Seasonal fluctuations in BDNF regulate hibernation and torpor in golden-mantled ground squirrels.

Aphagic hibernators such as the golden-mantled ground squirrel (GMGS; Callospermophilus lateralis) can fast for months and exhibit profound seasonal fluctuations in body weight, food intake, and behavior. Brain-derived neurotrophic factor (BDNF) regulates cellular and systemic metabolism via mechanisms that are conserved across mammalian species. In this study, we characterized regional changes in BDNF with hibernation, hypothermia, and seasonal cycle in GMGS. Analysis of BDNF protein concentrations by ELISA revealed overlapping seasonal patterns in the hippocampus and hypothalamus, where BDNF levels were highest in summer and lowest in winter. BDNF is the primary ligand for receptor tyrosine kinase B (TrkB), and BDNF/TrkB signaling in the brain potently regulates energy expenditure. To examine the functional relevance of seasonal variation in BDNF, hibernating animals were injected with the small molecule TrkB agonist 7,8-dihydroxyflavone (DHF) daily for 2 wk. When compared with vehicle, DHF-treated animals exhibited fewer torpor bouts and shorter bout durations. These results suggest that activating BDNF/TrkB disrupts hibernation and raise intriguing questions related to the role of BDNF as a potential regulatory mechanism or downstream response to seasonal changes in body temperature and environment.NEW & NOTEWORTHY Golden-mantled ground squirrels exhibit dramatic seasonal fluctuations in metabolism and can fast for months while hibernating. Brain-derived neurotrophic factor is an essential determinant of cellular and systemic metabolism, and in this study, we characterized seasonal fluctuations in BDNF expression and then administered the small molecule BDNF mimetic 7,8-dihydroxyflavone (DHF) in hibernating squirrels. The results indicate that activating BDNF/TrkB signaling disrupts hibernation, with implications for synaptic homeostasis in prolonged hypometabolic states.

The effects of neurectomy and hibernation on bone properties and the endocannabinoid system in marmots (Marmota flaviventris).

Hibernators have adapted a physiological mechanism allowing them to undergo long periods of inactivity without experiencing bone loss. However, the biological mechanisms that prevent bone loss are unknown. Previous studies found meaningful changes, between active and hibernating marmots, in the endocannabinoid system of many tissues, including bone. Cannabinoid receptors (CB1 and CB2) have divergent localization in bone. CB1 is predominately found on sympathetic nerve terminals, while CB2 is more abundant on bone cells and their progenitors. This study aimed to determine the contribution of innervation on endocannabinoid regulation of bone properties in hibernating (during torpor) and non-hibernating yellow-bellied marmots. Neurectomy, a model for disuse osteoporosis, was performed unilaterally in both hibernating and active marmots. Endocannabinoid concentrations were measured in bone marrow, cortical, and trabecular regions from fourth metatarsals of both hindlimbs using microflow chromatography-tandem quadrupole mass spectrometry. Trabecular bone architectural properties of fifth metatarsals were evaluated using micro-computed tomography. There were ligand-specific increases with neurectomy in active, but not hibernating, marmots. Trabecular bone architectural properties were not affected by neurectomy during hibernation, but did show some minor negative changes in active marmots. These findings suggest protection from bone loss in hibernating rodents is peripherally rather than centrally regulated. Furthermore, findings suggest even active marmots with normal metabolism are partially protected from disuse induced bone loss compared to laboratory rodents. Understanding the mechanism hibernators use to maintain bone density may guide development for novel bone loss prevention therapies.

Tissue-specific seasonal changes in mitochondrial function of a mammalian hibernator.

Mammalian hibernators, such as golden-mantled ground squirrels (Callospermophilus lateralis; GMGS), cease to feed while reducing metabolic rate and body temperature during winter months, surviving exclusively on endogenous fuels stored before hibernation. We hypothesized that mitochondria, the cellular sites of oxidative metabolism, undergo tissue-specific seasonal adjustments in carbohydrate and fatty acid utilization to facilitate or complement this remarkable phenotype. To address this, we performed high-resolution respirometry of mitochondria isolated from GMGS liver, heart, skeletal muscle, and brown adipose tissue (BAT) sampled during summer (active), fall (prehibernation), and winter (hibernation) seasons using multisubstrate titration protocols. Mitochondrial phospholipid composition was examined as a postulated intrinsic modulator of respiratory function across tissues and seasons. Respirometry revealed seasonal variations in mitochondrial oxidative phosphorylation capacity, substrate utilization, and coupling efficiency that reflected the distinct functions and metabolic demands of the tissues they support. A consistent finding across tissues was a greater influence of fatty acids (palmitoylcarnitine) on respiratory parameters during the prehibernation and hibernation seasons. In particular, fatty acids had a greater suppressive effect on pyruvate-supported oxidative phosphorylation in heart, muscle, and liver mitochondria and enhanced uncoupled respiration in BAT and muscle mitochondria in the colder seasons. Seasonal variations in the mitochondrial membrane composition reflected changes in the supply and utilization of polyunsaturated fatty acids but were generally mild and inconsistent with functional variations. In conclusion, mitochondria respond to seasonal variations in physical activity, temperature, and nutrient availability in a tissue-specific manner that complements circannual shifts in the bioenergetic and thermoregulatory demands of mammalian hibernators.

