Sleep loss tied to the gut microbiota changes in humans

 

Results from a new clinical study conducted at Uppsala University suggest that curtailing sleep alters the abundance of bacterial gut species that have previously been linked to compromised human metabolic health. The new article is published in the journal Molecular Metabolism.

Changes in the composition and diversity of the gut microbiota have been associated with diseases such as obesity and type-2 diabetes in humans. These diseases have also been linked with chronic sleep loss. However, it is not known whether sleep loss alters the gut microbiota in humans. With this in mind, Christian Benedict, associate professor of neuroscience, and Jonathan Cedernaes, M.D., Ph.D, both from Uppsala University, collaborated with researchers from the German Institute of Human Nutrition Potsdam-Rehbruecke. In their study, the researchers sought to investigate in nine healthy normal-weight male participants whether restricting sleep to about four hours per night for two consecutive days as compared with conditions of normal sleep (about 8 hours of sleep opportunity) may alter the gut microbiota in humans.
"Overall we did not find evidence that suggests that the diversity of the gut microbiota was altered by sleep restriction. This was somewhat expected given the short-term nature of the intervention and the relatively small sample size. In more specific analyses of groups of bacteria, we did however observe microbiota changes that parallel some of the microbiota changes observed when for instance obese subjects have been compared with normal-weight subjects in other studies, such as an increased ratio of Firmicutes to Bacteroidetes. Longer and larger clinical sleep interventions will be needed to investigate to what extent alterations of the gut microbiota may mediate negative health consequences attributed to sleep loss, such as weight gain and insulin resistance," says senior author Jonathan Cedernaes.
"We also found that participants were over 20 percent less sensitive to the effects of the hormone insulin following sleep loss. Insulin is a pancreatic hormone needed to bring down blood glucose levels. This decreased insulin sensitivity was however unrelated to alterations in gut microbiota following sleep loss. This suggests that changes in microbiota may not, at least in the short-term, represent a central mechanism through which one or several nights of curtailed sleep reduce insulin sensitivity in humans," says first author Christian Benedict.
"The gut microbiota is very rich and its functional role far from completely characterized. Future studies will hopefully be able to ascertain how the composition and functional role of the gut microbiota is able to modulate at the individual level how sensitive we humans are to negative metabolic, but also cognitive, effects of sleep loss," concludes senior author Jonathan Cedernaes.

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Story Source:
Materials provided by Uppsala University. Note: Content may be edited for style and length.

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Journal Reference:

1. Christian Benedict, Heike Vogel, Wenke Jonas, Anni Woting, Michael Blaut, Annette Schürmann, Jonathan Cedernaes. Gut Microbiota and Glucometabolic Alterations in Response to Recurrent Partial Sleep Deprivation in Normal-weight Young Individuals. Molecular Metabolism, 2016; DOI: 10.1016/j.molmet.2016.10.003 

 

Just 2 days of sleep loss may trigger changes in gut microbiota that are associated with poor metabolic health. This is the finding of a small study published in the journal Molecular Metabolism.

Researchers suggest sleep loss may alter gut microbiota.

For optimal health and well-being, the American Academy of Sleep Medicine recommend adults aged 18-60 years get at least 7 hours of sleep every night.
However, earlier this year, a report from the Centers for Disease Control and Prevention (CDC) revealed that more than a third of adults in the United States are not meeting these recommendations.
Lack of sleep has been linked to increased risk of numerous health problems, including high blood pressure, stroke, heart disease, obesity, and type 2 diabetes.
Previous research has also associated changes in gut microbiota - the community of microorganisms that reside in the digestive tract - with obesity and type 2 diabetes. Whether sleep loss plays a role in this relationship, however, has been unclear.
According to the researchers of the new study - including first author Christian Benedict of the Department of Neuroscience at Uppsala University in Sweden - some studies in mice and humans have suggested that gut bacteria have a circadian rhythm that might be disrupted by sleep loss.
"However, to date, there are no studies that have investigated the impact of insufficient sleep on the composition of the human gut microbiota," they add.
"Studies are therefore lacking that assess whether important adverse metabolic changes that may increase the risk of [type 2 diabetes] and obesity, such as impaired insulin sensitivity, are associated with changes in the gut microbiome and associated SCFAs [short chain fatty acids] that could result from recurrent sleep loss."

Restricted sleep linked to obesity-associated changes in gut microbiota

To find out more about the link between sleep loss, gut microbiota, and metabolic changes, Benedict and team enrolled nine healthy, normal-weight males to their study.
The researchers analyzed fecal samples from the men after two sleep conditions: 1 day of normal sleep (around 8 hours) and 2 days of restricted sleep (around 4 hours each night).
Meal times and food intake can affect the composition of gut bacteria, so these were kept consistent across both sleep conditions.
While the team found no evidence that sleep loss alters the diversity of gut bacteria, their analysis did identify changes in microbiota - such as an increase in the ratio of Firmicutes to Bacteriodetes - that previous studies have associated with obesity.
Additionally, the researchers found that following sleep restriction, subjects showed a 20 percent reduction in sensitivity to insulin - the hormone that regulates blood glucose levels.
"This decreased insulin sensitivity was however unrelated to alterations in gut microbiota following sleep loss," says Benedict. "This suggests that changes in microbiota may not, at least in the short-term, represent a central mechanism through which one or several nights of curtailed sleep reduce insulin sensitivity in humans."
While these findings suggest sleep loss can trigger changes in gut bacteria, the researchers say further investigation is warranted to better understand whether these changes influence metabolic health.
The authors add:

"Given our small sample size that only involved healthy young men, larger and more long-term studies are required to investigate to what extent these findings persist over longer time periods and whether these are observed in females, older or diseased patients and in other sleep restriction paradigms.
Nevertheless, our study is the first to provide evidence for sleep deprivation-induced changes in microbial families of bacterial gut species, which have previously been linked to metabolic pathologies."

