Novel technique helps explain why bright light keeps us awake

Source: Salk Institute
Date: 10/15/2019
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Researchers discover a way to make electron microscopy more detailed and precise by visualizing the activation of brain circuits over long distances

In recent decades, scientists have learned a great deal about how different neurons connect and send signals to each other. But it’s been difficult to trace the activity of individual nerve fibers known as axons, some of which can extend from the tip of the toe to the head. Understanding these connections is important for figuring out how the brain receives and responds to signals from other parts of the body.

Researchers at the Salk Institute and UC San Diego are reporting a novel technique for tracing these connections and determining how neurons communicate. The team used this technique to uncover details about how the brain responds to light signals received by the retina in mice, published October 15, 2019, in Cell Reports.

“This study is a breakthrough because no one could figure out how to study these connections before,” says Salk Professor Satchidananda Panda, co-corresponding author of the paper. “This new technique has enabled us to go well beyond the limitations of electron microscopy.”

The new method makes use of several different laboratory techniques to understand a type of neuron called intrinsically photosensitive retinal ganglion cells (ipRGCs). These cells, which are found in the retina, in the back of the eye, express a protein called melanopsin that senses blue light.

The Salk and UCSD teams used a virus to deliver a protein called a mini-singlet oxygen-generating protein (mini-SOG) to the ipRGCs, so that the cells could be viewed in more detail under election microscopy. The system was designed to tether the mini-SOG to the membranes of the light-sensitive cells so that the entire neuron, including its long axons that reach out to different parts of the brain, can be easily tracked under both light and electron microscope.

“Thanks to development and application of new genetically introduced probes for correlated multiscale light and electron microscopic imaging, our Salk and UCSD-based research teams were able to follow the small processes emanating from nerve cells over centimeters, all the way from the retina to multiple places where they connect to brain regions critical to circadian rhythms, eye reflexes and vision,” says Mark Ellisman, distinguished professor of neurosciences at UC San Diego and adjunct professor at Salk, who co-led the work. “We were able to obtain unprecedented three-dimensional information about the machinery required for these neuronal cells to signal the next neurons in the complex circuits.”

Most of the previous work with mini-SOGs has been done in cell lines, and using them in mice, to map how neurons from the retina wire the brain, was a first, according the researchers. The method enabled them to glean new information about the connections between ipRGCs and different parts of the brain.

The ipRGCs are known to connect to many brain regions that regulate very different tasks. The cells tell one part of the brain how bright it is outside so that our pupil can rapidly close—in less than a second. The same ipRGCs also connect to the master clock in the brain that regulates our sleep-wake cycle. “However, it takes several minutes of bright light to make us fully awake,” Panda says. “How the same ipRGCs do these very different tasks with different time scales was not clear until now.”

The investigators found that the difference has to do with the way that light detected by the retina reaches the brain. By delivering the mini-SOG to the eyes of the mice, they were able to trace the signal to the part of the brain that constricts the pupil in response to light.

“These connections were much stronger—similar to water pouring out of a garden hose,” Panda says. “Whereas the connection between the ipRGCs and the master clocks were weaker—more like drip irrigation.” Because the ipRGCs deliver the light signal to the circadian center through this slower drip system, it takes longer for any meaningful information to reach and reset the brain clock.

“This research helps explain why, when you get up in the night to get a drink of water and turn on the light for a few seconds, you’re usually able to go right back to sleep,” Panda says. “But if you hear a noise outside and end up walking around your house for half an hour with the lights on, it’s much harder. There will be enough light signal reaching the master clock neurons in the brain that ultimately wakes up the rest of the brain.”

Panda says that the new technique will be useful for studying other neural connections, as the researchers can essentially use the same viruses to express mini-SOGs in any neuron and ask how different neurons make connections to different appendages.

“These findings and methods open new opportunities for brain researchers studying the long-distance wiring of brains in normal and in animal models of human disease,” adds Ellisman.

Other researchers on the paper were Luis Rios, Hiep Le, Yu Hsin Liu, Masatoshi Hirayama, Ludovic Mure, and Megumi Hatori of Salk and Keun-Young Kim, Alex Perez, Sébastien Phan, Eric Bushong, Thomas Deerinck, Maya Ellisman, Varda Lev-Ram, Suyeon Ju, Sneha Panda, Sanghee Yoon, and Mark Ellisman of the University of California at San Diego.

The research was supported by National Institutes of Health grants EY 016807, P41GM103412, RO1 GM086197, and RO1 NS027177.