Fecal glucocorticoid metabolites in wild yellow-bellied marmots: experimental validation, individual differences and ecological correlates.

Natural selection is expected to shape phenotypic traits that permit organisms to respond appropriately to the environments in which they live. One important mechanism by which animals cope with changes in their environment is through physiological responses to stressors mediated by glucocorticoid hormones. Here we perform biological and physiological validations of a minimally-invasive technique for assessing fecal corticosterone metabolites (FCMs) in captive and wild groups of yellow-bellied marmots (Marmota flaviventris). Then we draw from ten years of data on these obligate hibernators at the Rocky Mountain Biological Laboratory in Colorado, USA to assess the extent to which seasonal and daily changes explain naturalistic variation in baseline levels of FCMs. Interestingly, beyond important population-level variation with respect to year, season, time of day, sex, age and reproductive state, we found repeatable inter-individual differences in FCMs, suggesting this hormonal trait might be a meaningful target of selection. FCM levels were 68% lower in captive than wild marmots, suggesting that the natural environment in which these animals occur is generally more challenging or less predictable than life in captivity. Most live-trapping events failed to represent stressors for wild marmots such that repeated measurements of traits were possible with minimal "stress" to subjects. We also document the natural ranges of annual and seasonal variation necessary for understanding the extent to which anthropogenic assaults represent stressors for wild mammals. Taken together, this study provides a foundation for understanding the evolution of hormonal traits and has important welfare and conservation implications for field biologists.

Peripheral ghrelin stimulates feeding behavior and positive energy balance in a sciurid hibernator.

Hibernators exhibit a robust circannual cycle of body mass gain and loss primarily mediated by food intake, but the pathways controlling food intake in these animals have not been fully elucidated. Ghrelin is an orexigenic hormone that increases feeding in all mammals studied so far, but has not until recently been studied in hibernators. In other mammals, ghrelin stimulates feeding through phosphorylation and activation of AMP-activated protein kinase (AMPK). Activation of AMPK phosphorylates and deactivates acetyl Co-A carboxylase (ACC), a committed step in fatty acid synthesis. In order to determine the effects of exogenous ghrelin on food intake and metabolic factors (i.e. non-esterified fatty acids (NEFAs), and hypothalamic AMPK and ACC) in hibernators, ghrelin was peripherally injected into ground squirrels in all four seasons. Changes in food intake and body mass were recorded over a 2-6 hour period post injections, and squirrels were euthanized. Brains and blood were removed, and Western blots were performed to determine changes in phosphorylation of hypothalamic AMPK and ACC. A colorimetric assay was used to determine changes in concentration of serum NEFAs. We found that food intake, body mass, and locomotor activity significantly increased with ghrelin injections versus saline-injected controls, even in animals injected during their aphagic winter season. Injected ghrelin was correlated with increased phosphorylation of AMPK, but didn't have an effect on ACC in winter. Ghrelin-injected animals also had increased levels of serum NEFAs compared with saline controls. This study is the first to show an effect of injected ghrelin on a hibernator.

AMPK and ACCchange with fasting and physiological condition in euthermic and hibernating golden-mantled ground squirrels (Callospermophilus lateralis).

AMP-activated protein kinase (AMPK) is a cellular energy sensor that responds to low endogenous energy by stimulating fatty acid oxidation (through inactivation of acetyl-CoA carboxylase (ACC)) and food intake. Fasting generally stimulates phosphorylation of AMPK (pAMPK) and ACC (pACC), but it is unclear how AMPK and ACC react to a long-term fast (i.e. hibernation). We performed Western blots for total and pAMPK and pACC on tissues from a species of hibernator (Callospermophilus lateralis) after short-term summer fasting (1-5 days) and long-term winter fasting (3 months). Winter animals were sacrificed during hibernation at low body temperature (torpid, T(b)~5°C) or at normal high T(b)(euthermic, T(b)~37°C). We found a general increase in pAMPK in most tissues (liver, muscle, and white adipose tissue (WAT), but not hypothalamus) and pACC in all tissues after a short-term summer fast. Response of AMPK and ACC to a long-term winter fast differed by tissue-in liver, there was no difference in total or pAMPK or pACC between groups, but in muscle, WAT and BAT, euthermic GMGS had lower relative abundance of pAMPK and pACC than torpid animals. Therefore, AMPK may be an important energy sensor at all points in hibernator's circannual cycles of food intake and T(b).

To eat or not to eat: the effect of AICAR on food intake regulation in yellow-bellied marmots (Marmota flaviventris).