Learn how gut bacteria may influence weight during childhood and adolescence.

Written by Honor Whiteman

 

Few health topics have garnered more interest in recent years than the human microbiome — the rich community of microorganisms that share our bodies, from the surface of our skin to the insides of our stomachs. Research is increasingly revealing the many complex ways that these little guys interact with and influence our bodily processes, and a growing scientific consensus suggests that we may need them just as much as they need us.
The microbes in our guts are proving themselves to be among the most essential, if also the most mysterious. Studies have suggested that they can influence everything from our weight to our mood, and disruptions in their natural, healthy structure can have noticeable impacts on our health. Entire diets have been constructed over this conceit.
Now, a growing body of research is suggesting that gut microbes may also be closely linked with our sleep quality, due to their close connection with our circadian rhythms, hormone expressions and other such processes. We’re still just beginning to understand these interactions, and many of the relevant studies so far have focused on mice instead of humans. Still, the research that does exist hints at some fascinating relationships between the little bugs in our stomachs and our nightly rest.

The Connection Between Microbiome and Circadian Rhythm

Some of the most intriguing recent research has focused on the connection between the gut microbiota and circadian rhythms — the 24-hour cycles the body goes through that regulate processes like sleeping and eating.
The circadian clock is what helps control when you fall asleep and when you wake up, and is also believed to be linked to the body’s metabolism, helping to control factors like when you’re hungry and how easily you gain or lose weight. Scientists now believe that gut microbes have a lot to do with the regulation of these processes.
One key to the connection is the liver, which helps express genes related to the regulation of the circadian clock. Past research in mice has found that mice specially raised to have no gut microbes at all have livers that function very differently from mice with normal microbiomes, said Eugene Chang, a professor of medicine at the University of Chicago.
“The liver is what is perhaps your major metabolic organ,” Chang explained. “And when we looked at the differences in the sort of profile of genes expressed in the liver of these two groups of mice, we found that a lot of these genes called circadian clock genes were really differentially expressed.”

The study “showed that gut microbes themselves actually have what we call diurnal, or ‘day versus night,’ patterns that are directly tied to our own circadian or diurnal patterns,”

Last year, Chang co-authored a study that built on these observations, revealing a link between diet, bacterial populations in the gut and the expression of genes that help control the circadian clock. He and his colleagues already knew that microbes in both mice and humans change their activity levels throughout the day in a way that suggests they respond to their own circadian rhythms. In addition, these microbes produce substances in the gut that help affect gene expression related to the circadian clock.
The new study showed that the microbes in mice who were fed a high-fat diet — aka relatively an unhealthy diet — did not exhibit the normal kinds of fluctuations throughout the day and produced different substances than those in mice who were fed a more normal diet. Additionally, mice who were specially raised to have no gut microbes did properly express the genes that control circadian rhythm -- and they also had trouble gaining weight, even when fed a high-fat diet.
In summary, the study “showed that gut microbes themselves actually have what we call diurnal, or ‘day versus night,’ patterns that are directly tied to our own circadian or diurnal patterns,” said the study’s lead author, Vanessa Leone, also of the University of Chicago. “And those interactions appear to be impacted by the type of diet that you take in.”
Other studies have suggested similar links. A 2014 paper, for instance, showed that mice who were both fed a high-fat diet and subjected to sleep disruptions showed significant changes in the structure of their gut microbiota.

How Gut Bacteria Messes with Hormones and Mood

It’s fairly well established that our hormones and our moods play a big role in the quality of our sleep. And these may also be significantly influenced by gut microbes.
Some previous research in mice has suggested that gut microbes are involved with the regulation of cortisol levels in the body, said Leone. When scientists reduced the amount of gut bacteria in these mice, levels went down.
Cortisol is involved in a number of bodily functions, including metabolic regulation and immune expression — and it’s also thought to be a key player in the sleep cycle. Cortisol levels have been shown to dip to their lowest levels at night when we’re sleeping, and they spike in the morning when we wake up. So disruptions in the microbiome that affect cortisol levels also have the potential to disturb the regular sleep cycle as well.

