How ground squirrels spend half of the year curled up without eating a thing and yet manage to maintain their protein balance, barely losing any muscle mass, has been a long-standing mystery. This is particularly remarkable given that prolonged periods of inactivity and fasting lead mammals’ bodies to get some energy by breaking down muscle proteins—a process known as muscle wasting.
A study published in Science today (January 27) offers a clue to this phenomenon: bacteria residing in the gut of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) convert urea, a molecule that accumulates during muscle wasting, into nitrogen that can be used to build new proteins, potentially counteracting muscle loss. In addition to highlighting the role of microbes in hibernation, these findings may also inspire the development of microbiota-based therapies to treat human conditions where muscle wasting is common, such as malnutrition.
University of Alaska Anchorage ecologist and bioinformatician Kirsten Grond, who studies host-microbe interactions in squirrels but was not involved in this work, says that one of its main highlights is demonstrating that microbes perform urea nitrogen salvage in the course of hibernation, helping the squirrels to “maintain their tissues during prolonged inactivity.” It is “fascinating” that the microbiome is “another important part of the overall health” of these mammals, she adds.
Amlan Patra, an animal nutritionist at the West Bengal University of Animal and Fishery Sciences in India who did not participate in the study, writes in an email to The Scientist that urea nitrogen salvage “is not a new phenomenon,” as it is known to occur “in many ruminant and non-ruminant animals.” But these new findings are interesting, he adds, because they explore “comprehensive mechanisms of urea recycling in hibernating mammals” and link gut microbiota to host metabolism.
Urea nitrogen recycling performed by microbes is particularly well understood in ruminants. Cattle, for instance, live on a relatively low-protein diet, says University of Wisconsin-Madison physiologist Hannah Carey, yet they “can grow big muscles” thanks to gut microbes that are able to convert urea into bioavailable forms of nitrogen. Carey has studied the gastrointestinal tract of hibernating animals for decades, but learned more about ruminant physiology while teaching a course for vet students. Inspired partly by the microbial tricks occurring inside the ruminants’ intestines, she set out to test whether similar microbes could be having a functional role in hibernating animals.
Earlier work by Carey and her colleagues had shown that muscle volume in thirteen-lined ground squirrels declines during the early phase of hibernation, only to later increase—as if their bodies begin to prepare for spring. Boosting “their lean mass and muscle proportion at the end of the hibernation . . . is especially odd considering that they are not taking up any protein,” says Felix Sommer, a microbiome researcher at the Kiel University in Germany who did not participate in either that previous study or the new one. Scientists have been wondering how these hibernators manage to do that, he notes.
Carey and others in the field hypothesized that this muscle gain could be occurring thanks to ureolytic bacteria inside the gut. In order to test this idea, she joined forces with microbiologists and biochemists with expertise in tracking metabolites inside animal bodies.
Most of the squirrels in the study were born at the UW-Madison campus to wild-caught females, while a few were wild-caught females themselves. The team divided the squirrels into three groups, one of them assessed in summer, during their active phase, and the other two in their early or late hibernating period in winter. Hibernation was stimulated by placing the squirrels in a cold room in constant darkness. Individuals within each group had either an intact microbiome or a depleted one due to treatment with antibiotics.
At a specific time point within each phase, the team injected stable isotope–labelled urea into each squirrel. In those undergoing hibernation, the procedure was performed after inducing arousal by moving the animal to a warmer lab. Carey and colleagues then followed the fate of the molecule.
Squirrels—like all vertebrates and many other animals—do not have ureases, the enzymes required to break down urea. Microbial species that have them are able to convert urea into ammonium and CO2. To detect whether such species were present and active, the team looked for any isotopically labelled CO2 in the breath of the animals. The tagged gas was only found in those squirrels with intact microbiomes, suggesting that, indeed, some of their bacteria were breaking down urea. After the squirrels were euthanized, metagenomic analyses of the contents of their cecum, a pouch connecting the small and large intestines, confirmed the presence of urease genes. The proportion of such genes, out of the total number of sequences, was higher in winter, though not significantly.
But what matters is whether the breakdown of urea actually benefits the animal. “Ultimately what you want to see,” says Carey, is “accumulation of this urea-derived nitrogen in actual protein.” And the team found it. After the squirrels were euthanized, liver tissue and skeletal muscle were collected, and in both, microbiome-intact individuals had significantly higher amounts of labelled nitrogen incorporated into their proteins than the microbiome-depleted animals. Moreover, labelled nitrogen levels were higher in the proteins from the two winter groups than in the proteins from the summer group. These key results represent, in essence, what the team wanted to test, says Carey. “We got super excited” about them, she says. The increased protein synthesis promoted by the ureolytic bacteria may be key to maintaining protein balance and muscle preservation in hibernating squirrels, she and the other authors suggest in their paper.
Sommer, who in 2016 reported the presence of seasonal bacteria potentially beneficial to hibernating brown bears, says that these new findings show that “there is an interplay” between the host and its intestinal microbiota “that allows specific adaptations” to hibernation. Yet there is more to it, and this discovery could also hold promise for treating muscle wasting in humans, he says—for example, in malnourished or elderly people. One idea would be to “genetically engineer other bacteria” currently used in probiotic formulations so they express the urease genes, which could give treated individuals the necessary material to build new protein. At this point, though, this is only speculation, he acknowledges.
Even if findings in hibernators turn out not to translate one-to-one to human physiology, Sommer notes that, as science history shows, “it’s always helpful to look into extremes to be able to uncover processes that otherwise may not be as obvious.” That is, he says, studying adaptations to hibernation, like the one reported in this study, may spark new ideas and serve as a model to analyze certain processes or challenges faced by humans.