Mammals that hibernate (hibernators) exhibit a circannual rhythm of food intake and body mass. In the laboratory during the winter hibernation period, many hibernators enter a series of multi-day torpor bouts, dropping their body temperature to near ambient, and cease to feed even if food is present in their cage. The mechanism(s) that regulates food intake in hibernators is unclear. Recently, AMP-activated protein kinase (AMPK) has been shown to play a key role in the central regulation of food intake in mammals. We hypothesized that infusing an AMPK activator, 5-aminoimidazole-4-carboxamide 1 B-D-ribofuranoside (AICAR), intracerebroventricularly (ICV) into the third ventricle of the hypothalamus would stimulate yellow-bellied marmots (Marmota flaviventris) to feed during their hibernation season. Infusion of AICAR ICV into marmots at an ambient temperature of 22 degrees C caused a significant (P<0.05) increase in food intake. In addition, animals stimulated to feed did not enter torpor during the infusion period. Marmots ICV infused with saline did not increase food intake and these animals continued to undergo torpor at an ambient temperature of 22 degrees C. Our results suggest that AICAR stimulated the food intake pathway, presumably by activating AMPK. These results support the hypothesis that AMPK may be involved in regulating food intake in hibernators and that there may be common neural pathways involved in regulating feeding and eliciting torpor.

Plasma ghrelin concentrations change with physiological state in a sciurid hibernator (Spermophilus lateralis).

Ghrelin is a recently discovered hormone which has profound effects on food intake and lipogenesis in mammals. In all mammals studied thus far, plasma ghrelin concentrations are increased before a meal and decrease immediately following a meal; ghrelin levels increase with fasting. The golden-mantled ground squirrel Spermophilus lateralis (also known as Callospermophilus lateralis (see Helgen et al., 2009) is a diurnal hibernator which has a robust annual cycle of body mass gain and loss that is primarily controlled by food intake. We hypothesized that in spring, summer, and autumn, the endogenous ghrelin concentrations of hibernators would be similar to those of non-hibernators, but that during the winter hibernation season, plasma ghrelin concentrations would be low or undetectable. We found that peripherally injected ghrelin significantly increased food intake in June. Plasma ghrelin concentrations were significantly increased through 5 days of fasting during a short-term fast in summer. Over a 24h period, ghrelin concentrations increased at night and decreased during the day with drops corresponding to times when squirrels were eating. In January, ghrelin concentrations are low but measurable even while animals are at low body temperature (Tb). This is the first report of ghrelin concentrations in a non-photoperiodic hibernator. We suggest that ghrelin may be important for the regulation of food intake and the body mass cycle in mammals that hibernate.

Seasonal Changes in Endocannabinoid Concentrations between Active and Hibernating Marmots (Marmota flaviventris).

Hibernation is a naturally occurring model for studying diseases such as obesity and osteoporosis. Hibernators, marmots (Marmota flaviventris) among them, are able to nearly double their body mass by increasing fat stores prior to hibernation without the negative consequences of obesity. They are also physically inactive for extended periods of time without experiencing negative effects on the skeleton. The endocannabinoid system is involved in modulating neural signaling, circannual rhythms, behavior, appetite, thermogenesis, and bone and energy metabolism. These systems are also altered to maintain homeostasis during hibernation. This study aims to better understand the involvement of the endocannabinoid system in the regulation of physiological processes during hibernation by quantifying the seasonal variation of endocannabinoids and endocannabinoid-like ligands in both active and hibernating marmots. We hypothesized that there would be significant changes in endocannabinoid concentrations at the tissue level in marmots between active and hibernating states. Concentrations were measured in brain, serum, brown adipose tissue, white adipose tissue, bone marrow, cortical bone, and trabecular bone using microflow chromatography coupled with tandem quadrupole mass spectrometry. Significant changes were found, such as a 30-fold decrease in 2-arachidonoyl glycerol (2-AG) in cortical bone during hibernation. Many endocannabinoid and endocannabinoid-like ligands decreased in brown adipose tissue, white adipose tissue, and cortical bone, while several ligands increased in bone marrow. This result supports our hypothesis and suggests the possibility of a peripherally controlled shift in energy metabolism, reduction in bone metabolism, and suppression of the immune system during hibernation.

Exploring the Bone Proteome to Help Explain Altered Bone Remodeling and Preservation of Bone Architecture and Strength in Hibernating Marmots.

Periods of physical inactivity increase bone resorption and cause bone loss and increased fracture risk. However, hibernating bears, marmots, and woodchucks maintain bone structure and strength, despite being physically inactive for prolonged periods annually. We tested the hypothesis that bone turnover rates would decrease and bone structural and mechanical properties would be preserved in hibernating marmots (Marmota flaviventris). Femurs and tibias were collected from marmots during hibernation and in the summer following hibernation. Bone remodeling was significantly altered in cortical and trabecular bone during hibernation with suppressed formation and no change in resorption, unlike the increased bone resorption that occurs during disuse in humans and other animals. Trabecular bone architecture and cortical bone geometrical and mechanical properties were not different between hibernating and active marmots, but bone marrow adiposity was significantly greater in hibernators. Of the 506 proteins identified in marmot bone, 40 were significantly different in abundance between active and hibernating marmots. Monoaglycerol lipase, which plays an important role in fatty acid metabolism and the endocannabinoid system, was 98-fold higher in hibernating marmots compared with summer marmots and may play a role in regulating the changes in bone and fat metabolism that occur during hibernation.