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.

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.

Arterial oxygen tensions in mechanically ventilated ICU patients and mortality: a retrospective, multicentre, observational cohort study.

BACKGROUND: Supplemental oxygen therapy is commonly required for respiratory failure requiring mechanical ventilation in the ICU. However, hyperoxaemia may be injurious and may increase mortality. We evaluated the relationship amongst the degree of hyperoxaemia and changes in fraction of inspired oxygen (Fio2) in response to hyperoxaemia, as well as associations with mortality in mechanically ventilated ICU patients. METHODS: We retrospectively identified all invasively mechanically ventilated patients admitted to five ICUs, and retrieved all oxygen tension (Pao2) and Fio2 data. We assessed the time between arterial blood gas (ABG) samples, proportions of patients with hyperoxaemia, and changes in Fio2 when hyperoxaemia was present. The primary outcome was the association between Pao2 (assessed by mechanically ventilated exposure-time-divided area under the curve [AUC]) and mortality (in-ICU and post-ICU discharge) using a multistate illness-death model with transition intensities estimated by Cox proportional hazards models. RESULTS: We assessed 177 769 ABG analyses obtained from 4998 patients between January 2012 and June 2016. The median time between ABGs was 3 h (inter-quartile range: 2-4 h); the median Pao2 was 11.3 kPa (9.8-13.6 kPa), and Fio2 was 0.40 (0.35-0.50). Hyperoxaemia (Pao2 >13.7 kPa) was present in 23.9% of the ABGs, and hyperoxaemia seemed to be disregarded when Fio2 was <0.40, as >50% of these Fio2 values were not subsequently reduced. AUC Pao2 >16.0 kPa was associated with increased ICU mortality (adjusted hazard ratio: 1.75; 95% confidence interval: 1.28-2.40). CONCLUSIONS: In mechanically ventilated ICU patients, hyperoxaemia was common. Although oxygen supplementation was often reduced when hyperoxaemia was observed, several patients remained hyperoxaemic. Hyperoxaemia was associated with increased ICU mortality in these patients.

Cell surface adhesion of pregnancy-associated plasma protein-A is mediated by four clusters of basic residues located in its third and fourth CCP module.

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A.

Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis.

Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.