How the gut and liver hibernate.

For hibernating mammals, the transition from summer active to winter hibernation seasons come with significant remodeling at cellular, organ and whole organism levels. This review summarizes and synthesizes what is known about hibernation-related remodeling in the gastrointestinal tract of the thirteen-lined ground squirrel, including intestinal and hepatic physiology and the gut microbiota. Hibernation alters intestinal epithelial, immune and cell survival pathways in ways that point to a protective phenotype in the face of prolonged fasting and major fluctuations in nutrient and oxygen delivery during torpor-arousal cycles. The prolonged fasting associated with hibernation alters lipid metabolism and systemic cholesterol dynamics, with both the gut and liver participating in these changes. Fasting also affects the gut microbiota, altering the abundance, composition and diversity of gut microbes and impacting the metabolites they produce in ways that may influence hibernation-related traits in the host. Finally, interventional studies have demonstrated that the hibernation phenotype confers resistance to experimental ischemia-reperfusion injury in both gut and liver, suggesting potential therapeutic roadmaps. We propose that the plasticity inherent to hibernation biology may contribute to this stress tolerance, and in the spirit of August Krogh, makes hibernators particularly valuable for study to identify solutions to certain problems.

Shifts in metabolic fuel use coincide with maximal rates of ventilation and body surface rewarming in an arousing hibernator.

It is well established that hibernating mammals rely predominantly on lipid stores to fuel metabolism throughout the hibernation season. However, it is unclear if other endogenous fuels contribute to the rapid, ~400-fold increase in metabolic rate during the early phase of arousal from torpor. To investigate this issue, we used cavity ring-down spectroscopy, a technique that provides a real-time indication of fuel use by measuring the ratio of 13C to 12C in the exhaled CO2 of arousing 13-lined ground squirrels (Ictidomys tridecemlineatus). We used infrared thermography to simultaneously measure ventilation and surface temperature change in various body regions, and we interpreted these data in light of changing plasma metabolite abundances at multiple stages of arousal from torpor. We found that hibernating squirrels use a combination of lipids and, likely, carbohydrates to fuel the initial ~60 min of arousal before switching to predominantly lipid oxidation. This fuel switch coincided with times of maximal rates of ventilation and rewarming of different body surface regions, including brown adipose tissue. Infrared thermography revealed zonal rewarming, whereby the brown adipose tissue region was the first to warm, followed by the thoracic and head regions and, finally, the posterior half of the body. Consistent with the results from cavity ring-down spectroscopy, plasma metabolite dynamics during early arousal suggested a large reliance on fatty acids, with a contribution from carbohydrates and glycerol. Because of their high oxidative flux rates and efficient O2 use, carbohydrates might be an advantageous metabolic fuel during the early phase of arousal, when metabolic demands are high but ventilation rates and, thus, O2 supply are relatively low.

The Hibernator Microbiome: Host-Bacterial Interactions in an Extreme Nutritional Symbiosis.

Animals that undergo seasonal cycles of feeding and fasting have adaptations that maintain integrity of organ systems when dietary nutrients are lacking. Food deprivation also challenges the gut microbiota, which relies heavily on host diet for metabolic substrates and the gastrointestinal tract, which is influenced by enteral nutrients and microbial activity. Winter fasting in hibernators shifts the microbiota to favor taxa with the capacity to degrade and utilize host-derived substrates and disfavor taxa that prefer complex plant polysaccharides. Microbiome alterations may contribute to hibernation-induced changes in the intestinal immune system, epithelial barrier function, and other host features that are affected by microbial short-chain fatty acids and other metabolites. Understanding mechanisms by which the hibernator host and its gut symbionts adapt to the altered nutritional landscape during winter fasting may provide insights into protective mechanisms that are compromised when nonhibernating species, such as humans, undergo long periods of enteral nutrient deprivation.

A place for host-microbe symbiosis in the comparative physiologist's toolbox.

