Circulating insulin-like growth factor system adaptations in hibernating brown bears indicate increased tissue IGF availability.

Brown bears conserve muscle and bone mass during 6 mo of inactive hibernation. The molecular mechanisms underlying hibernation physiology may have translational relevance for human therapeutics. We hypothesize that protective mechanisms involve increased tissue availability of insulin-like growth factors (IGFs). In subadult Scandinavian brown bears, we observed that mean plasma IGF-1 and IGF-2 levels during hibernation were reduced to 36 ± 10% and 56 ± 15%, respectively, compared with the active state (n = 12). Western ligand blotting identified IGF-binding protein (IGFBP)-3 as the major IGFBP in the active state, whereas IGFBP-2 was codominant during hibernation. Acid labile subunit (ALS) levels in hibernation were reduced to 41±16% compared with the active state (n = 6). Analysis of available grizzly bear RNA sequencing data revealed unaltered liver mRNA IGF-1, IGFBP-2, and IGFBP-3 levels, whereas ALS levels were significantly reduced during hibernation (n = 6). Reduced ALS synthesis and circulating levels during hibernation should prompt a shift from ternary IGF/IGFBP/ALS to smaller binary IGF/IGFBP complexes, thereby increasing IGF tissue availability. Indeed, size-exclusion chromatography of bear plasma demonstrated a shift to lower molecular weight IGF-containing complexes in the hibernating versus the active state. Furthermore, we note that the major IGF-2 mRNA isoform expressed in livers in both Scandinavian brown bears and grizzly bears was an alternative splice variant in which Ser29 is replaced with a tetrapeptide possessing a positively charged Arg residue. Homology modeling of the bear IGF-2/IGFBP-2 complex showed the tetrapeptide in proximity to the heparin-binding domain involved in bone-specific targeting of this complex. In conclusion, this study provides data which suggest that increased IGF tissue availability combined with tissue-specific targeting contribute to tissue preservation in hibernating bears.NEW & NOTEWORTHY Brown bears shift from circulating ternary IGF/IGFBP/ALS complexes in the active state to binary IGF/IGFBP complexes during hibernation, indicating increased tissue IGF-bioactivity. Furthermore, brown bears use a splice variant of IGF-2, suggesting increased bone-specific targeting of IGF anabolic signaling.

Hypothyroidism in hibernating brown bears.

Brown bears hibernate throughout half of the year as a survival strategy to reduce energy consumption during prolonged periods with scarcity of food and water. Thyroid hormones are the major endocrine regulators of basal metabolic rate in humans. Therefore, we aimed to determine regulations in serum thyroid hormone levels in hibernation compared to the active state to investigate if these are involved in the adaptions for hibernation.We used electrochemiluminescence immunoassay to quantify total triiodothyronine (T3) and thyroxine (T4) levels in hibernation and active state in paired serum samples from six subadult Scandinavian brown bears. Additionally, we determined regulations in the liver mRNA levels of three major thyroid hormone-binding proteins; thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin, by analysis of previously published grizzly bear RNA sequencing data.We found that bears were hypothyroid when hibernating with T4 levels reduced to less than 44% (P = 0.008) and T3 levels reduced to less than 36% (P = 0.016) of those measured in the active state. In hibernation, mRNA levels of TBG and albumin increased to 449% (P = 0.031) and 121% (P = 0.031), respectively, of those measured in the active state. TTR mRNA levels did not change.Hibernating bears are hypothyroid and share physiologic features with hypothyroid humans, including decreased basal metabolic rate, bradycardia, hypothermia, and fatigue. We speculate that decreased thyroid hormone signaling is a key mediator of hibernation physiology in bears. Our findings shed light on the translational potential of bear hibernation physiology to humans for whom a similar hypometabolic state could be of interest in specific conditions.

Plasma proteomics data from hibernating and active Scandinavian brown bears.

In this article, we present mass-spectrometry based plasma proteomics data from hibernating and active free-ranging Scandinavian brown bears (Ursus arctos). The brown bear hibernates for half the year. Despite obesity when entering the den and the prolonged period of inactivity, the bear shows no signs of the harmful effects associated with these conditions in humans. Thus, the hibernating bear is a potential translational model for addressing these complications in humans. We analyzed plasma samples from fourteen 2- to 3-year-old bears (6 males and 8 females) collected both during hibernation and the active state, and for some of the bears during two seasons, resulting in a total of 38 analyzed plasma samples. In triplicates, the plasma proteins were unfolded and reduced. To increase the chance of detecting low-molecular-weight proteins and peptides, we filtered the samples using a 50 K molecular weight cut-off filter with the aim to deplete larger abundant proteins, including albumin, and thereby increase the depth of the analysis. The proteins in the permeate were then tryptically digested, desalted, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed with the MaxQuant software searching against an Ursus arctos horribilis protein database. Here, we provide the raw data, a list with identified proteins in the plasma samples, and the databases applied for protein identification. Based on the provided data, differentially expressed proteins in hibernation compared to active state can be identified. These proteins may be involved in the bears' adaptions to hibernation physiology and hold potential as novel therapeutic targets.