Certain gut microbes have exhibited the ability to produce, convert or consume other hormones related to sleep and the circadian rhythm, including melatonin and serotonin

The hormone is also known to affect mood, with high levels of it associated with stress. Leone pointed out that past research has also shown that mice with no gut microbes exhibit higher cortisol levels when they’re put in stressful situations, for example, if they’re forced into small, restrained areas.
“In the absence of gut microbes, those mice are at a heightened level of anxiety,” Leone said. This research could provide another glimpse into the link between the microbiome and sleep, as research in humans has suggested that mood — including problems with stress, anxiety or depression — can heavily influence our sleep quality.
Leone also added that certain gut microbes have exhibited the ability to produce, convert or consume other hormones related to sleep and the circadian rhythm, including melatonin and serotonin. However, scientists are just starting to explore these interactions.
“You kind of have a situation where we know that those hormones are really important for regulating circadian rhythm, but clearly the gut microbes have their own capacity to utilize or produce those hormones as well,” Leone said. “How that feeds into our circadian system isn’t fully appreciated. We’re just kind of now starting to examine the microbial side of that and how that interaction occurs.”

The Two-Way Street

One especially interesting aspect of all the research described so far is that it suggests a two-way interaction between gut microbes and the factors that influence sleep.
Some studies have shown that disruptions to the microbiome — caused by diet, for instance, or other external factors — can produce changes in hormones or gene expression that could then affect the sleep cycle. Other studies have suggested that disruptions in sleep can feed back in the other direction, causing changes in the gut microbiota.

“The way I see the gut microbiome — it’s an organ of our body,” Chang said. “And that organ is highly sensitive to environmental and dietary changes."

“The way I see the gut microbiome — it’s an organ of our body,” Chang said. “And that organ is highly sensitive to environmental and dietary changes, and also it’s probably very responsive to cues or signals from the person or individual as well.”
On top of this two-way feedback, changes in sleep and the microbiome — caused by any factors — can also team up together to bring about changes in other aspects of the body.
A study earlier this year from Baylor College of Medicine, for instance, explored the connection between sleep apnea, disruptions in the microbiome and hypertension. That study, conducted in rats, found that rodents with both sleep apnea and an imbalanced microbiome were likely to develop hypertension, while those with sleep apnea and a normal microbiome tended to maintain normal blood pressure

The Human Connection  

It’s important to keep in mind that all of these studies have taken place in mice, rather than humans — so we can’t definitively say the same kinds of effects would take place in our own bodies. Given what’s known about the similarities between the functions of mouse microbes and human ones, however, researchers feel confident that there’s good reason to continue exploring the connections.
And, in fact, at least one group of researchers is in the process of conducting one of the first human studies to examine the link between gut microbes and sleep quality. That project is being led by Amy Reynolds, a research associate at CQ University in Australia, in collaboration with uBiome, a startup that sells microbiome sampling kits and analyzes the samples for consumers.
By analyzing microbiome samples from participants with varying degrees of sleep disruption, the project aims to explore the ways that sleep quality and the gut microbiota influence one another.

When people are sleep restricted, we see changes in their cortisol levels.” And as Leone pointed out before, cortisol is one of the hormones thought to be tightly tied up with healthy microbiome function.

Reynolds, whose background is in psychology, first developed an interest in the subject after studying the ways that shift work — and the unusual sleep schedules that tend to accompany it — affect metabolic health, which is also shown to be linked to the microbiome. “Surely there may be something going on in humans that explains this sleep-microbiota-metabolic relationship,” she says.
The research in mice and rats has already provided a good starting point, she added, so there are already some theories to go on when it comes to the link.
“We know…not getting enough sleep initiates a stress response in your body,” Reynolds said. “When people are sleep restricted, we see changes in their cortisol levels.” And as Leone pointed out before, cortisol is one of the hormones thought to be tightly tied up with healthy microbiome function.
For now, the human study, as Reynolds says, is “still in its infancy” so it will be a while yet before we see any published results. But she has high hopes that the project will help shed some light on what is unfolding into a complex and fascinating series of connections among our body’s processes.
 

New research on sleep and the world of the microbiome

There’s a lot of discussion these days about gut health—about how a healthy gut can support overall health, and about the ways a compromised gut may contribute to illness and disease. We’re learning continually more about the complexity of the vast, dense, microbial world of the human gut, and its influence over immune health, hormone balance, brain function, mental and physical equilibrium.

What relationship exists between sleep and this microbial ecosystem within the human body? Emerging science demonstrates that there is a very real, dynamic connection between the microbiome and sleep itself.

What is the microbiome?

The term microbiome can mean a couple of different things. Microbiome is sometimes used to describe the collection of all microbes in a particular community. In scientific terms, the microbiome can also refer to the genes belonging to all the microbes living in a community. The microbiome is often seen as a genetic counterpart to the human genome.

The genes that make up a person’s microbiome are far more numerous than human genes themselves—there are roughly 100 times more genes in the human microbiome than in the human genome. This makes sense when you consider that there are somewhere in the neighborhood of 100 trillion microbes living in (and on) each of us—a combination of many different types, including bacteria, fungi, viruses and other tiny organisms.

This vast array of microbial life lives on skin and throughout the body. The largest single collection of microbes resides in the intestine—hence the attention to “gut” health. Here, trillions of microscopic organisms live and die—and appear to exert a profound effect on human health.

The microbiome and sleep

The human microbiota is a complicated, dynamic ecosystem within the human body. The microbiome appears to interact in some important ways with another fundamental aspect of living: sleep. As with much about the microbiome, there is a tremendous amount we don’t know. That said, there are some fascinating possible connections and shared influences between the microbial world of the gut and sleep.