Although scientists have long appreciated that metazoans evolved in a microbial world, we are just beginning to appreciate the profound impact that host-associated microbes have on diverse aspects of animal biology. The enormous growth in our understanding of host-microbe symbioses is rapidly expanding the study of animal physiology, both technically and conceptually. Microbes associate functionally with various body surfaces of their hosts, although most reside in the gastrointestinal tract. Gut microbes convert dietary and host-derived substrates to metabolites such as short-chain fatty acids, thereby providing energy and nutrients to the host. Bacterial metabolites incorporated into the host metabolome can activate receptors on a variety of cell types and, in doing so, alter host physiology (including metabolism, organ function, biological rhythms, neural activity and behavior). Given that host-microbe interactions affect diverse aspects of host physiology, it is likely that they influence animal ecology and, if they confer fitness benefits, the evolutionary trajectory of a species. Multiple variables - including sampling regime, environmental parameters, host metadata and analytical methods - can influence experimental outcomes in host-microbiome studies, making careful experimental design and execution crucial to ensure reproducible and informative studies in the laboratory and field. Integration of microbiomes into comparative physiology and ecophysiological investigations can reveal the potential impacts of the microbiota on physiological responses to changing environments, and is likely to bring valuable insights to the study of host-microbiome interactions among a broad range of metazoans, including humans.

Animals in a bacterial world, a new imperative for the life sciences.

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal-bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other's genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal-bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

Prioritization of skeletal muscle growth for emergence from hibernation.

Mammalian hibernators provide an extreme example of naturally occurring challenges to muscle homeostasis. The annual hibernation cycle is characterized by shifts between summer euthermy with tissue anabolism and accumulation of body fat reserves, and winter heterothermy with fasting and tissue catabolism. The circannual patterns of skeletal muscle remodelling must accommodate extended inactivity during winter torpor, the motor requirements of transient winter active periods, and sustained activity following spring emergence. Muscle volume in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) calculated from MRI upper hindlimb images (n=6 squirrels, n=10 serial scans) declined from hibernation onset, reaching a nadir in early February. Paradoxically, mean muscle volume rose sharply after February despite ongoing hibernation, and continued total body mass decline until April. Correspondingly, the ratio of muscle volume to body mass was steady during winter atrophy (October-February) but increased (+70%) from February to May, which significantly outpaced changes in liver or kidney examined by the same method. Generally stable myocyte cross-sectional area and density indicated that muscle remodelling is well regulated in this hibernator, despite vastly altered seasonal fuel and activity levels. Body composition analysis by echo MRI showed lean tissue preservation throughout hibernation amid declining fat mass by the end of winter. Muscle protein synthesis was 66% depressed in early but not late winter compared with a summer fasted baseline, while no significant changes were observed in the heart, liver or intestine, providing evidence that could support a transition in skeletal muscle regulation between early and late winter, prior to spring emergence and re-feeding.

Hibernation alters the diversity and composition of mucosa-associated bacteria while enhancing antimicrobial defence in the gut of 13-lined ground squirrels.

The gut microbiota plays important roles in animal nutrition and health. This relationship is particularly dynamic in hibernating mammals where fasting drives the gut community to rely on host-derived nutrients instead of exogenous substrates. We used 16S rRNA pyrosequencing and caecal tissue protein analysis to investigate the effects of hibernation on the mucosa-associated bacterial microbiota and host responses in 13-lined ground squirrels. The mucosal microbiota was less diverse in winter hibernators than in actively feeding spring and summer squirrels. UniFrac analysis revealed distinct summer and late winter microbiota clusters, while spring and early winter clusters overlapped slightly, consistent with their transitional structures. Communities in all seasons were dominated by Firmicutes and Bacteroidetes, with lesser contributions from Proteobacteria, Verrucomicrobia, Tenericutes and Actinobacteria. Hibernators had lower relative abundances of Firmicutes, which include genera that prefer plant polysaccharides, and higher abundances of Bacteroidetes and Verrucomicrobia, some of which can survive solely on host-derived mucins. A core mucosal assemblage of nine operational taxonomic units shared among all individuals was identified with an average total sequence abundance of 60.2%. This core community, together with moderate shifts in specific taxa, indicates that the mucosal microbiota remains relatively stable over the annual cycle yet responds to substrate changes while potentially serving as a pool for 'seeding' the microbiota once exogenous substrates return in spring. Relative to summer, hibernation reduced caecal crypt length and increased MUC2 expression in early winter and spring. Hibernation also decreased caecal TLR4 and increased TLR5 expression, suggesting a protective response that minimizes inflammation.