Two studies explore whether time of day can affect the body’s response to exercise

Source: Cell Press
Date: 04/18/2019
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Two papers appearing April 18 in the journal Cell Metabolism confirm that the circadian clock is an important factor in how the body responds to physical exertion. The studies focused on different components of exercise, thereby complementing each other. Based on this work alone, it’s too early to say when the best time is for you to go for a jog. But at least in the lab, exercise in the evening seems to be more productive, although human lifestyles are much more complicated and so this area of research is only just beginning.

“It’s quite well known that almost every aspect of our physiology and metabolism is dictated by the circadian clock,” says Gad Asher of the Department of Biomolecular Sciences at the Weizmann Institute of Science, who is senior author of one of the studies. “This is true not only in humans but in every organism that is sensitive to light. We decided to ask whether there is a connection between the time of day and exercise performance.”

“Circadian rhythms dominate everything we do,” adds Paolo Sassone-Corsi of the Center for Epigenetics and Metabolism at the University of California, Irvine, who is senior author of the other paper. “Previous studies from our lab have suggested that at least 50% of our metabolism is circadian, and 50% of the metabolites in our body oscillate based on the circadian cycle. It makes sense that exercise would be one of the things that’s impacted.”

Both research teams looked at the association between time of day and exercise performance primarily in mice. Because mice are nocturnal, one thing they had to do was translate mouse timing to human timing, by distinguishing between the active phase and resting phase of the mice rather than using numbers on the clock.

Asher’s group started by putting mice in treadmills at different times of day within their active phase. They examined the exercise capacity of mice upon different exercise intensities and regimens and found that overall exercise performance is substantially better (about 50% on average and more in some protocols) in the “mouse evening” (toward the end of their active time) compared to the morning hours. These daily differences were diminished in mice that had mutant clocks–supporting a potential role of the clock in the observed variance in exercise performance.

To identify a potential determinant of daily variance in exercise performance, they applied high-throughput transcriptomics and metabolomics on muscle tissue. The researchers found that in response to exercise in the “mouse evening,” there were higher levels of a metabolite called ZMP (5-aminoimidazole-4-carboxamide ribonucleotide). ZMP is known to activate metabolic pathways that are related to glycolysis and fatty acid oxidation through activation of AMPK, which is a master cellular metabolic regulator. Therefore, it is likely to contribute to the increased exercise capacity in the evening. “Interestingly, ZMP is an endogenous analog of AICAR [aminoimidazole carboxamide riboside], a compound that some athletes use for doping,” Asher says.

The researchers also studied 12 humans and found similar effects. Overall, the people in the study had lower oxygen consumption while exercising in the evening compared with the morning; this translated to better exercise efficiency.

Sassone-Corsi’s team also put mice on treadmills, but they had a different approach. Using high-throughput transcriptomics and metabolomics to look at a wide range of possible factors, they characterized the changes in the mice’s muscle tissue that occur in response to exercise. This allowed them to look at processes like glycolysis (which contributes to sugar metabolism and energy production) and lipid oxidation (fat burning).

They found that a protein called hypoxia-inducible factor 1-alpha (HIF-1α) plays an important role and that it is activated by exercise in different ways depending on the time of day. HIF-1α is a transcription factor that is known to stimulate certain genes based on oxygen levels in tissue. “It makes sense that HIF-1α would be important here, but until now we didn’t know that its levels fluctuate based on the time of day,” Sassone-Corsi says. “This is a new finding.”

Based on the work from the UC Irvine team, exercise seemed to have the most beneficial impact on the metabolism at the beginning of the active phase phase (equivalent to late morning in humans) compared with the resting phase (evening).

The researchers note that even though circadian clocks have been conserved throughout evolution, translating the findings to humans is not so straightforward. One reason is that humans have more variation in their chronotypes than mice living in a lab. “You may be a morning person, or you may be a night person, and those things have to be taken into account,” Sassone-Corsi says.

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Cell Metabolism, Ezagouri, Zwighaft, and Sobel et al.: “Physiological and Molecular Dissection of Daily Variance in Exercise Capacity” https://www.cell.com/cell-metabolism/fulltext/S1550-4131(19)30141-X DOI: 10.1016/j.cmet.2019.03.012

This study was supported by the European Research Council and an EMBO Young Investigator Award. It was also supported by Fonds de Dotation AGIR pour les Maladies Chroniques and a fellowship from the Placid Nicod Foundation.