Scientists investigating the relationship between sleep and the microbiome are finding that this microbial ecosystem may affect sleep and sleep-related physiological functions in a number of different ways: shifting circadian rhythms, altering the body’s sleep-wake cycle, affecting hormones that regulate sleep and wakefulness. Sleep, in turn, may affect the health and diversity of the human microbiome.

The microbial life within our bodies is in perpetual flux, with microbes constantly being generated and dying. Some of this decay and renewal naturally occurs during sleep. There’s no answer yet, but the question is very important: what role does sleep itself play in maintaining the health of the microbial world that lives inside us, and appears to contribute so significantly to our health?

There are some important signs of a significant connection between the microbiome and sleep. We’ve seen significant research demonstrating that circadian rhythm disruptions can have negative effects on gut microbiota. (More on this shortly.) There’s also evidence that the disordered breathing associated with obstructive sleep apnea, a common sleep disorder, may disrupt the health of the microbiome. Scientists put mice through a pattern of disrupted breathing that mimicked the effects of OSA. They found the mice that lived with periods of OSA-like breathing for six weeks showed significant changes to the diversity and makeup of their microbiota.

Sleep and the gut-brain connection

A significant, fast-growing body of research illustrates the far-reaching effects of the microbiome over brain function and brain health—as well as the influence of the brain over gut health and the microbiome. This “gut-brain axis” is being shown to have what is likely a profound influence over nearly every aspect of human health and physiological function, including sleep.

The constant communication and interplay between the gut and the brain has the potential to influence and intersect with sleep both directly and indirectly. Let’s take a closer look at some of the ways that might occur.

Mood. Studies indicate that the health and balance of the gut microbiota has a significant influence over mood and emotional equilibrium. Disruptions and imbalance of gut microbes have been strongly connected to anxiety and depression. This has potentially significant implications for sleep, as both anxiety and depression can trigger or exacerbate sleep disruptions.

Stress. Research is also revealing a complicated, two-way relationship between stress and gut health that also may exert an influence over sleep and sleep architecture. Stress is an extremely common obstacle to healthy, sufficient sleep.

Pain. Studies link gut health to pain perception, specifically for visceral pain. An unhealthy microbiome appears to increase sensitivity to this form of pain. Like so many others, this connection travels the communication pathway between the gut and the brain. The connection between sleep and physical pain or discomfort is significant—the presence of pain can make falling asleep and staying asleep much more difficult.

Hormones. Several hormones and neurotransmitters that play important roles in sleep also have significant influence over gut health and function. The intestinal microbiome produces and releases many of the same neurotransmitters—dopamine, serotonin, and GABA among them—that help to regulate mood, and also help to promote sleep.

Melatonin, the “darkness hormone,” which is essential to sleep and a healthy sleep-wake cycle, also contributes to maintaining gut health. Deficiencies in melatonin have been linked to increased permeability of the gut—the so-called “leaky gut,” which is increasingly associated with a range of diseases. Melatonin is produced in the gut as well as the brain, and evidence suggests intestinal melatonin may operate on a different cyclical rhythm than the pineal melatonin generated in the brain.

Cortisol is another hormone critical to the human sleep-wake cycle. Rising levels of cortisol very early in the day help to promote alertness, focus and energy. Cortisol levels are influenced in several ways within gut-brain axis: this hormone is central to the stress and inflammatory response, and it also exerts an effect on gut permeability and microbial diversity. The changes to cortisol that occur amid the interplay of the gut and brain are likely to have an effect on sleep.

‘Circadian rhythms’ of the gut?

There is some pretty fascinating research connecting the gut microbiome to circadian rhythms, the 24-hour biological rhythms that regulate sleep and wake cycles, in addition to many other important physiological processes. A growing number of studies now suggest that the vast and diverse microbial ecosystem of the gut has its own daily rhythms. These microbiome rhythms appear to be deeply entwined with circadian rhythms—research suggests that both circadian and microbial rhythms are capable of influencing and disrupting the other, with consequences for health and sleep.

The rhythms of gut microbes are affected by diet, both the timing of eating and the composition of foods consumed, according to research. A recent study using mice found that mice eating a healthy diet generated more beneficial gut microbes, and that the collective activity of microbial life in the gut followed a daily—or diurnal—rhythm. That rhythm in turn supported circadian rhythms in the animal. Mice that were fed a high-fat, stereotypically “Western” diet, on the other hand, produced less optimal microbial life. The gut microbes of these mice did not adhere to a daily rhythm themselves, and also sent signals that disrupted circadian rhythms. These mice gained weight and became obese, while the mice that ate healthfully did not.

Scientists bred a third group of mice without any gut microbes at all. These mice had no signals to send from a gut microbiome. Circadian disruption occurred in these mice—but they did not gain weight or suffer metabolic disruption, even when fed the high-fat diet. This suggests a couple of important conclusions. First, that microbial activity is key to normal circadian function—and therefore to sleep. Second, that the microbiome is a key player along with diet in the regulation of weight and metabolism.

