Cholesterol and lipoprotein dynamics in a hibernating mammal

Hibernating mammals cease feeding during the winter and rely primarily on stored lipids to fuel alternating periods of torpor and arousal. How hibernators manage large fluxes of lipids and sterols over the annual hibernation cycle is poorly understood. The aim of this study was to investigate lipid and cholesterol transport and storage in ground squirrels studied in spring, summer, and several hibernation states. Cholesterol levels in total plasma, HDL and LDL particles were elevated in hibernators compared with spring or summer squirrels. Hibernation increased plasma apolipoprotein A-I expression and HDL particle size. Expression of cholesterol 7 alpha-hydroxylase was 13-fold lower in hibernators than in active season squirrels. Plasma triglycerides were reduced by fasting in spring but not summer squirrels. In hibernators plasma β-hydroxybutyrate was elevated during torpor whereas triglycerides were low relative to normothermic states. We conclude that the switch to a lipid-based metabolism during winter, coupled with reduced capacity to excrete cholesterol creates a closed system in which efficient use of lipoproteins is essential for survival.     

Seasonal climate effects on the survival of a hibernating mammal

Global climate change and associated regional climate variability is impacting the phenology of many species, ultimately altering individual fitness and population dynamics. Yet, few studies have considered the effects of pertinent seasonal climate variability on phenology and fitness. Hibernators may be particularly susceptible to changes in seasonal climate since they have a relatively short active season in which to reproduce and gain enough mass to survive the following winter. To understand whether and how seasonal climate variability may be affecting hibernator fitness, we estimated survival from historical (1964–1968) and contemporary (2014–2017) mark–recapture data collected from the same population of Uinta ground squirrels (UGS, Urocitellus armatus), a hibernator endemic to the western United States. Despite a locally warming climate, the phenology of UGS did not change over time, yet season‐specific climate variables were important in regulating survival rates. Specifically, older age classes experienced lower survival when winters or the following springs were warm, while juveniles benefited from warmer winter temperatures. Although metabolic costs decrease with decreasing temperature in the hibernacula, arousal costs increase with decreasing temperature. Our results suggest that this trade‐off is experienced differently by immature and mature individuals. We also observed an increase in population density during that time period, suggesting resources are less limited today than they used to be. Cheatgrass is now dominating the study site and may provide a better food source to UGS than native plants did historically.     

Fluorescence anisotropy of kidney lipids and membranes of a hibernating mammal

The fluorescence anisotropy of lipids and membranes isolated from kidneys of European hamsters (Cricetus cricetus L.) has been estimated using 1,6-diphenyl-1,3,5-hexatriene as a probe. We have compared in this study the results obtained for two critical periods for a hibernator: winter (torpid state), and summer (active state). The differences were of very low magnitude. A slight increase in anisotropy was noticed in the kidney lipids and microsomal membrane preparations from torpid animals. In contrast, a small decrease in anisotropy was observed in the microsomal lipid extracts of torpid animals. A difference in triglyceride content of winter and summer total kidney lipids was detected, as well as a difference in microsomal protein content between winter and summer membrane preparations. It is hypothesized that the latter observations may explain why the behavior of kidney total lipids and microsomal preparations were different from that presented by kidney microsomal lipids in respect to fluorescence anisotropy. Therefore, only a little, if any, homeoviscous adaptation is exhibited by kidney membranes during hibernation of this mammal.     

Differential plasticity of size and mass to environmental change in a hibernating mammal

