Hibernation microbiota: bacteria work while the animal sleeps

17th May 2023

Translated from the original article in Catalan (13th April 2023)

HIBERNATION

Hibernation is a state of minimal activity and slowing down of the metabolism carried out by some animals —basically mammals —, usually during the winter, although there are some that do “aestivation”. When they hibernate, their temperature, heart and breathing rates and overall metabolism drop. This physiological body state of dormancy is known as torpor.

For example, ground squirrels (Figure 1) or marmots hibernate for about 6 months, from autumn to spring, breathing once per minute, with about 5 heartbeats per minute and the temperature drops to only 5-10^oC. This state of torpor is not constant, but every 10-12 days there are phases of interruption, where they revive a little for about 12 hours, their temperature rises (Figure 2), they shiver a little as if to warm up and the metabolism is activated (Carey et al. 2003), but they neither eat nor drink nor defecate (Wilke 2020).

Figure 1. A hibernating ground squirrel (Ictidomys tridecemlineatus). Image of R. Streiffer, taken from Garcia de Jesús (2022). 

Figure 2. Body temperature (T_b) of a ground squirrel throughout the year, from June to May. In the hibernation period, cycles of long phases of torpor (10-12 days) with short phases of interruption (IBA, 12 hours) are observed (Adapted from Carey et al. 2003).

Hibernation serves to maintain and save body energy when there is not enough food available. For this reason, before hibernation and in general all the warmer months, these animals eat a lot, accumulating enough energy reserves for the winter period. All of them hibernate in dens or caves, somewhat sheltered from the cold and other inclement weather outside.

The hibernation period is very variable, from a few weeks to months, and can be mandatory or optional. Different levels of hibernation are also distinguished, from deep hibernation such as many rodents, the mouse lemur, the hedgehog and many marsupials, to lighter hibernation such as bears. In these, the body temperature only drops by about 5^oC, while in some rodents it drops by more than 30^oC.

On the other hand, ectothermic vertebrate animals —cold-blooded— such as fish, amphibians and reptiles, also lower their metabolic activity and body temperature and enter a state of torpor, but it is not considered hibernation because these animals do not activate it voluntarily but is a function of the external environment.

METABOLISM OF HIBERNATION

Hibernation is a mammalian strategy that uses metabolic plasticity to reduce energy demands and allow long-term fasting. To allow for these long periods without food, many of these animals eat much more before hibernation, and accumulate body fat. Brown bears, for example, double their fat reserves and consume the lipids stored during hibernation. Although these bears become seasonally obese, they remain metabolically healthy, which contrasts with the strong relationship between obesity and insulin resistance and other metabolic problems seen in humans (Sommer et al. 2016).

Fasting mitigates winter food shortages but removes nitrogen from the diet, thereby compromising the body’s protein balance (Regan 2022). Muscle atrophy due to fasting —or inactivity— releases nitrogen compounds such as ammonium, which can be toxic, and the body must eliminate its excess, usually in the form of urea, through the urine. However, hibernating animals such as the much-studied ground squirrels maintain or lose very little muscle mass over the winter, and even in the final phase of hibernation the rate of protein synthesis increases to levels like when they will be active. Below we will see the role of the microbiota in this nitrogen recovery.

The knowledge of how these animals maintain muscle mass can help find a remedy in the case of people with muscle loss due to malnutrition, forced sedentarism or muscle wasting diseases (García de Jesús 2022).

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THE INTESTINAL MICROBIOTA OF HIBERNANTS

One of the most well-known and studied wintering mammals is the brown bear (Ursus arctos). Sommer et al. (2016) conducted a study with 16 free-ranging Eurasian brown bears in the forests of central and northern Sweden, taking faecal and blood samples for metabolic study, both during hibernation (February–March) and during active time (June). The faecal microbiota was analysed by metagenomics, extracting the genomic DNA, amplifying the V2-V3 region of the 16S rRNA gene, sequencing it by 454 pyrosequencing and analysing it with the MacQIIME program to obtain its identification. The observed results were a clear difference between winter and summer, demonstrable with a PCA analysis (Figure 3, left). They also saw a greater diversity of microbiota in summer, 4 times compared to winter, which was more homogeneous than summer (results not shown). The heterogeneity in summer may be related to a more varied diet. The lower winter diversity is linked to a reduction mainly of Firmicutes (now renamed Bacillota, see my post on Bacterial main phyla) and Actinobacteria (now renamed Actinomycetota), and instead there is an increase of Bacteroidetes (now renamed Bacteroidota) (Figure 3, right).

