In the early 2000s, Stefano Schiaffino, a muscle physiologist at the University of Padova in Italy, was faced with puzzling results: two seemingly identical experiments involving hind leg muscles in rats had yielded different findings.
Schiaffino and his team were investigating nuclear factor of activated T cells (NFAT), a transcription factor that responds to the level of muscle activity. Despite using similar procedures, the researchers found that in the tissues from one set of animals, NFAT had moved from the cytoplasm into the nucleus in a large proportion of cells, while in tissues from another experiment, this change had not occurred.
The explanation for this difference turned out to be simple: timing. The researcher responsible for one trial had sacrificed the nocturnal animals in the evening, while another had conducted the same procedure for the second trial in the morning. This meant that the first group of animals was more active at the time of measurement than the second. When the scientists repeated the second experiment late in the day, when the animals were more likely to be awake, they observed high levels of NFAT in the nuclei of the muscle cells, essentially replicating the first experiment. “At that time, I’d been working for many years on muscle, but had never thought about the circadian rhythms,” recalls Schiaffino, whose research now focuses on this aspect of muscle biology.
Around the same time, on other side of the globe, muscle physiologist Karyn Esser, then at the University of Illinois at Chicago, also stumbled upon a surprising discovery: genes encoding essential elements of biological clocks being expressed in rat muscle tissue. Esser had been studying how muscles adapt to physical activity, but the unexpected finding piqued her interest—so much so that she decided to take a sabbatical at Northwestern University to investigate it further with geneticist Joseph Takahashi, a pioneer in the field of circadian rhythms. “It was a sort of 90-degree tangent from what I’d been doing,” recalls Esser, now at the University of Florida. “But the more I read about circadian rhythms and clocks, the more [the findings] just made sense. I couldn’t back away once I started.”
I think there has evolved a fairly clear picture that the clock is segregating . . . aspects of metabolism to fit with the rest and activity cycles of the day.—Karyn Esser, University of Florida
A growing body of evidence now points to these cyclical dynamics as mediators of metabolism. Disrupting them may have consequences for health, predisposing individuals to conditions such as diabetes. Research also indicates that these clocks may influence muscle strength and structure, and may even regulate neurological processes such as sleep.
“Clock systems are a sort of core, primordial part of our genome that instruct and prepare cells for the work of using nutrients, moving around, breathing, and [other] fundamental processes,” says Joseph Bass, a clinical endocrinologist at Northwestern University. “This is a story that’s evolving across a lot of different experimental systems—and muscle is now a new experimental system on the block.”
The study of circadian rhythms, the daily cycles that regulate tissue and cell function, was once focused primarily on the suprachiasmatic nucleus, the “master clock” in the brain. Beginning in the late 1990s, scientists began uncovering peripheral clocks—timekeepers located throughout the body—and in 2007, Esser, Takahashi, and their colleagues confirmed their presence in muscles.
Using microarrays to examine the transcriptomes in mouse tissue, the researchers found a number of genes expressed in a rhythmic fashion in muscle.1 These included the clock genes Bmal1 and Per2, as well as genes involved in a variety of functions, such as transcription and metabolism.2
By the time Schiaffino pivoted the course of his research to focus on circadian biology, Esser and her colleagues had published these results. “We were lucky that some pioneering groups had started to do circadian transcriptomics on skeletal muscle,” says Kenneth Dyar, a postdoc at the Institute for Diabetes and Obesity of Helmholtz Zentrum München in Germany who joined Schiaffino’s lab as a graduate student in 2006. “So we had a short list of probable circadian clock–dependent genes because they were cycling over 24 hours.”
Schiaffino and his colleagues decided to knock out Bmal1, a core clock gene, from the muscles of mice. Upon doing so, they discovered that the tissue’s ability to take up glucose in response to insulin was impaired. Further analyses revealed that this was due to decreased levels of proteins such as GLUT4, an insulin-dependent glucose transporter, and TBC1D1, a factor involved in the movement of GLUT4 to the plasma membrane of cells. The researchers also found reduced activity of pyruvate dehydrogenase, an enzyme involved in metabolizing glucose in muscles.3
These findings implied that “the intrinsic muscle clock is an important controller of glucose metabolism,” Schiaffino says. This makes sense, he adds, because a muscle can become a “sponge for glucose” when insulin is released in a healthy animal—after a meal, for example. In fact, skeletal muscle is the body’s major glucose storage unit, responsible for around 70 percent of the body’s uptake of the sugar.