Circadian rhythms and microbiome: a two-way street

Research in humans has returned similar results. The human microbiome appears to follow daily rhythms influenced by timing of eating and the types of foods consumed, and to exert effects over circadian rhythms. Research has also found that the relationship between these different biological rhythms works both ways. Scientists have discovered that disruptions to circadian rhythms—the kind that occurs through jet lag, whether through actual travel or from “social” jet lag—disrupts microbial rhythms and the health of the microbial ecosystem. People who experience these changes to microbial rhythms as a result of circadian disruption suffer metabolic imbalance, glucose intolerance, and weight gain, according to research. And there’s preliminary evidence suggesting that gender may play some role in the relationship of gut microbial health, metabolism, and circadian function: a study using mice found that females had more pronounced microbiome rhythms than males.

New understanding of circadian role in metabolism?

We’ve known for some time about the relationship of sleep, circadian rhythms, and metabolic health. Disrupted sleep and misaligned circadian rhythms have been strongly tied to higher rates of obesity, and to metabolic disorders including type 2 diabetes. This emerging knowledge of the microbiome and its relationship to circadian function may in time deliver to us a deeper understanding of how health is influenced by sleep and circadian activity.

Science has really only just begun to delve into the world of the microbiome, and its relationship to sleep as well as to health more broadly. All the early signs suggest that this is a profoundly important area of research, but it’s not clear yet what unlocking more fully the secrets of the microbiome will mean for preventive health and for the treatment of disease, including disorders of sleep. It will be fascinating to see where this takes us, and what it means for sleep.

Highlights

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Possibly the first results of how short sleep impacts the human gut microbiota.

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Two nights of short sleep do not significantly impact beta diversity.

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The Firmicutes to Bacteroidetes ratio is significantly affected by sleep loss.

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Fecal short-chain fatty acid levels do not change depending on sleep duration.

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Increased insulin resistance after sleep loss is unrelated to alterations in the microbiota.

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Abstract

Objective

Changes to the microbial community in the human gut have been proposed to promote metabolic disturbances that also occur after short periods of sleep loss (including insulin resistance). However, whether sleep loss affects the gut microbiota remains unknown.

Methods

In a randomized within-subject crossover study utilizing a standardized in-lab protocol (with fixed meal times and exercise schedules), we studied nine normal-weight men at two occasions: after two nights of partial sleep deprivation (PSD; sleep opportunity 02:45–07:00 h), and after two nights of normal sleep (NS; sleep opportunity 22:30–07:00 h). Fecal samples were collected within 24 h before, and after two in-lab nights, of either NS or PSD. In addition, participants underwent an oral glucose tolerance test following each sleep intervention.

Results

Microbiota composition analysis (V4 16S rRNA gene sequencing) revealed that after two days of PSD vs. after two days of NS, individuals exhibited an increased Firmicutes:Bacteroidetes ratio, higher abundances of the families Coriobacteriaceae and Erysipelotrichaceae, and lower abundance of Tenericutes (all P < 0.05) – previously all associated with metabolic perturbations in animal or human models. However, no PSD vs. NS effect on beta diversity or on fecal short-chain fatty acid concentrations was found. Fasting and postprandial insulin sensitivity decreased after PSD vs. NS (all P < 0.05).

Discussion

Our findings demonstrate that short-term sleep loss induces subtle effects on human microbiota. To what extent the observed changes to the microbial community contribute to metabolic consequences of sleep loss warrants further investigations in larger and more prolonged sleep studies, to also assess how sleep loss impacts the microbiota in individuals who already are metabolically compromised.

Keywords

• Bacteroidetes;
• Firmicutes;
• Insulin resistance;
• Intestinal microbiome;
• Short-chain fatty acid;
• Sleep restriction

Abbreviations

• d2, day 2;
• F:B, Firmicutes:Bacteroidetes (ratio);
• HDL, high-density lipoprotein;
• HOMA-IR, homeostatic assessment model of insulin resistance;
• LDL, low-density lipoprotein;
• NS, normal sleep;
• OGTT, oral glucose tolerance test;
• OTU, Operational Taxonomic Units;
• PERMANOVA, permutational analysis of variance;
• PSD, partial sleep deprivation;
• SCFA, short-chain fatty acid;
• T2DM, type-2 diabetes mellitus

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1. Introduction

The last two decades have revealed an increasingly important role of the gut microbiome for human health [1], [2] and [3]. Differences in intestinal microbiota composition have been uncovered between healthy controls and those suffering from various diseases, including metabolic pathologies such as obesity, the metabolic syndrome and type-2 diabetes mellitus (T2DM) [4], [5] and [6]. An important role of the gut microbiome for energy homeostasis is supported by studies showing that germfree mice – which lack a functional microbiome – are resistant to diet-induced obesity on a western diet [7]. Consistently, transplanting the microbiota of genetically obese ob/ob mice or obese humans to lean germfree mice increases the adiposity of the recipient mice [8] and [9], indicating an increased capacity to harvest energy from gut nutrients in the obese state when identical nutrients are provided [8] and [10]. Furthermore, similar transplantation of the microbiome from the obese but not normal-weight twin, to healthy germ-free mice, also promotes adiposity and perturbs glucose metabolism [9]. Providing evidence for the importance of a healthy microbiota, beneficial effects concerning insulin sensitivity have been observed in human trials of patients with metabolic syndrome who received the transplants from lean donors [4].