Cell Metabolism, Sato et al.: “Time of Exercise Specifies the Impact on Muscle Metabolic Pathways and Systemic Energy Homeostasis” https://www.cell.com/cell-metabolism/fulltext/S1550-4131(19)30183-4 DOI: 10.1016/j.cmet.2019.03.013

This study was supported by the Novo Nordisk Foundation, the Swedish Diabetes Foundation, the Swedish Research Council, the National Institutes of Health, INSERM, and the Della Martin Foundation.

Clinical trial shows alternate-day fasting a safe alternative to caloric restriction

Source: Cell Press
Date: 08/27/2019
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In recent years there has been a surge in studies looking at the biologic effects of different kinds of fasting diets in both animal models and humans. These diets include continuous calorie restriction, intermittent fasting, and alternate-day fasting (ADF). Now the largest study of its kind to look at the effects of strict ADF in healthy people has shown a number of health benefits. The participants alternated 36 hours of zero-calorie intake with 12 hours of unlimited eating. The findings are reported August 27 in the journal Cell Metabolism.

“Strict ADF is one of the most extreme diet interventions, and it has not been sufficiently investigated within randomized controlled trials,” says Frank Madeo, a professor of the Institute of Molecular Biosciences at Karl-Franzens University of Graz in Austria. “In this study, we aimed to explore a broad range of parameters, from physiological to molecular measures. If ADF and other dietary interventions differ in their physiological and molecular effects, complex studies are needed in humans that compare different diets.”

In this randomized controlled trial, 60 participants were enrolled for four weeks and randomized to either an ADF or an ad libitum control group, the latter of which could eat as much as they wanted. Participants in both groups were all of normal weight and were healthy. To ensure that the people in the ADF group did not take in any calories during fast days, they underwent continuous glucose monitoring. They were also asked to fill in diaries documenting their fasting days. Periodically, the participants had to go to a research facility, where they were instructed on whether to follow ADF or their usual diet, but other than that they lived their normal, everyday lives.

Additionally, the researchers studied a group of 30 people who had already practiced more than six months of strict ADF previous to the study enrollment. They compared them to normal, healthy controls who had no fasting experience. For this ADF cohort, the main focus was to examine the long-term safety of the intervention.

“We found that on average, during the 12 hours when they could eat normally, the participants in the ADF group compensated for some of the calories lost from the fasting, but not all,” says Harald Sourij, a professor at the Medical University of Graz. “Overall, they reached a mean calorie restriction of about 35% and lost an average of 3.5 kg [7.7 lb] during four weeks of ADF.”

The investigators found several biological effects in the ADF group:

* The participants had fluctuating downregulation of amino acids, in particular the amino acid methionine. Amino acid restriction has been shown to cause lifespan extension in rodents.

  • They had continuous upregulation of ketone bodies, even on nonfasting days. This has been shown to promote health in various contexts.
  • They had reduced levels of sICAM-1, a marker linked to age-associated disease and inflammation.
  • They had lowered levels of triiodothyronine without impaired thyroid gland function. Previously, lowered levels of this hormone have been linked to longevity in humans.
  • They had lowered levels of cholesterol.
  • They had a reduction of lipotoxic android trunk fat mass–commonly known as belly fat.

“Why exactly calorie restriction and fasting induce so many beneficial effects is not fully clear yet,” says Thomas Pieber, head of endocrinology at the Medical University of Graz. “The elegant thing about strict ADF is that it doesn’t require participants to count their meals and calories: they just don’t eat anything for one day.”

The investigators point to other benefits that ADF may have, compared with continuous calorie restriction. Previous studies have suggested calorie-restrictive diets can result in malnutrition and a decrease in immune function. In contrast, even after six months of ADF, the immune function in the participants appeared to be stable.

“The reason might be due to evolutionary biology,” Madeo explains. “Our physiology is familiar with periods of starvation followed by food excesses. It might also be that continuous low-calorie intake hinders the induction of the age-protective autophagy program, which is switched on during fasting breaks.”

Despite the benefits, the researchers say they do not recommend ADF as a general nutrition scheme for everybody. “We feel that it is a good regime for some months for obese people to cut weight, or it might even be a useful clinical intervention in diseases driven by inflammation,” Madeo says. “However, further research is needed before it can be applied in daily practice. Additionally, we advise people not to fast if they have a viral infection, because the immune system probably requires immediate energy to fight viruses. Hence, it is important to consult a doctor before any harsh dietary regime is undertaken.”