Figure 3. Brown bear faecal microbiota compared in winter (blue) and summer (red). Left: PCA (principal component analysis); right: relative abundance of the most abundant phyla (Sommer et al. 2016).

The increase in Bacteroidetes —especially Bacteroides fragilis— during hibernation can be explained by their ability to degrade host glycans in the absence of dietary polysaccharides and because Bacteroidetes can metabolize proteins and fats from the intestinal epithelium. Firmicutes such as Streptococcus probably decrease because they require more dietary fibre (Sommer et al. 2016).

It has been seen that the hibernation of brown bears also affects the metabolism, especially the lipidic one. Furthermore, transplanting the microbiota of wintering or active bears in June to mice with no microbiota results in the mice acquiring the corresponding seasonal metabolic traits of winter or summer, respectively (Sommer et al. 2016).

It should be noted that the lowest body temperatures during hibernation are well below the optimal temperatures of most gut microbes, but as mentioned above, the torpor is periodically interrupted by short phases of thermal rise that allow —in addition to activate the animal’s metabolism— increase the bacterial metabolism, with the consequent degradation of the substrates present in the intestinal lumen (Carey et al. 2013).

The animals most studied in relation to hibernation and their microbiota are ground squirrels, and specifically the thirteen-lined ground squirrel, Ictidomys tridecemlineatus (formerly Spermophilus tridecemlineatus) (Figure 4). They have a striking coloration, with stripes and spots on the dorsal fur that make it very easy to distinguish them from other species. These ground squirrels, along with the more well-known arboreal ones, groundhogs, marmots, flying squirrels and prairie dogs, make up the family of squirrels, within the order Rodentia, the rodents.

Figure 4. The thirteen-lined ground squirrel (Ictidomys tridecemlineatus). Image of Cheryl Gorske, taken from Pinterest.

The effect of the annual hibernation cycle on the intestinal microbiota of ground squirrell has been studied (Carey et al. 2013) by metagenomics in a similar way to that discussed for brown bears, by sequencing the 16 S rRNA genes from samples of the intestinal cecum of I. tridecemlineatus.

In this study the most abundant bacterial groups found, and which had more variation in the state of hibernation compared to when the animals were active, were Firmicutes, Bacteroidetes and Verrucomicrobiota —such as Akkermansia muciniphila, known to be beneficial to the human microbiota—. As we can see (Figure 5), in the active season there are more Firmicutes while with hibernation the other two groups increase. As I mentioned for bears, the Firmicutes prefer a diet rich in polysaccharides while the Bacteroidetes—and the Verrucomicrobiota in this case—consume mucins produced by the intestine. There is also less diversity of bacteria in winter. The microbiota differences between active and hibernation season are statistically significant, much more so than those observed for age, pre-hibernation diet, and other conditions. All in all, the microbiota of these ground squirrels is restructured every year, reflecting the differences in the preferences of the microbes regarding the substrates of the diet or the host and the survival capacities of the different bacterial taxa in the altered environment of the intestine in hibernation (Carey et al. 2013).

Figure 5. Illustrative diagram of the main changes in the microbiota of the ground squirrel throughout the annual hibernation cycle (Taken from Carey et al. 2013)

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THE MICROBIOTA OF HIBERNATION ALLOWS THE RECOVERY OF NITROGEN

As we have seen before, with hibernation there is a lack of nitrogen in the diet, and the minimum maintenance metabolism leads to a consumption of protein reserves —which can lead to muscle atrophy— and with this consumption, nitrogen compounds are released, such as ammonium which is toxic, and the body removes it in the form of urea by the urine.

But these animals do not lose muscle mass during the winter, and they do not excrete urea, since they do not urinate when they hibernate. How is it explained ? Well, the microbiota allows the survival of the animal during hibernation. Indeed, it has been seen that the microbiota of the 13-lined ground squirrels —Ictidomys tridecemlineatus— recycle the urea produced by protein catabolism, in this process called urea nitrogen salvage, where the microbial urease has a key role.

In fact, this mechanism was already known in ruminants, since the very complex microbiota of the rumen includes the recycling of protein nitrogen from the same microorganisms through urea, and even this compound is used as a nitrogen alternative in the diet of stabled ruminants, given the great urease capacity of the rich and diverse ruminant microbiota (Patra et al. 2018).