The gut microbiome regulates nutrient availability through complex interactions with dietary factors, and, consequently, the composition of the microbiota has been found to regulate the metabolic response to various such factors in humans [11] and [12]. Diet is a key pathophysiological and treatment component for diseases such as T2DM [13], and diet has a major impact on the gut microbiome as it is the most important energy source for intestinal bacteria. Both acute [12], [14], [15] and [16] and long-term [16] and [17] changes in diet have been demonstrated to alter the abundance of bacterial species in the gut, as well as their functional capacity to process various nutrients, with such changes being detectable within days of dietary interventions in humans [15] and [16].

Macronutrients in our diets enable bacteria in the large intestine to produce short-chain fatty acids (SCFAs), which include acetate, propionate, and butyrate. SCFAs can constitute about 2.2% of daily caloric intake [18], but also exert effects locally in the gut, as well as on hepatic glucose and lipid metabolism [19]. Butyrate and propionate have recently been demonstrated to play a role in intestinal gluconeogenesis, which has beneficial effects on glucose and energy homeostasis [20]. T2DM patients have been found to have a reduction in butyrate-producing bacterial species, and lower levels of butyrate biosynthesis when the microbiomes of T2DM patients are compared with those of non-diabetic controls [6]. Instead, obesity has been associated with an increase in total levels of SCFAs [10], and dietary factors can also alter these levels [19] and [21]. Indeed, increased acetate production by gut bacteria has been found recently to promote insulin secretion, hyperphagia, and obesity [22], although there is also evidence that acetate is able to decrease appetite [23].

The risk of T2DM and obesity – metabolic pathologies that have been linked to dysregulated gut microbiotas – has been found to be increased in subjects suffering from chronic sleep loss [24], which has become increasingly common in modern stressful 24/7 lifestyle [25]. Merely curtailing sleep to half the recommended amount for a single night acutely impairs fasting insulin sensitivity [26]. When prolonged, sleep restriction can promote weight gain [27], possibly by altering energy expenditure as well as food choices and the behavioral response to especially hedonic food stimuli [24].

There is currently some evidence in both mice and humans that the gut microbiota exhibits a circadian rhythm [28], [29] and [30] and, correspondingly, that this may be perturbed following circadian misalignment [28]. However, to date, there are no studies that have investigated the impact of insufficient sleep on the composition of the human gut microbiota. Studies are therefore lacking that assess whether important adverse metabolic changes that may increase the risk of T2DM and obesity, such as impaired insulin sensitivity, are associated with changes in the gut microbiome and associated SCFAs that could result from recurrent sleep loss. To this end, we conducted a study in which the impact of sleep loss on the human gut microbiota was assessed, in healthy, young normal-weight individuals.

2. Methods

2.1. Participants

Out of a total of 16 male individuals recruited for the present study, nine were able to provide fecal samples for both the baseline (see below) and second day measurement for both sessions, and were thus included for further analysis. All nine individuals had self-reported 24-hr sleep-wake within the recommended range as defined by the National Sleep Foundation (self-reported habitual sleep duration 7–9 h during nocturnal hours; sleep onset latency <30 min) and meal patterns (regular breakfast, lunch and dinner as main meals) as assessed by sleep diaries (data not shown). Participant data is shown in Table 1. Written and oral interviews of participants' current and prior physical and mental health were conducted by a medical doctor (J.C.) before participants were enrolled in the study, to ensure that participants were in general good health, did not use any medication or supplements, and that the participants did not suffer from or had been diagnosed with any psychiatric conditions, sleep disorders, or allergies. Participants reported not to suffer from any gastrointestinal disorder and did not experience regular or intermittent gastrointestinal discomfort. Furthermore, none of the participants had suffered from any diarrheal or infectious gastrointestinal disease in the last two years. All participants consumed a mixed normal diet, and none was a strict vegetarian. Furthermore, none of the participants reported having been born via caesarian section, and none of the participants had been treated with antibiotics during the previous twelve months.

Table 1. Baseline data for the study participants (n = 9).

Parameter	Mean ± S.E.M.
Age	23.3 ± 0.6 years
Body mass index	23.1 ± 0.6 kg/m2
Waist circumference	79.1 ± 2.0 cm
Hip circumference	98.2 ± 1.6 cm
Waist:hip ratio	0.80 ± 0.02

Table options

During the morning screening visit, fasting glucose (<6.1 mmol/L) and normal blood count values were verified, followed by a 2-h oral glucose tolerance test (OGTT), which confirmed that all participants had normal glycemic control. Finally, about a week prior to the first session, participants underwent a night-long laboratory polysomnography, in order to habituate them to the experimental setting. All participants provided informed consent; the study was approved by the Regional Ethical Review Board in Uppsala (EPN 2014/242/1) and was conducted in accordance with the Helsinki Declaration.