Elucidating nature's solutions to heart, lung, and blood diseases and sleep disorders.

Evolution has provided a number of animal species with extraordinary phenotypes. Several of these phenotypes allow species to survive and thrive in environmental conditions that mimic disease states in humans. The study of evolved mechanisms responsible for these phenotypes may provide insights into the basis of human disease and guide the design of new therapeutic approaches. Examples include species that tolerate acute or chronic hypoxemia like deep-diving mammals and high-altitude inhabitants, as well as those that hibernate and interrupt their development when exposed to adverse environments. The evolved traits exhibited by these animal species involve modifications of common biological pathways that affect metabolic regulation, organ function, antioxidant defenses, and oxygen transport. In 2006, the National Heart, Lung, and Blood Institute released a funding opportunity announcement to support studies that were designed to elucidate the natural molecular and cellular mechanisms of adaptation in species that tolerate extreme environmental conditions. The rationale for this funding opportunity is detailed in this article, and the specific evolved mechanisms examined in the supported research are described. Also highlighted are past medical advances achieved through the study of animal species that have evolved extraordinary phenotypes as well as the expectations for new understanding of nature's solutions to heart, lung, blood, and sleep disorders through future research in this area.

Animal-microbial symbioses in changing environments.

The environments in which animals have evolved and live have profound effects on all aspects of their biology. Predictable rhythmic changes in the physical environment are arguably among the most important forces shaping the evolution of behavior and physiology of animals, and to anticipate and prepare for these predictable changes, animals have evolved biological clocks. Unpredictable changes in the physical environment have important impacts on animal biology as well. The ability of animals to cope with and survive unpredictable perturbations depends on phenotypic plasticity and/or microevolution. From the time metazoans first evolved from their protistan ancestors they have lived in close association with a diverse array of microbes that have influenced, in some way, all aspects of the evolution of animal structure, function and behavior. Yet, few studies have addressed whether daily or seasonal rhythms may affect, or be affected by, an animal's microbial symbionts. This survey highlights how biologists interested in the ecological and evolutionary physiology of animals whose lifestyles are influenced by environmental cycles may benefit from considering whether symbiotic microbes have shaped the features they study.

Seasonal restructuring of the ground squirrel gut microbiota over the annual hibernation cycle.

Many hibernating mammals suspend food intake during winter, relying solely on stored lipids to fuel metabolism. Winter fasting in these species eliminates a major source of degradable substrates to support growth of gut microbes, which may affect microbial community structure and host-microbial interactions. We explored the effect of the annual hibernation cycle on gut microbiotas using deep sequencing of 16S rRNA genes from ground squirrel cecal contents. Squirrel microbiotas were dominated by members of the phyla Bacteroidetes, Firmicutes, and Verrucomicrobia. UniFrac analysis showed that microbiotas clustered strongly by season, and maternal influences, diet history, host age, and host body temperature had minimal effects. Phylogenetic diversity and numbers of operational taxonomic units were lowest in late winter and highest in the spring after a 2-wk period of refeeding. Hibernation increased relative abundance of Bacteroidetes and Verrucomicrobia, phyla that contain species capable of surviving on host-derived substrates such as mucins, and reduced relative abundance of Firmicutes, many of which prefer dietary polysaccharides. Hibernation reduced cecal short-chain fatty acid and ammonia concentrations, and increased and decreased concentrations of acetate and butyrate, respectively. These results indicate that the ground squirrel microbiota is restructured each year in a manner that reflects differences in microbial preferences for dietary vs. host-derived substrates, and thus the competitive abilities of different taxa to survive in the altered environment in the hibernator gut.