Morphological changes following changes in species' distribution and phenology have been suggested to be the third universal response to global environmental change. Although structural size and body mass result from different genetic, physiological, and ecological mechanisms, they are used interchangeably in studies evaluating population responses to environmental change. Using a 22‐year (1991–2013) dataset including 1768 individuals, we investigated the coupled dynamics of size and mass in a hibernating mammal, the Alpine marmot (Marmota marmota), in response to local environmental conditions. We (i) quantified temporal trends in both traits, (ii) determined the environmental drivers of trait dynamics, and (iii) identified the life‐history processes underlying the observed changes. Both phenotypic traits were followed through life: we focused on the initial trait value (juvenile size and mass) and later‐life development (annual change in size [Δsize] and mass [Δmass]). First, we demonstrated contrasting dynamics between size and mass over the study period. Juvenile size and subsequent Δsize showed significant declines, whereas juvenile mass and subsequent Δmass remained constant. As a consequence of smaller size associated with a similar mass, individuals were in better condition in recent years. Second, size and mass showed different sensitivities to environmental variables. Both traits benefited from early access to resources in spring, whereas Δmass, particularly in early life, also responded to summer and winter conditions. Third, the interannual variation in both traits was caused by changes in early life development. Our study supports the importance of considering the differences between size and mass responses to the environment when evaluating the mechanisms underlying population dynamics. The current practice of focusing on only one trait in population modeling can lead to misleading conclusions when evaluating species' resilience to contemporary climate change.     

Fluorescence polarization study of lipids and membranes prepared from brain hemispheres of a hibernating mammal

The physical behavior of total lipids, microsomes and microsomal lipids prepared from brain hemispheres of European Hamsters ($\text{Cricetus cricetus}$) was approached by the measure of the fluorescence polarization of the probe 1,6-diphenyl 1,3,5-hexatriene. We compare in this study the results obtained for two critical periods for a hibernator: winter (torpid state) and summer (active state). An increase in fluidity was noticed in the winter lipid and membrane preparations. The difference was however of very low magnitude, suggesting that only the microenvironment of some proteins was involved, rather than the bulk membrane fluidity.     

Identification of proteins from non-model organisms using mass spectrometry: application to a hibernating mammal

A major challenge in the life sciences is the extraction of detailed molecular information from plants and animals that are not among the handful of exhaustively studied "model organisms." As a consequence, certain species with novel phenotypes are often ignored due to the lack of searchable databases, tractable genetics, stock centers, and more recently, a sequenced genome. Characterization of phenotype at the molecular level commonly relies on the identification of differentially expressed proteins by combining database searching with tandem mass spectrometry (MS) of peptides derived from protein fragmentation. However, the identification of short peptides from nonmodel organisms can be hampered by the lack of sufficient amino acid sequence homology with proteins in existing databases; therefore, a database search strategy that encompasses both identity and homology can provide stronger evidence than a single search alone. The use of multiple algorithms for database searches may also increase the probability of correct protein identification since it is unlikely that each program would produce false negative or positive hits for the same peptides. In this study, four software packages, Mascot, Pro ID, Sequest, and Pro BLAST, were compared in their ability to identify proteins from the thirteen-lined ground squirrel (Spermophilus tridecemlineatus), a hibernating mammal that lacks a completely sequenced genome. Our results show similarities as well as the degree of variability among different software packages when the identical protein database is searched. In the process of this study, we identified the up-regulation of succinyl CoA-transferase (SCOT) in the heart of hibernators. SCOT is the rate-limiting enzyme in the catabolism of ketone bodies, an important alternative fuel source during hibernation.     

Expression of mRNA for 3HADH in manipulated embryos to produce germline chimeric chickens

Germline chimeric chickens were produced by the transfer of primordial germ cells (PGCs) or blastoderm cells. The hatchability of eggs produced by transfer of exogenous PGCs is usually low. The purpose of the present study was investigated to express (3-hydroxyacyl CoA dehydrogenase) 3HADH which is a limiting enzyme in the beta-oxidation of fatty acids for hatching energy. Manipulations of both donor and recipient eggshells were as follows. A window approximately 10 mm in diameter was opened at the pointed end of the eggs at stage 12–15 days incubation. Donor PGCs, taken from the blood vessels of donor embryos from fertilized eggs at the same stage of development, were injected into the blood vessels of recipient embryos. The muscles of chicks in the eggs with transferred PGCs were removed after 20 days of incubation. A cDNA was prepared from the total RNA. The expression of 3HADH in the manipulated embryos was investigated using real-time PCR analysis. Real-time PCR analysis showed that expression of 3HADH was reduced in the muscles of manipulated embryos.     