Esser posits that this daily cycle helps muscles prepare to transition from rest (and fasting), when the cells tend to store fuels, to a wakeful period, when the animal is eating and its cells burn fuels to generate energy for activity. In a 2015 study, she and her colleagues discovered that, in mice, genes involved in metabolism were primarily expressed before the rodents entered their active phase in the evening. Prior to sleep, on the other hand, the expression of genes involved in storing glucose and lipids peaked.4
Earlier this year, Charna Dibner of the University of Geneva and her colleagues reported similar outcomes in human muscle cells: disrupting the clock in vitro altered the expression of a number of genes, including those encoding proteins involved in glucose transport and lipid metabolism, and impaired the muscle cells’ ability to take up glucose in response to insulin.5 “It was very consistent with what we see in the mouse, suggesting a very conserved function for the muscle clock,” Esser says.
The muscle clock also appears to regulate the type of fuel that the cell burns. Although active tissues require more energy, cells still need some fuel during sleep, but rather than rely predominately on glucose, which powers contractile activity during waking hours, they burn lipids and amino acids while at rest. By examining mouse tissues at various time points, Schiaffino’s team observed that Bmal1 and its target gene, REV-ERBα, play a key role in this fuel selection process.6
“I think there has evolved a fairly clear picture that the clock is segregating . . . aspects of metabolism to fit with the rest and activity cycles of the day,” says Esser.
Muscle clocks and health
Those who have flown overseas or from one North American coast to another will likely understand what it feels like when the body’s rhythms are out of sync. Traveling long distances across multiple time zones throws off the usual clock-setting cues, or zeitgebers, such as the daily light-dark cycle. Jet lag can cause a variety of temporary symptoms, including dizziness, irritability, and indigestion.
Longer-term perturbations of these rhythms can have lasting effects on the body. Researchers have also found that, in rodents, mutations in circadian clock genes can cause obesity, metabolic syndrome (a cluster of conditions that includes high sugar and low insulin levels in the blood), and diabetes.7,8 A number of epidemiological studies have shown that people who work night shifts are at a higher risk for these conditions as well.9,10
Together, these findings imply that disrupting the muscle clock may predispose individuals to metabolism-related ailments. However, the specific mechanisms underlying this connection have yet to be determined. “If we succeed in showing that the [muscle] clock is involved in this passage from insulin-sensitive to insulin-resistant muscle, then we can imagine therapeutic directions, like using clock modulators,” Dibner says. “But what happens in type 2 diabetes is still a remaining question in humans.”
Muscle clocks may affect aspects of health other than metabolism as well. Esser and her colleagues have found that knocking out tissue-specific timekeepers leads to weaker muscles in mice.11 Her team has also observed, in unpublished work, that perturbing these clocks can influence the physiology of sarcomeres, the basic units of muscle tissue. “When we disrupt the clock, we’re starting to see variations in the length of the sarcomere along a single fiber,” Esser says. “[This can] affect force generation, and the prediction is that you might make this muscle more susceptible to injury.”
Muscle clocks can also influence rodents’ slumber. Neuroscientist Ketema Paul of the University of California, Los Angeles, and his colleagues revealed that removing Bmal1 from the muscles of mice increased the amount of time the rodents spent in non-REM sleep.12 Moreover, the researchers found that, while knocking out this gene in the brain or muscles impaired the rodents’ ability to regain normal sleep patterns after six hours of forced wakefulness, increasing Bmal1 expression in muscle made them better at bouncing back after sleep deprivation. In addition to providing a better understanding of the mechanisms that mediate sleep homeostasis, Paul says, these findings hint at potential treatments that target nonbrain tissues for sleep disorders or improve recovery after sleep loss.