Characterization and comparison of recombinant full-length ursine and human sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) regulates the bioavailability of sex steroid hormones in the blood. Levels of SHBG increase markedly in brown bears (Ursus arctos) during hibernation, suggesting that a key regulatory role of this protein is to quench sex steroid bioavailability in hibernation physiology. To enable characterization of ursine SHBG and a cross species comparison, we established an insect cell-based expression system for recombinant full-length ursine and human SHBG. Compared with human SHBG, we observed markedly lower secretion levels of ursine SHBG, resulting in a 10-fold difference in purified protein yield. Both human and ursine recombinant SHBG appeared as dimeric proteins in solution, with a single unfolding temperature of ~ 58 °C. The thermal stability of ursine and human SHBG increased 5.4 and 9.5 °C, respectively, in the presence of dihydrotestosterone (DHT), suggesting a difference in affinity. The dissociation constants for [3 H]DHT were determined to 0.21 ± 0.04 nm for human and 1.32 ± 0.10 nm for ursine SHBG, confirming a lower affinity of ursine SHBG. A similarly reduced affinity, determined from competitive steroid binding, was observed for most steroids. Overall, we found that ursine SHBG had similar characteristics to human SHBG, specifically, being a homodimeric glycoprotein capable of binding steroids with high affinity. Therefore, ursine SHBG likely has similar biological functions to those known for human SHBG. The determined properties of ursine SHBG will contribute to elucidating its potential regulatory role in hibernation physiology.

Differential Changes in Circulating Steroid Hormones in Hibernating Brown Bears: Preliminary Conclusions and Caveats.

Brown bears are obese when they enter the den, and after 6 mo of hibernation and physical inactivity, bears show none of the adverse consequences of a sedentary lifestyle in humans, such as cardiovascular disease, type 2 diabetes, and kidney failure. The metabolic mechanisms that drive hibernation physiology in bears are poorly defined, but systemic endocrine regulators are likely involved. To investigate the potential role of steroid hormones, we quantified the total levels of 12 steroid hormones, the precursor cholesterol, sex hormone-binding globulin (SHBG), and corticosterone-binding globulin (CBG) in paired serum samples from subadult free-ranging Scandinavian brown bears during the active and hibernation states. During hibernation, androstenedione and testosterone were significantly decreased in subadult female bears (n=13), whereas they increased in all males but one (n=6) and therefore did not reach a significant difference. Despite this difference, SHBG increased more than 20-fold during hibernation for all bears. Compared with SHBG concentrations in humans, bear levels were very low in the active state, but during hibernation, levels equaled high levels in humans. The increased SHBG levels likely maintain a state of relative quiescence of the reproductive hormones in hibernating bears. Interestingly, the combination of SHBG and testosterone levels results in similar free bioavailable testosterone levels of 70-80 pM in both subadult and adult sexually active male bears, suggesting a role for SHBG in controlling androgen action during hibernation in males. Dehydroepiandrosterone sulfate, dihydrotestosterone, and estradiol levels were below the detection limit in all but one animal. The metabolically active glucocorticoids were significantly higher in both sexes during hibernation, whereas the inactive metabolite cortisone was reduced and CBG was low approaching the detection limit. A potential caveat is that the glucocorticoid levels might be affected by the ketamine applied in the anesthetic mixture for hibernating bears. However, increased hibernating cortisol levels have consistently been reported in both black bears and brown bears. Thus, we suggest that high glucocorticoid activity may support the hibernation state, likely serving to promote lipolysis and gluconeogenesis while limiting tissue glucose uptake to maintain a continuous glucose supply to the brain.

Expression of Paracrine Effectors in Human Adipose-Derived Mesenchymal Stem Cells Treated With Plasma From Brown Bears (Ursus arctos).

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates for novel cell therapeutic applications. Hibernating brown bears sustain tissue integrity and function via unknown mechanisms, which might be plasma borne. We hypothesized that plasma from hibernating bears may increase the expression of favorable factors from human ADSCs. In an experimental study, ADSCs from patients with ischemic heart disease were treated with interventional media containing plasma from hibernating and active bears, respectively, and with control medium. Extracted RNA from the ADSCs was sequenced using next generation sequencing. Statistical analyses of differentially expressed genes were performed using fold change analysis, pathway analysis, and gene ontology. As a result, we found that genes associated with inflammation, such as IGF1, PGF, IL11, and TGFA, were downregulated by > 10-fold in ADSCs treated with winter plasma compared with control. Genes important for cardiovascular development, ADM, ANGPTL4, and APOL3, were upregulated in ADSCs when treated with winter plasma compared with summer plasma. ADSCs treated with bear plasma, regardless if it was from hibernating or active bears, showed downregulation of IGF1, PGF, IL11, INHBA, IER3, and HMOX1 compared with control, suggesting reduced cell growth and differentiation. This can be summarized in the conclusion that plasma from hibernating bears suppresses inflammatory genes and activates genes associated with cardiovascular development in human ADSCs. Identifying the involved regulator(s) holds therapeutic potential.