Overcoming muscle atrophy in a hibernating mammal despite prolonged disuse in dormancy: proteomic and molecular assessment

Prolonged disuse of skeletal muscle causes significant loss of myofibrillar contents, muscle tension, and locomotory capacity. However, hibernating mammals like bats appear to deviate from this trend. Although low functional demands during winter dormancy has been implicated as a factor contributing to reduced muscle loss, the precise mechanism that actively prevents muscle atrophy remains unclear. We explored proteomic and molecular assessments of bat muscle to test a hypothesis that expression levels of major myofibrillar proteins are retained during hibernation, with periodic arousals utilized as a potential mechanism to prevent disuse atrophy. We examined changes in myofibrillar contents and contractile properties of the pectoral or biceps brachii muscles of the bat Murina leucogaster in summer active (SA), hibernation (HB) and early phase of arousal (AR) states. We found the bat muscles did not show any sign of atrophy or tension reduction over the 3-month winter dormancy. Levels of most sarcomeric and metabolic proteins examined were maintained through hibernation, with some proteins (e.g., actin and voltage dependent anion channel 1) 1.6- to 1.8-fold upregulated in HB and AR compared to SA. Moreover, expression levels of six heat shock proteins (HSPs) including glucose-regulated protein 75 precursor were similar among groups, while the level of HSP70 was even 1.7-fold higher in HB and AR than in SA. Thus, considering the nature of arousal with strenuous muscle shivering and heat stress, upregulation or at least balanced regulation of the chaperones (HSPs) would contribute to retaining muscle properties during prolonged disuse of the bat.     

Assessing seasonal demographic covariation to understand environmental‐change impacts on a hibernating mammal

Natural populations are exposed to seasonal variation in environmental factors that simultaneously affect several demographic rates (survival, development and reproduction). The resulting covariation in these rates determines population dynamics, but accounting for its numerous biotic and abiotic drivers is a significant challenge. Here, we use a factor‐analytic approach to capture partially unobserved drivers of seasonal population dynamics. We use 40 years of individual‐based demography from yellow‐bellied marmots (Marmota flaviventer) to fit and project population models that account for seasonal demographic covariation using a latent variable. We show that this latent variable, by producing positive covariation among winter demographic rates, depicts a measure of environmental quality. Simultaneously, negative responses of winter survival and reproductive‐status change to declining environmental quality result in a higher risk of population quasi‐extinction, regardless of summer demography where recruitment takes place. We demonstrate how complex environmental processes can be summarized to understand population persistence in seasonal environments.     

Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast

Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.     

Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo.

The subclass of catalytic RNAs termed ribozymes cleave specific target RNA sequences in vitro. Only circumstantial evidence supports the idea that ribozymes may also act in vivo. In this study, ribozymes with a hammerhead motif directed against a target sequence within the mRNA of the neomycin phosphotransferase gene (npt) were embedded into a functional chimeric gene. Two genes, one containing the ribozyme and the other producing the target, were cotransfected into plant protoplasts. Following in vivo expression, a predefined cleavage product of the target mRNA was detected by ribonuclease protection. Expression of both the ribozyme gene and the target gene was driven by the CaMV 35S promoter. Concomitant with the endonucleolytic cleavage of the target mRNA, a complete reduction of NPT activity was observed. An A to G substitution within the ribozyme domain completely inactivates ribozyme-mediated hydrolysis but still shows a reduction in NPT activity, albeit less pronounced. Therefore, the reduction of NPT activity produced by the active ribozyme is best explained by both hydrolytic cleavage and an antisense effect. However, the mutant ribozyme--target complex was more stable than the wildtype ribozyme--target complex. This may result in an overestimation of the antisense effect contributing to the overall reduction of gene expression.     