But in monogastric animals such as these squirrels, this ability to salvage N from urea, recently demonstrated by Regan et al., was not known (2022). The urea resulting from the catabolism of the animal’s proteins (Figure 6) is transformed by the urease of the microbes in the intestinal cecum into CO₂ and ammonium, and this is used by other microbes to generate amino acids that are finally transported to the liver to synthesize proteins for the different organs.

Figure 6. Proposed mechanism of N salvage from urea by the caecal microbiota of the large intestine during hibernation in the 13-lined ground squirrell Ictidomys tridecemlineatus (Figure taken from Regan et al. 2022).

Regan et al. (2022) have also seen that N salvage from urea is greatest at the end of hibernation, just before the squirrel wakes up and enters the active season, where it will need the muscles to have well recovered from torpor. This increase in N recirculation is reflected in the greater abundance of urea transporters and urease genes in this period (Figure 7). The urease genes are one transporter, 2 structural and 4 accessory genes.

Figure 7. Proportion of urease genes in the gut bacterial metagenome of Ictidomys tridecemlineatus, in summer, early winter and late winter (Regan et al. 2022)

Analysing the metagenomics of the urease genes has also shown that the most abundant bacterial taxa are different depending on which of these 3 periods (Figure 8). As we can see, the genus Alistipes (a Bacteroidetes) has the highest proportion of urease genes during hibernation, where it is also the most abundant genus, with populations in late winter six times higher than in summer (Regan et al. 2022).

Figure 8. The 10 most abundant bacterial taxa for urease genes in Ictidomys tridecemlineatus gut bacterial metagenome sequences, in summer, early winter, and late winter (Regan et al. 2022)

Furthermore, nitrogen salvage from urea may facilitate water conservation in wintering squirrels by diverting urea from the kidneys, thereby requiring less water for urine production, as is also the case in camels when lack of water (Mousa et al. 1983).

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AND BEYOND HIBERNATION

This mechanism of nitrogen salvage by the microbiota has implications that go beyond hibernation. For example, muscle atrophy affects millions of humans due to nitrogen-limited diets or sarcopenia in elderly people. A better understanding of the mechanisms of how the hibernation microbiota mitigates the effects of nitrogen limitation could provide strategies for muscle preservation in humans (Regan et al. 2022).

On the other hand, as we have seen, brown bears—and other animals—accumulate a lot of fat before hibernation and instead remain metabolically healthy. Understanding the underlying physiological mechanisms and the possible relationship with the microbiota could provide clues for new obesity therapies in humans (Sommer 2016).

And from a more general point of view, it is clear that the coevolution of mammals —and other animals— with intestinal microbes has produced very complex relationships that benefit both symbiont parties. Microbes shape the biology of their hosts in multiple ways, increasing resistance to colonization by pathogens, influencing gastrointestinal function and structure, directing immune system development, and increasing energy uptake from the diet. In turn, animal hosts provide a nutrient-rich environment that allows the development of well-diverse microbial communities (Carey et al. 2013).

BIBLIOGRAPHY

Carey HV, Andrews MT, Martin SL (2003) Mammalian Hibernation: Cellular and Molecular Responses to Depressed Metabolism and Low Temperature. Physiol Rev 83: 1153-1181.

Carey HV, Walters WA, Rob Knight R (2013) Seasonal restructuring of the ground squirrel gut microbiota over the annual hibernation cycle. Amer J Physiol-Reg It Comp Physiol 304, R33-R42 

Garcia de Jesús E (2022) Gut microbes help some squirrels stay strong during hibernation. ScienceNews 27 Jan 2022. 

Hibernation (2023, April 4). Wikipedia

Mousa HM, Ali KE, Hume ID (1983) Effects of water deprivation on urea metabolism in camels, desert sheep and desert goats fed dry desert grass. Comp Biochem Physiol A Comp Physiol 74:715-20

Patra AK, Aschenbach JR (2018) Ureases in the gastrointestinal tracts of ruminant and monogastric animals and their implication in urea-N/ammonia metabolism: A review. J Adv Res 13, 39-50. 

Regan MD et al (2022) Nitrogen recycling via gut symbionts increases in ground squirrels over the hibernation season. Science 375, 6579, 460-463

Sommer F et al. (2016) The Gut Microbiota Modulates Energy Metabolism in the Hibernating Brown Bear Ursus arctos. Cell Reports 14, 1655-1661. 

Thirteen-lined_ground_squirrel (2023, April 4) Wikipedia

Wilke C (2020) These Arctic squirrels recycle bits of their own bodies to survive winter. ScienceNews 16 Dec 2020.