Hibernation is an adaptation that helps many animals to conserve energy during food shortage in winter. Brown bears double their fat depots during summer and use these stored lipids during hibernation. Although bears seasonally become obese, they remain metabolically healthy. We analyzed the microbiota of free-ranging brown bears during their active phase and hibernation. Compared to the active phase, hibernation microbiota had reduced diversity, reduced levels of Firmicutes and Actinobacteria, and increased levels of Bacteroidetes. Several metabolites involved in lipid metabolism, including triglycerides, cholesterol, and bile acids, were also affected by hibernation. Transplantation of the bear microbiota from summer and winter to germ-free mice transferred some of the seasonal metabolic features and demonstrated that the summer microbiota promoted adiposity without impairing glucose tolerance, suggesting that seasonal variation in the microbiota may contribute to host energy metabolism in the hibernating brown bear.

Here, we investigated how hibernation in free-ranging brown bears affects the gut microbiota and plasma metabolites, and whether a seasonally altered microbiota contributes to the healthy obesity phenotype during summer. We used 16S rRNA profiling and next-generation sequencing to comprehensively analyze the fecal microbiota of free-ranging brown bears captured during hibernation (February) and during the active period (June) of the same year ( Figure 1 A). We showed that the winter microbiota comprised fewer bacterial taxa ( Figure S1 A) and was more homogenous than the summer microbiota ( Figure S1 B), which may reflect the varied diet among bears during the summer.

Free-ranging brown bears (Ursus arctos) undergo cycles of intense eating and weight gain during the summer followed by prolonged dormant hypometabolic fasting for up to 6 months during the winter (). Despite the large fat accumulation before hibernation, bears remain metabolically healthy (), which contrasts with the strong association between obesity and insulin resistance in humans. Thus, the brown bear may constitute a model for healthy obesity and studying hibernation might be a promising approach to develop novel therapies for obesity. The intestines of mammals harbor diverse microbial ecosystems that have profound effects on host physiology (). The gut microbiota contributes to energy harvest from the diet () and is altered in obesity and type 2 diabetes (). Furthermore, diet, which is seasonably variable in bears (), strongly affects the gut microbiota () and both fasting and hibernation alter the gut microbiota composition ().

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

Seasonal changes in food composition of the brown bear (Ursus arctos) from the edge of its occurrence – Eastern Carpathians (Slovakia).

Results and Discussion

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Gerok W. Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties--studies on isolated hepatocytes and lipid membrane vesicles. Table 1 Signs of Dehydration and Reduced Hemolysis in Blood of Hibernating Brown Bears Parameter Unit Summer Mean (Range) Winter Mean (Range) Ratio W/S p Value Total bile acids nM 1,762 (137–4,379) 606 (171–1,177) 0.3 <0.01 White blood cells 109/l 7.7 (3.4–15.7) 6.2 (3.8–15.6) 0.8 ns Red blood cells 1012/l 6.6 (6.2–7) 8.6 (7.7–9.4) 1.3 <0.001 Hemoglobin g/l 161 (132–176) 203 (183–223) 1.3 <0.001 Hematocrit % 42.6 (36.6–46.1) 54.2 (48–60) 1.3 <0.001 Platelets 109/l 310 (251–359) 184 (65–265) 0.6 <0.001 Neutrophils 109/l 3.3 (2.4–4.4) 3.5 (2.1–4.4) 1.1 ns Lymphocytes 109/l 1.2 (0.8–1.9) 1.5 (0.9–2.7) 1.2 ns Monocytes 109/l 0.3 (0.2–0.5) 0.4 (0.3–0.7) 1.3 ns Eosinophils 109/l 0.003 (0–0.01) 0 (0–0) 0.0 <0.05 Basophils 109/l 0.01 (0–0.04) 0.001 (0–0.01) 0.2 ns Alkaline phosphatase U/l 134 (100–174) 19.1 (13–27) 0.1 <0.001 Alanine transaminase U/l 36 (23–60) 11.4 (9–14) 0.3 <0.001 Aspartate transaminase U/l 90 (57–148) 53.1 (39–85) 0.6 <0.05 Bilirubin μM 18 (9.9–30.9) 10.6 (5–23) 0.6 0.08 Lactate dehydrogenase μkat/l 13.3 (13.3–13.3) 9.1 (7.2–11.2) 0.7 <0.001 Gamma glutamyltransferase μkat/l 0.5 (0.3–0.7) 0.3 (0.2–0.5) 0.5 <0.001 C-reactive protein mg/l 0.003 (0–0.01) 0.014 (0–0.04) 5.1 ns Hematology analysis was performed on blood samples from brown bears during summer and winter, and marker enzymes were measured. Data show mean and range with n = 11–15 for summer and n = 7 for winter. ns, nonsignificant. We also observed that total bile acid levels in the serum were lower in the winter with large reductions in primary and conjugated bile acids ( Figure 2 G; Table S2 ). Notably, expression of the rate-limiting enzyme of bile acid production CYP7A1 is reduced in the liver of hibernating mammals (), and the microbiota contributes to modifications of bile acids (). Bile acids promote lipid uptake and respond to food intake. Bears do not eat for up to 6 months during hibernation, which likely explains the reduced bile acid levels in the winter. Levels of deoxycholic acid and lithocholic acid, both of which are dependent on the microbiota, are known to have hemolytic activity () and were reduced during hibernation ( Table S2 ). Notably, several blood parameters that are linked to hemolysis and dehydration were also altered during hibernation ( Table 1 ). For example, levels of red blood cells and hemoglobin were higher in the winter, whereas lactate dehydrogenase (marker of hemolysis) and bilirubin (used during recycling of hemoglobin) levels decreased during hibernation. Thus, microbiota-dependent changes in the bile acid profile might contribute to the reduced hemolysis during hibernation.