Bigger is not always better: Viability selection on body mass varies across life stages in a hibernating mammal

Body mass is often viewed as a proxy of past access to resources and of future survival and reproductive success. Links between body mass and survival or reproduction are, however, likely to differ between age classes and sexes. Remarkably, this is rarely taken into account in selection analyses. Selection on body mass is likely to be the primary target accounting for juvenile survival until reproduction but may weaken after recruitment. Males and females also often differ in how they use resources for reproduction and survival. Using a long‐term study on body mass and annual survival in yellow‐bellied marmots (Marmota flaviventer), we show that body mass was under stabilizing viability selection in the first years of life, before recruitment, which changed to positive directional selection as age increased and animals matured. We found no evidence that viability selection across age classes on body mass differed between sexes. By investigating the link between running speed and body mass, we show that the capacity to escape predators was not consistent across age classes and followed a quadratic relationship at young ages only. Overall, our results indicate that mature age classes exhibit traditional patterns of positive viability selection on body mass, as expected in a hibernating mammal, but that mass in the first years of life is subjected to stabilizing selection which may come from additional predation pressures that negate the benefits of the largest body masses. Our study highlights the importance to disentangle selection pressures on traits across critical age (or life) classes.     

Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.     

Expression of chimeric genes in Caenorhabditis elegans

We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system. Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C. elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays. We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences. The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera. Expression is at a very low level, and seems to be constitutive. We have used histochemical techniques to visualize the enzyme activity in embryos.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Brain energy metabolism and neurotransmission at near-freezing temperatures: in vivo (1)H MRS study of a hibernating mammal

The brain of a hibernating mammal withstands physiological extremes that would result in cerebral damage and death in a non-hibernating species such as humans. To examine the possibility that this neuroprotection results from alterations in cerebral metabolism, we used in vivo(1)H NMR spectroscopy at high field (9.4 T) to measure the concentration of 18 metabolites (neurochemical profile) in the brain of 13-lined ground squirrels (Spermophilus tridecemlineatus) before, during, and after hibernation. Resolved in vivo(1)H NMR spectra were obtained even at low temperature in torpid hibernators ( approximately 7 degrees C). The phosphocreatine-to-creatine ratio was increased during torpor (+143%) indicating energy storage, and remained increased to a lesser extent during interbout arousal (IBA) (+83%). The total gamma-aminobutyric acid concentration was increased during torpor (+135%) and quickly returned to baseline during IBA. Glutamine (Gln) was decreased (-54%) during torpor but quickly returned to normal levels during IBA and after terminal arousal in the spring. Glutamate (Glu) was also decreased during torpor (-17%), but remained decreased during IBA (-20% compared with fall), and returned to normal level in the spring. Our observation that Glu and Gln levels are depressed in the brain of hibernators suggests that the balance between anaplerosis and loss of Glu and Gln (because of glutamatergic neurotransmission or other mechanisms) is altered in hibernation.     

Hormonal control of lipolysis from the white adipose tissue of hibernating jerboa (Jaculus orientalis)

1. Plasma glucose, glycerol, free fatty acids and total lipid content of the white adipose tissue were measured in euthermic and hibernating jerboa. 2. During hibernation, plasma glucose and glycerol were low compared to the euthermic animals, whereas there was no obvious difference in plasma free fatty acids. The white adipose tissue lipid content was strongly reduced in the hibernating state. 3. The effect of lipolytic hormones (norepinephrine and glucagon) and antilipolytic hormone (insulin) on in vitro glycerol release by adipose tissue isolated from hibernating or euthermic jerboa has been studied. 4. The white adipose tissue from hibernating jerboa presented a higher sensitivity to norepinephrine and glucagon than that of euthermic jerboa; insulin did not modify either basal glycerol release or lipolysis induced by the two lipolytic hormones at low temperatures (7 degrees C) and during the rewarming (from 7 degrees C to 37 degrees C) of the tissue slices. 5. These results suggested that white adipose tissue constitutes an important source of substrates derived from lipolysis during hibernation.     

Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats

A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RII alpha, and RII beta) in P(40) fraction (sedimenting at 40,000g), but not in I(40) fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P(40) fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P(40) but not in I(40) fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes.