Shedding light on the dark hours

Why do asthma, heart attacks and many other health conditions tend to strike in the early hours of the morning? One possible explanation for this mysterious phenomenon has been discovered by researchers from Prof. Gad Asher’s laboratory at the Weizmann Institute of Science’s Biomolecular Sciences Department. In a study published in Cell Metabolism, the scientists found that a key component of our circadian clock – the 24-hour internal molecular clock that ticks away in every single cell – also regulates the body’s response to oxygen deficiency. This component, which undergoes changes over the course of the day and night, could affect the timing of outbreaks of diseases that are influenced by the body’s oxygen cycle.

As breathing creatures, our ability to sense and respond to a shortage of oxygen is as vital to us as the air we breathe. The 2019 Nobel Prize in Physiology or Medicine was awarded to three researchers who had discovered the hypoxia-inducible factor 1-alpha (HIF-1α), the key protein that determines how each cell responds to a lack of oxygen. As long as there is plenty of oxygen, the protein remains unstable and breaks down rapidly; but when there is a shortage of oxygen, it stabilizes, accumulates and enters the cells’ nuclei where it activates numerous genes vital for responding to oxygen deficiency.

It turns out, however, that HIF-1α is not the only key player. In a new study conducted in Asher’s lab, led by doctoral student Vaishnavi Dandavate and Dr. Nityanand Bolshette, the team discovered that the BMAL1 protein, a key component of our circadian clocks, also plays an important role in the body’s response to oxygen deficiency and is necessary for stabilizing and activating the HIF-1α protein. Moreover, the study also suggests that BMAL1 is more than just a “reinforcement” and that it plays a role independent of HIF-1α in activating the body’s plan for dealing with oxygen shortage. These new findings could explain why the body’s response to oxygen deficiency and its coping with various medical conditions change over the course of the day and night.

Day protein, night protein

Researchers from Asher’s lab, which for years has been studying the connection between metabolism and circadian clocks, had previously discovered that liver tissue responds differently to oxygen shortage at different times of the day. To deepen their understanding of the relationship between oxygen, liver tissue and the circadian clocks, they created three groups of genetically engineered mice that could not produce either one or both of the above-mentioned proteins in their liver tissue: The first group did not produce HIF-1α, the protein that regulates the response to oxygen deficiency; the second group did not produce BMAL1, the key component of the circadian clock; and the third one did not produce either of them. The researchers then examined what happened to each group when oxygen levels were reduced. They found that, in the absence of BMAL1, the HIF-1α protein failed to accumulate as it does in a normal response to oxygen shortage. Moreover, they discovered that these two proteins – separately and together – are largely responsible for activating the genetic response needed to deal with oxygen shortage.

“The mechanism we discovered, which combines both proteins, is probably the main mechanism by which mammals cope with oxygen deficiency,” says Asher. “These and other findings helped us understand that the circadian clock not only responds to oxygen deficiency, as was already known, but that it actually activates the body’s mechanism for dealing with oxygen deficiency.”

The scientists were especially surprised to discover that, unlike the mice in the control group and those whose liver tissue failed to produce one of the proteins, either HIF-1α or BMAL1, the mice that lacked both of these proteins had very low survival rates under oxygen deficiency conditions in a time-dependent manner: Their mortality rates were high during the hours of darkness but not under identical conditions during daylight hours. These findings indicate that the combination of HIF-1α and BMAL1 plays a significant, time-dependent role in dealing with oxygen deficiency.

"The circadian clock not only responds to oxygen deficiency but also activates the body’s mechanism for dealing with it"

“We know that BMAL1 undergoes changes in the course of the natural circadian cycle, which could explain why mortality rates vary throughout the day and perhaps also why oxygen deficiency-related diseases are time-dependent,” Asher says.

The next stage of the study was to clarify the cause of death in those mice that had been genetically engineered to produce neither of the two proteins in their liver. The researchers were surprised to discover only slight damage to the tissue, which was not enough to explain the mortality on its own. They also found that these mice had low blood oxygen levels to begin with, even before they were exposed to oxygen shortage conditions. These findings led to the suspicion that the cause of death was connected to damage to the lungs’ ability to absorb oxygen and not to the liver’s response to oxygen deficiency. Many people with liver disease, of all levels of severity, also develop a pathological condition called hepatopulmonary syndrome, in which blood vessels in the lungs dilate, leading to accelerated blood flow in the lungs that reduces the ability to absorb oxygen. The researchers discovered the same phenomenon in mice lacking both the HIF-1α and the BMAL1 in their livers. These mice are now being used as the first genetic research model of its kind for the hepatopulmonary syndrome, in studies that might shed light on the mechanisms involved in this condition.

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At least 25 percent of people with liver disease also develop lung disease.

“We identified increased production of nitric oxide in the lungs, which causes the blood vessels to dilate. As a result, blood flows through the lungs much more quickly and does not supply oxygen efficiently,” Asher adds. “We still do not know through which mechanisms the liver damage affects lung function, but the initial findings from our genetic mouse model point to an interesting group of proteins that could be part of the communication between the liver and the lungs. In mice that developed the hepatopulmonary syndrome, this communication was disrupted. If these proteins are also produced in human patients and are indeed connected to the syndrome, they might serve as a target for a future therapy.”

Also participating in the study were Rachel Van Drunen, Dr. Gal Manella, Dr. Ippei Kawano, Dr. Marina Golik and Dr. Yaarit Adamovich from Weizmann’s Biomolecular Sciences Department; and Dr. Hanna Bueno-Levy and Mirie Zerbib from Weizmann’s Veterinary Resources Department.

Prof. Gad Asher's research is supported by the Dr. Barry Sherman Institute for Medicinal Chemistry.