiasmatic nucleus of mammals is light, whose information is transmitted from the retina through a direct neuronal pathway called the retinohypothalamic tract. The subordinate clocks of the rest of the body can also be synchronized by other secondary signals, including food or exercise. 

When all these clocks are perfectly aligned with each other and with the environment, the circadian system optimizes the differential coordination of physiology and metabolism between day and night. However, it has been proven that disruption of circadian rhythms caused by a mismatch between the different clocks and the environment can severely affect our health, contributing to the development of neurodegenerative or metabolic diseases, and cancer, among others. 

OUR INTERNAL CLOCKS ARE ENCODED IN EVERY CELL OF THE BODY BY A MOLECULAR MECHANISM THAT ASSIGNS THE TIME AT WHICH EACH FUNCTION IS EXECUTED

THE MOLECULAR CLOCK
Our internal clocks are encoded in every cell of the body by a molecular mechanism that assigns the time at which each function is executed. The molecular clock is essential for separating incompatible cellular functions, such as DNA replication and oxidative metabolism, into different times of the day. During the day, cells generate energy through mitochondrial respiration, which produces reactive oxygen species (ROS) that may damage DNA. DNA replication, on the other hand, is the process that leads to the copying of essential genetic material during cell division. This process is highly vulnerable to ROS damage, which increases the risk of introducing mutations detrimental to the cell. The molecular clock ensures the temporal separation of both functions, assigning DNA replication and repair to the organism’s rest period (the night for humans) and energy production to the activity period (day).

This type of temporal separation allows the cell to perform its functions safely and efficiently. It is interesting to notice that, although circadian rhythms are found in most living organisms—from archaebacteria, fungi, protozoa, plants, insects, to humans—the molecular clock that governs them diverges over the evolutionary scale.

The discovery of the molecular mechanisms that control circadian rhythms was a turning point in our understanding of the control of diurnal physiology, to the point that, in 2017, the Nobel Prize in Physiology or Medicine was awarded to scientists Jeffrey C. Hall, Michael Rosbash, and Michael W. Young [see box in p. 226] for their pioneering research on the molecular control of circadian rhythms in the fruit fly (Drosophila melanogaster). Encoded in the genome of cells identified in many eukaryotic organisms, molecular clock genes direct the timing of each cellular function. In mammals, these genes encode transcription factors (TFs), a type of highly specialized protein that can control the expression of multiple genes in the genome.

The TFs of the circadian clock can be classified as activators or repressors of gene expression. The activators (CLOCK and BMAL₁) are proteins that work together to drive the expression of thousands of clock-controlled genes (GCGs, figure 1). Together, they promote the expression of the Period (PER_1-3) and Cryptochrome (CRY_1-2) genes, which encode the TF as repressor of the circadian clock, the Period complex. This molecular interplay of activation-repression cycles is unique in that it lasts approximately 24 hours, generating diurnal rhythms of expression of hundreds or thousands of genes depending on the tissue or cell type.

Figure 1. Transcription factors

The activating transcription factors CLOCK/BMAL₁ act together to drive the expression of genes related to metabolism, repair, and cell signaling, among other cellular functions. They also drive the expression of their repressors, PER and CRY, which, when translocated to the cell nucleus, act as repressors, halting gene expression. This cycle of activation and repression of thousands of genes occurs every 24 hours in almost all cells of the body. 

IMPLICATIONS OF THE MOLECULAR CLOCK IN TISSUE PHYSIOLOGY
The self-regulated transcriptional oscillations resulting from the action of the molecular clock coordinate complex gene expression programs throughout the day that are specific to each tissue in the organism. For example, in the brain, clock genes are involved in controlling synaptic plasticity and memory consolidation in the hippocampus, and they regulate astrocyte (non-neural cells in the central nervous system) function and neurotransmitter availability depending on the time-of day.

In the skeletal muscle’s tissue, the clock regulates processes such as insulin sensitivity, mitochondrial biogenesis, and oxidative metabolism. This results in diurnal variations in strength, muscle fatigue, and the adaptive response to exercise. In the bowel, aspects such as intestinal transit, interaction with the microbiota, and epithelial renewal are orchestrated by the molecular clock, resulting in diurnal changes in digestive function. In the heart, the molecular clock directs gene expression programs related to contractility, vulnerability to ischemic events (reduction of blood flood), sympathetic signaling, and the expression of ion channels essential for contractile function (the cell’s ability to modify its size and move). 
In the liver one of the most complex and coordinated temporal segmentations can be found, derived from both nutrition and the molecular clock, which provide efficient management of metabolism throughout the day (figure 2). In this way, energy storage and release are optimally distributed during the day.

Figure 2. Molecular clock coordination

The molecular clock and feeding rhythms coordinate the temporal separation of different liver functions to optimize energy production by anticipating the needs that arise at different times of the day.

Overall, the molecular clock regulates fundamental functions for the body through its diurnal control of gene expression programs. 

CHRONOTHERAPIES: USING CIRCADIAN RHYTHMS TO OPTIMIZE TREATMENTS
The circadian clock organizes physiological, cellular, and molecular processes based on the time-of-day. In medicine, this has direct clinical implications: drug efficacy also varies throughout the day, depending on endogenous rhythms that affect the presence or functionality of their different therapeutic targets. This is the biological basis of chronotherapies, which consider the impact that biological rhythms have on the response to a therapy to optimize its action, maximize health benefits, and minimize potential adverse effects. An example is statins, which are used to lower cholesterol and are most effective when administered at night, particularly short-acting ones. This is because cholesterol synthesis in the liver occurs at night, which is when the drug can most effectively inhibit it.

Also, nighttime administration of antihypertensives such as angiotensin-converting enzyme inhibitors or angiotensin receptor blockers improves blood pressure control during sleep and reduces cardiovascular events, compared to morning therapy.

CHRONOTHERAPIES CONSIDER THE IMPACT THAT BIOLOGICAL RHYTHMS HAVE ON THE RESPONSE TO A THERAPY TO OPTIMIZE ITS ACTION

In our lab, we have shown that therapies based on elevating nicotinamide adenine dinucleotide levels are more effective for the treatment of obesity and prediabetes if administered at the beginning of the active phase in mice, compared to treatment during their rest period. Adjusting the time of drug administration to the time of greatest efficacy also makes it possible to reduce the dose, potentially reducing its side effects.

Undoubtedly, advances in chronobiology are transforming our understanding of human physiology. As we incorporate biological time into clinical practice, a new therapeutic dimension emerges: treating at the right time may be as important as the drug itself.

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