In the future, the researchers plan to study the effects of strict ADF in different groups of people including people with obesity and diabetes. They also plan to compare ADF to other dietary interventions and to further explore the molecular mechanisms in animal models.

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The research was primarily funded by the Austrian Science Fund; the Austrian Federal Ministry of Education, Science and Research; the University of Graz, and the field of excellence program BioHealth. Additional funding and declarations of interests can be found in the study.

Cell Metabolism, Stekovic, Hofer, and Tripolt et al.: “Alternate day fasting improves physiological and molecular markers of aging in healthy, non-obese humans.” https://www.cell.com/cell-metabolism/fulltext/S1550-4131(19)30429-2

Researchers identify a gene linked to needing less sleep

Source: Cell Press
Date: 08/28/2019
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The genetics of circadian rhythms have been well studied in recent years, but much less is known about other types of genes that play a role in sleep, specifically those that regulate how much sleep our bodies require. Now, by studying a family with several members who require significantly less sleep than average, a team of researchers has identified a new gene that they believe has a direct impact on how much someone sleeps. They report their findings on August 28 in the journal Neuron.

“It’s remarkable that we know so little about sleep, given that the average person spends a third of their lives doing it,” says Louis Ptáček, a neurologist at the University of California, San Francisco (UCSF), and one of the paper’s two senior authors. “This research is an exciting new frontier that allows us to dissect the complexity of circuits in the brain and the different types of neurons that contribute to sleep and wakefulness.”

The family whose DNA led to the identification of this gene is one of several that Ptáček and UCSF geneticist Ying-Hui Fu, the paper’s other senior author, are studying and includes several members who function normally on only six hours of sleep. The gene, ADRB1, was identified using genetic linkage studies and whole-exome sequencing, which revealed a novel and very rare variant.

The first step in deciphering the role of the gene variant involved studying its protein in the test tube. “We wanted to determine if these mutations caused any functional alterations compared with the wild type,” Fu says. “We found that this gene codes for ß1-adrenergic receptor, and that the mutant version of the protein is much less stable, altering the receptor’s function. This suggested it was likely to have functional consequences in the brain.”

The researchers then conducted a number of experiments in mice carrying a mutated version of the gene. They found that these mice slept on average 55 minutes less than regular mice. (Humans with the gene sleep two hours less than average.) Further analysis showed that the gene was expressed at high levels in the dorsal pons, a part of the brain stem involved in subconscious activities such as respiration and eye movement as well as sleep.

Additionally, they discovered that normal ADRB1 neurons in this region were more active not only during wakefulness, but also during REM (rapid eye movement) sleep. However, they were quiet during non-REM sleep. Furthermore, they found that the mutant neurons were more active than normal neurons, likely contributing to the short sleep behavior.

“Another way we confirmed the role of the protein was using optogenetics,” Fu explains. “When we used light to activate the ADRB1 neurons, the mice immediately woke up from sleep.”

Ptáček acknowledges some limitations of using mice to study sleep. One of these is that mice exhibit different sleep patterns than humans, including, for example, sleeping in a fragmented pattern, rather than in a single continuous period. “But it’s challenging to study sleep in humans, too, because sleep is a behavior as well as a function of biology,” he says. “We drink coffee and stay up late and do other things that go against our natural biological tendencies.”

The investigators plan to study the function of the ADRB1 protein in other parts of the brain. They also are looking at other families for additional genes that are likely to be important. “Sleep is complicated,” Ptáček notes. “We don’t think there’s one gene or one region of the brain that’s telling our bodies to sleep or wake. This is only one of many parts.”

Fu adds that the work may eventually have applications for developing new types of drugs to control sleep and wakefulness. “Sleep is one of the most important things we do,” she says. “Not getting enough sleep is linked to an increase in the incidence of many conditions, including cancer, autoimmune disorders, cardiovascular disease, and Alzheimer’s.”

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This work was funded by the National Institute of Neurological Disorders and Stroke Informatics Center for Neurogenetics and Neurogenomics, the National Institutes of Health (NIH), and the William Bowes Neurogenetics Fund.

