Why does a rapid change in time zone or seasonally setting clocks forward or back throw people for such a loop? We can chalk this up to internal timepieces. In animals and humans, biological “circadian clocks” regulate behaviors and bodily processes—including sleep/wake cycles, changes in blood pressure, and body temperature fluctuations—to harmonize these activities with daily, rhythmic changes in the environment, most notably day/night cycles. One commonly observed sign of the strong influence of circadian rhythms is jet lag, the sleep disturbances and other symptoms that occur after flying across multiple time zones. However, disrupting circadian rhythms can have more insidious consequences.
In humans, misaligning normal circadian rhythms with behaviors such as sleep and eating—for example, by working the night shift—increases vulnerability to diabetes, obesity, and other metabolic problems. One likely reason for these problems is the fact that the circadian clock has a critical relationship with metabolic pathways important to maintaining normal energy balance. For example, the synthesis of glucose (sugar) and fats and the release of glucose into the blood by the liver are governed by the circadian clock. Understanding how circadian rhythm and metabolism are linked therefore could help in the design of strategies to reduce vulnerability to metabolic diseases, and is an area of intense investigation.
Researchers working on the link between the circadian clock and metabolism are actually faced with an additional layer of complexity. Animals and humans possess two types of clocks—a “core” circadian clock, located in the brain, and “peripheral,” tissue specific circadian clocks. The core or master clock responds to cues such as light and dark, nutrient uptake, and temperature. For this clock, a set of core genes has been identified. Some of these clock genes encode activator proteins that help “turn on” target genes, and some of them encode repressor proteins that help “turn off” target genes. Cleverly, the clock genes interact to generate cyclical oscillations in the levels of proteins they encode—e.g., products of activator genes “turn on” repressor genes, and products of repressor genes “turn off” activator genes, so that levels of activator and repressor proteins wax and wane. This leads to downstream effects that occur with regularity within each 24-hour period. The master clock also synchronizes the tissue specific clocks, which use much of the same genetic machinery. However, the clocks present in each cell can also act and respond on their own, setting up local, tissue specific rhythms governing gene activation or repression, and subsequent cellular processes—including metabolic activities.
Intriguingly, researchers have found that some of the factors regulating tissue specific clocks are shared with pathways regulating metabolism. But is there a single factor (or factors) that acts as a key molecular link between circadian rhythms and metabolism? The Rev-erb-α protein has emerged as a candidate. Rev-erb-α is a nuclear receptor protein, meaning that one part of the protein functions as a sensor for a specific signal molecule, or ligand, while another part of the protein functions to bind to DNA in the cell’s nucleus and regulate gene activation. The ligand for Rev-erb-α is heme, a small molecule that is integral to many metabolic pathways and whose cellular levels oscillate in a circadian manner. When bound by heme, Rev-erb-α binds to specific target DNA sequences to repress activation of nearby genes. In the context of the circadian clock, Rev-erb-α is believed to regulate how much protein is made by a master clock gene called Bmal1. In the context of metabolism, Rev-erb-α regulates genes involved in glucose metabolism, regulates lipid and bile acid production in the liver, and is necessary for the maturation of fat cells from precursor cells. But, because mice genetically engineered to lack Rev-erb-α do not display a strongly disrupted circadian rhythm, a question has remained as to whether Rev-erb-α plays an accessory role in the circadian clock or is truly a central factor that could link the clock with metabolism.
A recent report has helped to clarify the role of Rev-erb-α. Through a series of experiments in mice, researchers found evidence suggesting Rev-erb-α doesn’t just regulate the Bmal1 gene, but acts cooperatively with the BMAL1 protein at numerous DNA target sites in the liver to regulate the activity of both metabolic and circadian clock genes. Moreover, they found that there is overlap in activity between Rev-erb-α and the closely related protein Rev-erb-β, which could explain why lacking only Rev-erb-α doesn’t result in disturbed circadian rhythms. To test this idea further, the researchers used a wheel running behavior test, a standard method to ascertain circadian dysfunction in mice. In this test, mice first have their rest and active periods artificially synchronized to a cycle of 12 hours of light alternating with 12 hours of darkness. During this initial step, baseline measures of wheel running rhythms are established; unlike humans, mice are nocturnal and will normally show the most activity during the dark. Then, the mice are put in constant darkness, and their wheel running behavior is assessed for changes. In the absence of the alternating light/dark cues, normal mice will maintain a circadian rhythm of rest and activity, although the total cycle length will shrink to slightly less than 24 hours. When shifted to constant darkness, mice lacking only one Rev-erb protein showed little or no change in wheel running behavior compared to normal mice, although mice lacking Rev-erb-α experienced a further shortening of the cycle length. In contrast, mice lacking both the Rev-erbs throughout their bodies showed weak synchronization during the light/dark cycle and, when put into total darkness, decreased and severely fragmented activity and other features indicating circadian dysfunction. As these features are also found in mice lacking BMAL1, their findings suggest both that the Rev-erbs can compensate for each other and that there is a much more central role for Rev-erbs in rhythmic behaviors.