2.2. Experimental procedure

A within-subject design was utilized for the current study: all participants took part in two separate experimental conditions (normal sleep, NS; vs. partial sleep deprivation, PSD) in which participants had an 8.5-h long sleep opportunity (22:30–07:00 h) in the normal sleep condition and a 4.25-h long sleep opportunity (2:45–07:00 h) in the PSD condition. Fecal samples were collected before and after 1) two nights of normal sleep and 2) after two nights of PSD. All experiments were conducted at our sleep laboratories at the Biomedical Center at Uppsala University, Sweden. Participants were randomly assigned which experimental condition they would start with (NS vs. PSD); this was counterbalanced for the total number of subjects included in the larger study set, of which results have previously been reported [31]. It should be noted, therefore, that in the present study, out of nine participants six started with their PSD condition, while three instead started with their normal sleep condition.

Participants maintained sleep diaries the week prior to admission to each of the two sessions and were instructed to try to go to bed around 22:00–24:00 h each night, and to get up after 7–9 h of sleep, around 6:00–8:00 h. The sleep diaries preceding each intervention did not reveal any significant differences for self-reported total sleep duration (P = 0.19, two-tailed). Participants were also instructed to maintain similar dietary and activity habits prior to each sleep intervention, including maintaining intake of breakfast, lunch and dinner around their previously documented regular hours. For each condition, participants came to the laboratory at 17:30 h on the arrival day (day 0). Participants were provided with a standardized dinner (33% of each participant's estimated daily calorie requirement), and were prepared for sleep (recorded by polysomnography). During the additional hours of wakefulness in the PSD condition (22:30–02:45 h), room lights were kept at <6 lux at eye-level.

As meal timing and nutrient composition can influence the gut microbiota [30], these were kept constant across the two study conditions. To further isolate the solitary impact of sleep on the microbiota – separate from the previously documented impact of e.g. high-fat diet [32] – participants were provided with a non-sugar sweetened, low-fat diet throughout the three meals, the exception being the oral glucose tolerance (OGTT) test on day 2; the OGTT was employed to test the participants' insulin sensitivity using the WHO-defined gold standard [33]. Participants were provided with yoghurt (Arla, 3 g fat/100 g) and natural muesli (ICA, 7 g fat/100 g) for breakfast; pasta bolognese for lunch (Findus, 2 g fat/100g); and a mix of potatoes, beef, rapeseed oil and onion (Findus, 5.5 g fat/100 g) for dinner. Both lunch and dinner were microwave-heated from frozen food packages within 15 minutes of each respective meal. On day 1, participants were provided with isocaloric meals (breakfast at 09:00 h, lunch at 13:00 h and dinner at 20:00 h; each providing 33% of each participant's estimated daily calorie requirement). On day 2, participants were first provided with a glucose solution (for the OGTT, at 08:30 h), and were then provided with a snack (400 kcal) around 11:00 h, followed by meal timing as on day 1, i.e. lunch at 13:00 h, and dinner at 20:00 h. Each meal had to be consumed in its entirety within 20 minutes of being served. Participants were allowed ad lib intake of regular water throughout the study except for during the OGTT in order to avoid dilution-induced differences in the rate of glucose uptake from the intestine.

To avoid a sedentary activity profile, participants were taken on three supervised walks at a standardized slow pace (walking pace ∼4 km/h; 10 min at 11:15 h, 30 min at 14:00 h and 20 min at 17:00 h) on day 1 and 2. Participants were otherwise confined to their rooms, where they could carry out activities at a sedentary level (e.g. reading, watching movies, playing board games) while being monitored by the experimenters.

On the morning of day 2, blood was collected through an indwelling catheter in the forearm, starting at around 08:30 h (fasting state), after which participants ingested the glucose solution for the OGTT (75 g glucose in 250 ml of water) within 60 s. After lying on their right side for five minutes (to ensure equal anatomical distribution of the glucose solution across all subjects; [34]), additional blood samples were collected every 30 min up to 2.5 h after ingestion of the glucose solution. Participants remained in a semi-recumbent position throughout the OGTT blood collection (∼08:15 h to ∼11:00 h).

Plasma lipids were obtained in the fasting state after another night of PSD or NS, this was followed by cognitive tests which have been reported elsewhere [31].

2.3. Collection of fecal samples

Sterile stool collection kits (Cat #K708, WA Products, UK) were handed out to the participants to collect fecal samples at home; these were also provided to the participants during their sessions in our sleep laboratory for continued fecal collection, which participants were encouraged to do whenever possible. Participants were provided with written and oral instructions on how to collect fecal samples before coming into the lab. These instructions were also repeated on the day that subjects were to collect their baseline samples, as well as when subjects arrived in the lab on each session. For baseline fecal samples, which were collected at home, participants were instructed to collect a sample as close as possible (within 24 h) to arriving at the sleep laboratory for each session. The participants were instructed to freeze the sample (to −18 °C or colder) in the provided sterile container, and to do so in a container filled with ice, enabling maintenance of sample integrity during the brief transport to the sleep laboratory, where samples were transferred to −80 °C freezer storage. Samples collected during the laboratory sessions were frozen at −80 °C as soon as possible; all samples were stored at −80 °C until further analyses. A total of 36 samples were utilized for the present analysis (nine subjects, two conditions, baseline and day 2 time points); these were aliquoted for subsequent analysis of microbial composition and SCFA content.