Induction of torpor: mimicking natural metabolic suppression for biomedical applications.

Mammalian hibernation consists of periods of depressed metabolism and reduced body temperature called "torpor" that are interspersed by normothermic arousal periods. Numerous cellular processes are halted during torpor, including transcription, translation, and ion homeostasis. Hibernators are able to survive long periods of low blood flow and body temperature followed by rewarming and reperfusion without overt signs of organ injury, which makes these animals excellent models for application of natural protective mechanisms to human medicine. This review examines efforts to induce torpor-like states in non-hibernating species using pharmacological compounds. Elucidating the underlying mechanisms of natural and pharmacologically induced torpor will speed the development of new clinical approaches to treat a variety of trauma and stress states in humans.

Characterization of captive and wild 13-lined ground squirrel cecal microbiotas using Illumina-based sequencing.

BACKGROUND: Hibernating animals experience extreme changes in diet that make them useful systems for understanding host-microbial symbioses. However, most of our current knowledge about the hibernator gut microbiota is derived from studies using captive animals. Given that there are substantial differences between captive and wild environments, conclusions drawn from studies with captive hibernators may not reflect the gut microbiota's role in the physiology of wild animals. To address this, we used Illumina-based sequencing of the 16S rRNA gene to compare the bacterial cecal microbiotas of captive and wild 13-lined ground squirrels (TLGS) in the summer. As the first study to use Illumina-based technology to compare the microbiotas of an obligate rodent hibernator across the year, we also reported changes in captive TLGS microbiotas in summer, winter, and spring. RESULTS: Wild TLGS microbiotas had greater richness and phylogenetic diversity with less variation in beta diversity when compared to captive microbiotas. Taxa identified as core operational taxonomic units (OTUs) and found to significantly contribute to differences in beta diversity were primarily in the families Lachnospiraceae and Ruminococcaceae. Captive TLGS microbiotas shared phyla and core OTUs across the year, but active season (summer and spring) microbiotas had different alpha and beta diversities than winter season microbiotas. CONCLUSIONS: This is the first study to compare the microbiotas of captive and wild rodent hibernators. Our findings suggest that data from captive and wild ground squirrels should be interpreted separately due to their distinct microbiotas. Additionally, as the first study to compare seasonal microbiotas of obligate rodent hibernators using Illumina-based 16S rRNA sequencing, we reported changes in captive TLGS microbiotas that are consistent with previous work. Taken together, this study provides foundational information for improving the reproducibility and experimental design of future hibernation microbiota studies.

Nitrogen recycling via gut symbionts increases in ground squirrels over the hibernation season.

Hibernation is a mammalian strategy that uses metabolic plasticity to reduce energy demands and enable long-term fasting. Fasting mitigates winter food scarcity but eliminates dietary nitrogen, jeopardizing body protein balance. Here, we reveal gut microbiome-mediated urea nitrogen recycling in hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Ureolytic gut microbes incorporate urea nitrogen into metabolites that are absorbed by the host, with the nitrogen reincorporated into the squirrel's protein pool. Urea nitrogen recycling is greatest after prolonged fasting in late winter, when urea transporter abundance in gut tissue and urease gene abundance in the microbiome are highest. These results reveal a functional role for the gut microbiome during hibernation and suggest mechanisms by which urea nitrogen recycling may contribute to protein balance in other monogastric animals.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) show microstructural bone loss during hibernation but preserve bone macrostructural geometry and strength.