The increasing evidence that Rev-erbs play a central role in both circadian rhythm and metabolism makes them promising therapeutic targets. Knowing that the natural ligand for Rev-erbs is heme, scientists recently synthesized and tested two small molecules for their ability to stimulate Rev-erb effects on circadian rhythms and metabolic outputs in mice. When administered to mice, these molecules were able to repress Rev-erb responsive metabolic genes in the liver and the oscillation of a core circadian clock gene in the brain. Interestingly, when subjected to the wheel running behavior test, mice that received single injections of either of the two drugs during the total darkness phase experienced transient but drastic disruptions in running behavior. However, the drugs were much less disruptive when tested under normal light/dark conditions, resulting in only a delay in activity. Drug administration to normal weight mice caused a loss in fat weight and an increase in metabolic rate without any change in food intake and a decrease in activity levels. This appeared to be due to an increase in the levels of enzymes that burn fat. When administered to mice with diet-induced obesity, the drugs improved their metabolic profile, with much greater weight loss compared to normal weight mice, a drop in triglycerides, and cholesterol levels cut nearly in half. The results in both normal weight and obese mice suggest that the drugs exert their effects through Rev-erbs by modifying genetic programs in a way that leads to increased burning of fatty acids and glucose, improving the metabolic profile.
Another potential therapeutic target that has emerged from the study of circadian rhythm and metabolism is a protein called HDAC3. HDAC3 is an enzyme that causes transitory structural changes along the chromosomes called “histone modifications,” which affect gene activation. When HDAC3 is recruited to sites in the genome, genes at those sites tend to be turned “off,” and when it is absent, those genes are free to be turned “on.” Scientists have studied the activity of HDAC3 in the liver and found that, directed by Rev-erb-α, HDAC3 drives circadian oscillations in the activation of genes controlling fat synthesis in liver cells. Now, a team of researchers has made another intriguing discovery. They found that while depleting HDAC3 in adult mouse livers leads to potentially harmful accumulation of fat in the liver, the fat is sequestered within little droplets surrounded by protective coatings and the mice actually have better insulin sensitivity than mice with normal levels of HDAC3, without any changes in body weight. By examining molecular pathways in the liver involved in the generation, storage, and burning of fat and in the generation of glucose, the researchers determined a likely mechanism: it appears that the constant, rather than rhythmic, activation of lipid-synthesizing and sequestering genes caused by the absence of HDAC3 redirects precursor molecules away from making glucose and toward the synthesis and storage of fat. These findings uncover a previously unknown means for regulating glucose generation and insulin sensitivity in the liver. By distinguishing liver fat accumulation caused by disruption of circadian control of metabolism from liver fat accumulation caused by diet, the findings also suggest that there are multiple pathways by which the liver can end up storing fat, and their potential for harm—or relative benefit—will need to be considered during therapeutic development and application.
In addition to identifying key molecular aspects of circadian control of metabolism, there is another facet to this problem: can we use the knowledge that there is circadian control of metabolism to develop behavioral strategies as well as pharmacologic ones to thwart metabolic disease? Researchers recently tested in mice whether timing of feeding, even with a high-fat diet, can influence weight gain and related metabolic problems. They compared four groups of mice: one group was given a normal diet and allowed to eat at any time (“ad lib”); another group was given a normal diet, but only during an 8-hour window at night, the natural feeding time for mice (“time-restricted”); a third group was given a high fat diet ad lib; and the fourth group was given a high-fat diet, but time-restricted. At the end of 100 days, the researchers found that, while the mice in all four groups consumed the same number of calories, mice on the time-restricted feeding regimens appeared to have better metabolic profiles. Strikingly, despite having the same diet, mice on the high-fat diet but time-restricted feeding regimen were leaner than their ad lib counterparts. At the molecular level, it appears that, by imposing a feeding rhythm, the time restricted feeding regimen “reprogrammed” activation of pathways governing glucose and fat metabolism in the liver and prevented the circadian and metabolic dysfunction that can occur with a high-fat diet.
As the understanding of the role of factors such as the Rev-erbs and HDAC3 in the circadian control of metabolism continues to evolve, it is helping researchers to test new and existing therapeutic compounds that may help in the fight against diseases such as diabetes and obesity. At the same time, the improved glucose tolerance, protection from obesity, and protection from fatty liver disease seen in mice on a time-restricted, high-fat diet—as well as the metabolic improvements seen in mice on a time-restricted, normal diet—is encouraging, and provides hope that behavioral strategies based on understanding the relationship between circadian rhythms and metabolism can also be developed to prevent metabolic disease.
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