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et al. Metabolic changes in summer active and anuric hibernating free-ranging brown bears (Ursus arctos). 11 and winter 2.55 ± 1.03 × 1011 16S rDNA copies/g cecal content, p = 0.6). Despite their increased fat mass, mice colonized with summer bear microbiota showed no differences or even a slight improvement in glucose metabolism compared to mice colonized with a winter microbiota ( Figure 3 Metabolic Programming by the Seasonal Bear Microbiota Show full caption (A) Experimental scheme. Germ-free mice were colonized with a bear summer or winter microbiota and followed for 2 weeks. (B) Principal coordinate analysis of the cecal microbiota of mice colonized with a bear fecal microbiota from summer or winter. 1104, 1202, 1303, and 1304 denote bear fecal donors. (C–E) Weight gain (C), body-fat gain (D), and epididymal white adipose tissue (EWAT) (D) weight were determined. (F) Glucose metabolism was assessed via intraperitoneal glucose tolerance test (IPGTT). (G) Concentrations of triglycerides in blood of mice colonized with seasonal bear microbiota. Data are mean ± SEM of four experiments (n = 4) with each five animals per colonization. ∗p < 0.05. To test whether the seasonal differences in the bear microbiota affect host physiology, we colonized germ-free mice with a summer or winter bear microbiota ( Figure 3 A). 16S rRNA profiling of the colonized mice confirmed successful colonization ( Figures 3 B and S4 ). There was no seasonal difference in alpha diversity, possibly because all mice received the same food. Mice colonized with a summer bear microbiota trended toward a greater weight (p = 0.09) and showed a greater fat gain than mice colonized with a winter bear microbiota ( Figures 3 C and 3D) but did not display a significant difference in epididymal white adipose tissue weight ( Figure 3 E). In humans, adiposity is associated with reduced insulin sensitivity (). In contrast, brown bears seem to become only temporarily insulin resistant with mild hyperglycemia during hibernation but remain insulin sensitive during the rest of the seasonal cycle independent of fat accumulation (L. Nelson, personal communication;). The increased weight and adiposity of the mice colonized with a summer bear microbiota were not due to higher bacterial abundance as tested by 16S rDNA qPCR (summer 2.27 ± 1.16 × 10and winter 2.55 ± 1.03 × 1016S rDNA copies/g cecal content, p = 0.6). Despite their increased fat mass, mice colonized with summer bear microbiota showed no differences or even a slight improvement in glucose metabolism compared to mice colonized with a winter microbiota ( Figure 3 F). By performing targeted metabolomics, we showed that the seasonal metabolic phenotype of the bears could be partially transferred to germ-free mice by colonization with a bear microbiota. For example, mice colonized with a winter bear microbiota trended toward slightly higher serum levels of cholesteryl esters ( Table S2 ) and triglycerides ( Figure 3 G) compared with mice that were colonized with summer microbiota.