Lack of activity causes bone loss In most animals. Hibernating bears have physiological processes to prevent cortical and trabecular bone loss associated with reduced physical activity, but different mechanisms of torpor among hibernating species may lead to differences in skeletal responses to hibernation. There are conflicting reports regarding whether small mammals experience bone loss during hibernation. To investigate this phenomenon, we measured cortical and trabecular bone properties in physically active and hibernating juvenile and adult 13-lined ground squirrels (Ictidomys tridecemlineatus, previous genus name Spermophilus). Cortical bone geometry, strength and mineral content were similar in hibernating compared with active squirrels, suggesting that hibernation did not cause macrostructural cortical bone loss. Osteocyte lacunar size increased (linear regression, P=0.001) over the course of hibernation in juvenile squirrels, which may indicate an osteocytic role in mineral homeostasis during hibernation. Osteocyte lacunar density and porosity were greater (+44 and +59%, respectively; P<0.0001) in hibernating compared with active squirrels, which may reflect a decrease in osteoblastic activity (per cell) during hibernation. Trabecular bone volume fraction in the proximal tibia was decreased (-20%; P=0.028) in hibernating compared with physically active adult squirrels, but was not different between hibernating and active juvenile squirrels. Taken together, these data suggest that 13-lined ground squirrels may be unable to prevent microstructural losses of cortical and trabecular bone during hibernation, but importantly may possess a biological mechanism to preserve cortical bone macrostructure and strength during hibernation, thus preventing an increased risk of bone fracture during remobilization in the spring.

Cholecystokinin activation of central satiety centers changes seasonally in a mammalian hibernator.

Hibernators that rely on lipids during winter exhibit profound changes in food intake over the annual cycle. The mechanisms that regulate appetite changes in seasonal hibernators remain unclear, but likely consist of complex interactions between gut hormones, adipokines, and central processing centers. We hypothesized that seasonal changes in the sensitivity of neurons in the nucleus tractus solitarius (NTS) to the gut hormone cholecystokinin (CCK) may contribute to appetite regulation in ground squirrels. Spring (SPR), late summer (SUM), and winter euthermic hibernating (HIB) 13-lined ground squirrels (Ictidomys tridecemlineatus) were treated with intraperitoneal CCK (100 µg/kg) or vehicle (CON) for 3h and Fos expression in the NTS was quantified. In CON squirrels, numbers of Fos-positive neurons in HIB were low compared to SPR and SUM. CCK treatment increased Fos-positive neurons in the NTS at the levels of the area postrema (AP) and pre AP during all seasons and at the level of the rostral AP in HIB squirrels. The highest absolute levels of Fos-positive neurons were found in SPR CCK squirrels, but the highest relative increase from CON was found in HIB CCK squirrels. Fold-changes in Fos-positive neurons in SUM were intermediate between SPR and HIB. Thus, CCK sensitivity falls from SPR to SUM suggesting that seasonal changes in sensitivity of NTS neurons to vagally-derived CCK may influence appetite in the active phase of the annual cycle in hibernating squirrels. Enhanced sensitivity to CCK signaling in NTS neurons of hibernators indicates that changes in gut-brain signaling may contribute to seasonal changes in food intake during the annual cycle.

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation.

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast.

Effect of stevia on the gut microbiota and glucose tolerance in a murine model of diet-induced obesity.

Artificial sweeteners have been shown to induce glucose intolerance by altering the gut microbiota; however, little is known about the effect of stevia. Here, we investigate whether stevia supplementation induces glucose intolerance by altering the gut microbiota in mice, hypothesizing that stevia would correct high fat diet-induced glucose intolerance and alter the gut microbiota. Mice were split into four treatment groups: low fat, high fat, high fat + saccharin and high fat + stevia. After 10 weeks of treatment, mice consuming a high fat diet (60% kcal from fat) developed glucose intolerance and gained more weight than mice consuming a low fat diet. Stevia supplementation did not impact body weight or glucose intolerance. Differences in species richness and relative abundances of several phyla were observed in low fat groups compared to high fat, stevia and saccharin. We identified two operational taxonomic groups that contributed to differences in beta-diversity between the stevia and saccharin groups: Lactococcus and Akkermansia in females and Lactococcus in males. Our results demonstrate that stevia does not rescue high fat diet-induced changes in glucose tolerance or the microbiota, and that stevia results in similar alterations to the gut microbiota as saccharin when administered in concordance with a high fat diet.