Neuron, Shi et al. “A rare mutation of β1-adrenergic receptor affects sleep/wake behaviors.” https://www.cell.com/neuron/fulltext/S0896-6273(19)30652-X

Pilot study finds time-restricted eating has benefits for people at risk for diabetes

Source: Cell Press
Date: 12/05/2019
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Previous studies have looked at employing time-restricted eating (TRE), a form of intermittent fasting, as a way to lose weight and improve health measures such as blood sugar and blood pressure in mice and healthy people. But in a study publishing December 5 in the journal Cell Metabolism, researchers for the first time looked at the effects of TRE in people who had been diagnosed with metabolic syndrome and therefore were at a higher risk of diabetes, heart disease, and stroke. The investigators found that when participants restricted their eating to 10 hours or less over a period of 12 weeks, they lost weight and some symptoms of metabolic syndrome improved.

“There has been a lot of discussion about intermittent fasting and what time window people should eat within to get the benefits of this kind of diet,” says co-corresponding author Satchidananda Panda, a Professor at the Salk Institute. “Based on what we’ve observed in mice, a 10-hour time window seems to convey these benefits. At the same time, it’s not so restrictive that people can’t follow it long-term.”

Metabolic syndrome is characterized by having three or more of five specific risk factors: high fasting blood sugar, high blood pressure, high triglyceride levels, low HDL (“good”) cholesterol, and abdominal obesity. People with metabolic syndrome are at greatly increased risk of developing more severe health problems, including diabetes, heart disease, and stroke.

“As a preventive cardiologist, I try to work with my patients and encourage them to make lifestyle changes, but it is very hard to get them to make lasting and meaningful changes,” says co-corresponding author Pam Taub, a cardiologist and Associate Professor of Medicine at the University of California San Diego School of Medicine. “When someone has been diagnosed with metabolic syndrome, this is a critical window for intervention. Once people become diabetic or are on multiple medications such as insulin, it’s very hard to reverse the disease process.”

In the study, 19 individuals with metabolic syndrome were recruited to participate in a program of TRE for three months. They were told they could decide what time to eat and how much to eat as long as all food consumption occurred within a 10-hour window. Most of the people in the study were obese and 84% were taking at least one medication, like a statin or antihypertensive.

At the end of the 12 weeks, the participants had an average of a 3% reduction in their weight and body mass index (BMI) and a 3% reduction in abdominal/visceral fat. Many also had reductions in cholesterol and blood pressure and improvements in fasting glucose.

Participants in the study used an app created by Panda called myCircadianClock (mCC) to log the times they ate and also to track their sleep. They also wore activity monitors that measured their sleeping and waking patterns and a glucose monitor that continuously tracked their glucose levels.

“We told people that they could choose when they ate their meals, as long as they remained within the 10-hour window,” Panda says. “We found that universally, they chose to eat breakfast later, about two hours after waking, and to eat dinner earlier, about three hours before going to bed.” He notes that in addition to the improvements seen in body weight and measures of metabolic syndrome, 70% of the participants also reported an increase in sleep satisfaction or in the amount they slept.

Taub says that the participants, about half of whom were already her patients, also reported generally having more energy, and some were able to have their medications lowered or stopped after completing the study. Overall, they told her that the plan was easier to follow than counting calories or embarking on an exercise program. More than two-thirds of participants continued with TRE for up to a year after the study was over, at least part of the time, she says.

Based on this pilot study, Taub and Panda have already begun a randomized, controlled clinical trial funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) to confirm the benefits of TRE in people with metabolic syndrome. They plan to recruit more than 100 participants–half for each arm. They also intend to conduct additional research to look at other physiological responses to TRE, including effects on the mitochondria in skeletal muscle and changes in liver function.

For anyone considering trying TRE, Taub recommends they first consult with a physician. This is especially important for anyone with metabolic syndrome who is already taking medication, she notes. “Any time someone is losing weight, they need to check with their doctor about whether their medications need to be adjusted,” she says. “For instance, if a patient is on blood pressure medications and they lose a significant amount of weight their blood pressure medication needs to be lowered.”

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This study was funded by a University of California San Diego Public Health Pilot Grant, an American College of Cardiology (ACC)/Merck Research Fellowship Award, a Larry L. Hillblom Foundation Postdoctoral Fellowship, a Salk Women in Science Fellowship, the Department of Homeland Security, the Department of Defense, the Leona M. and Harry B. Helmsley Charitable Trust, the Robert Wood Johnson Foundation, and the National Institutes of Health.

Cell Metabolism, Wilkinson et al.: “Ten-hour time-restricted eating reduces weight, blood pressure, and atherogenic lipids in patients with metabolic syndrome” https://www.cell.com/cell-metabolism/fulltext/S1550-4131(19)30611-4