Many questions remain about the way muscle keeps time, such as how external signals are incorporated. Studies, primarily in rodents, suggest that feeding and exercise may serve as primary environmental cues. Oxygen levels may also play a role. A recent study by Bass and his colleagues at Northwestern University revealed that muscle clocks interact with the hypoxia-inducible factor (HIF) pathway, which responds to the availability of oxygen.13 When the muscles are rapidly depleted of oxygen—during exercise, for example—HIF helps the body transition from metabolizing glucose through mitochondrial respiration, an aerobic process, to anaerobic glycolysis, which takes place in the cytoplasm and produces lactic acid.
“The clock seems to be gating the capacity of the skeletal muscle to activate HIF and HIF-dependent metabolism at different times of day,” says Clara Peek, a biochemist at Northwestern University who took part in that work. “I think [this] new molecular connection in mice possibly explains some of the connections between metabolism and the clock in muscle.”
How the muscle clocks interact with other peripheral clocks also remains an open question. The muscles do not act alone. With metabolism, for example, researchers have found that other peripheral timekeepers, such as those in the liver, pancreas, and adipose tissue, also play a part. Studies by Dibner’s group14 and others15 have shown that the clocks cycling within the pancreas’ islet cells are crucial for maintaining proper insulin secretion.
See “Out of Sync”
If we succeed in showing that the muscle clock is involved in this passage from insulin-sensitive to insulin-resistant muscle, then we can imagine therapeutic directions, like using clock modulators.—Charna Dibner, University of Geneva
“In animals with a central nervous system, clocks are organized—to the best of our approximation—in a hierarchical manner,” says Bass. “At the top of the pyramid is the so-called master clock, which is aligned with the light/dark cycle, and through mechanisms that are still not fully known, it orchestrates the alignment of peripheral tissue clocks with the environmental cycle.”
And when it comes to uncovering the molecular pathways that keep time in muscles and control that clock’s effects on the body, Esser says, researchers have only just begun. “There’s a lot still to learn.”
- B.H. Miller et al., “Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation,” PNAS, 104:3342–47, 2007.
- J.J. McCarthy et al., “Identification of the circadian transcriptome in adult mouse skeletal muscle,” Physiol Genomics, 31:86–95, 2007.
- K.A. Dyar et al., “Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock,“ Mol Metab, 3:29–41, 2014.
- B.A. Hodge et al, “The endogenous molecular clock orchestrates the temporal separation of substrate metabolism in skeletal muscle,” Skeletal Muscle, 5:17, 2015.
- L. Perrin et al., “Transcriptomic analyses reveal rhythmic and CLOCK-driven pathways in human skeletal muscle,” eLife, 7:e34114, 2018.
- K.A. Dyar et al., “Transcriptional programming of lipid and amino acid metabolism by the skeletal muscle circadian clock,” PLOS Biol, doi:10.1371/journal.pbio.2005886, 2018.
- F.W. Turek et al., “Obesity and metabolic syndrome in circadian clock mutant mice,” Science, 308:1043–45, 2005.
- B. Marcheva et al., “Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes,” Nature, 466:627:31, 2010.
- C. Vetter et al., “Night shift work, genetic risk, and type 2 diabetes in the UK Biobank,” Diabetes Care, 41:dc171933, 2018.
- S. Fun et al., “Meta-analysis on shift work and risks of specific obesity types,” Obes Rev, 19:28–40, 2018.
- E.A. Schroder et al., “Intrinsic muscle clock is necessary for musculoskeletal health,” J Physiol, 593:5387–404, 2015.
- J.C. Ehlen et al., “Bmal1 function in skeletal muscle regulates sleep,” eLife, 6:e26557, 2017.
- C.B. Peek et al., “Circadian clock interaction with HIF1α mediates oxygenic metabolism and anaerobic glycolysis in skeletal muscle,” Cell Metab, 25:86–92, 2017.
- C. Saini et al., “A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells,” Diabetes Obes Metab, 18:355–65, 2016.
- M. Perelis et al., “Pancreatic cell enhancers regulate rhythmic transcription of genes controlling insulin secretion,” Science, 350:aac4250, 2015.
Correction (September 10): The original version of this post contained the wrong subhead. The Scientist regrets the error.