2.4. Microbial sequencing

For assessing the microbiota composition of the obtained fecal samples, nucleic isolation from fecal aliquots was carried out by Second Genome (San Francisco, CA, USA). The MoBio PowerMag^® Microbiome kit (Carlsbad, CA, USA) was utilized according to manufacturer guidelines, optimized for high-throughput processing. Extracted sample DNA content was quantified with the Qubit^® Quant-iT dsDNA High Sensitivity Kit (Invitrogen, Life Technologies, Grand Island, NY, USA). For subsequent library preparation, enrichment of bacterial 16S V4 rDNA region was achieved by amplifying DNA with the use of fusion primers, designed to target the surrounding conserved regions, incorporating Illumina (San Diego, CA, USA) adapters and indexing barcodes. PCR amplification was done with two different barcoded V4 fusion primers for each sample. Samples were then concentrated with a solid-phase reversible immobilization method for the purification of PCR products and qPCR quantified. Samples were finally pooled for 16S V4-enriched, amplified, barcoded sample sequencing, using MiSeq (Illumina, San Diego, CA), and run for 250 cycles with custom primers designed for paired-end sequencing. Samples were processed in a Good Laboratory Practices (GLP) compliant service laboratory, with Quality Management Systems for tracking of samples and data.

2.5. Analysis of short-chain fatty acids

Fecal SCFA levels were analyzed in duplicates and determined as previously described [35] with an HP 5890 series II gas chromatograph, but equipped with an HP-FFAP column. Helium served as carrier gas at a flow rate of 1 mL/min. SCFA concentrations were calculated using iso-butyric acid (12 mM) as internal standard.

2.6. Biochemical analyses

Plasma concentrations of glucose, triglycerides, as well as cholesterol levels (LDL, HDL, and total levels), were analyzed with an Architect C16000 chemistry analyzer (Abbott Laboratories, Chicago, USA). Serum insulin and cortisol were analyzed using commercially available ELISA kits (Cortisol Parameter Assay Kit, R&D Systems, Abington, UK).

2.7. Sleep assessment

To record sleep, Embla A10 recorders (Flaga hf, Reykjavik, Iceland) were employed, and EEG signals were derived from C3, C4, Fp1, Fp2 (referenced to the contralateral mastoid). An experienced scorer blinded to the study conditions adhered to standardized criteria for subsequent sleep analysis [36]. The polysomnographic analysis revealed that both on the first and second night, subjects slept 7 h:47min ± 13 min and 8 h:00 min ± 7 min, respectively, in the NS condition (i.e. over the minimum 7 h per night as recommended for adults by the US National Sleep Foundation). Instead, in the PSD condition, participants slept 3 h:59 min ± 4 min on the first night, and 4 h:3 min ± 3 min on the second night.

2.8. Statistical analysis

Second Genome's analysis software package was used for statistical analysis composition and diversity of the microbiome; this utilized permutational analysis of variance (PERMANOVA). Differential abundance of Operational Taxonomic Units (OTUs) was tested with a negative binomial noise model and an intrinsic Poisson process; this took into account both technical and biological variability between experimental conditions. For this analysis, DESeq was run with default settings and false discovery rate (FDR; q-values) was calculated with the Benjamini–Hochberg procedure. Wilcoxon signed rank test was performed for paired comparisons of microbial composition at the phylum or family level, whereas Student's t-test was used for other normally distributed parameters. Analyses of changes in the phyla, families, or OTUs of sequenced gut bacteria were analyzed for the eight most abundant features to minimize multiple comparisons. Repeated measures ANOVA was conducted to investigate the effects of PSD vs. NS on (a) glucose values (day 2), (b) insulin values (day 2), (c) baseline vs. day 2 differences across the sleep conditions for SCFAs, as well as for (d) the ratio of Firmicutes:Bacteroidetes. The factors sleep (PSD vs. NS) and time (baseline vs. day 2) were utilized for these ANOVAs; where applicable, normally distributed parameters were post-hoc analyzed using Student's t-test. Normal distribution of variables was assessed by the Shapiro–Wilk's test for normality; parameters not passing the normality test were log2-transformed before analysis. The Greenhouse–Geisser method was used to correct ANOVA analyses for sphericity deviations. Correlations were conducted using Pearson's correlation as all tested values were found to be normally distributed.

Two-sided P-values were utilized for the PERMANOVA, as we did not have any a priori hypothesis regarding the influence of sleep loss on beta diversity. One-sided P-values below 0.05 were considered significant for microbiota-derived parameters (such as the Firmicutes:Bacteroidetes ratio; [37], [38], [39] and [40]) that have previously been associated with metabolic perturbations that also occur after acute sleep loss, including insulin resistance and dysregulation of lipid metabolism [34] and [41]. Finally, as previous data have demonstrated increased insulin resistance following one or several nights of sleep loss, with increased postprandial but not baseline glucose levels [34] and [41], one-sided P-values were used for postprandial markers of insulin resistance and for post-OGTT glucose values. Data are presented as means ± standard deviation (for all analyses of microbial composition) or standard error of the mean (S.E.M.; for all other analyses).