Hibernation slows epigenetic ageing in yellow-bellied marmots.

Species that hibernate generally live longer than would be expected based solely on their body size. Hibernation is characterized by long periods of metabolic suppression (torpor) interspersed by short periods of increased metabolism (arousal). The torpor-arousal cycles occur multiple times during hibernation, and it has been suggested that processes controlling the transition between torpor and arousal states cause ageing suppression. Metabolic rate is also a known correlate of longevity; we thus proposed the 'hibernation-ageing hypothesis' whereby ageing is suspended during hibernation. We tested this hypothesis in a well-studied population of yellow-bellied marmots (Marmota flaviventer), which spend 7-8 months per year hibernating. We used two approaches to estimate epigenetic age: the epigenetic clock and the epigenetic pacemaker. Variation in epigenetic age of 149 samples collected throughout the life of 73 females was modelled using generalized additive mixed models (GAMM), where season (cyclic cubic spline) and chronological age (cubic spline) were fixed effects. As expected, the GAMM using epigenetic ages calculated from the epigenetic pacemaker was better able to detect nonlinear patterns in epigenetic ageing over time. We observed a logarithmic curve of epigenetic age with time, where the epigenetic age increased at a higher rate until females reached sexual maturity (two years old). With respect to circannual patterns, the epigenetic age increased during the active season and essentially stalled during the hibernation period. Taken together, our results are consistent with the hibernation-ageing hypothesis and may explain the enhanced longevity in hibernators.

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.

Correlates of maternal glucocorticoid levels in a socially flexible rodent.

While it is generally accepted that social isolation has detrimental effects on social species, little is known about the importance of social interactions in less social species-particularly for wild reproductive females. We studied socially-flexible yellow-bellied marmots (Marmota flaviventer) and asked whether features of the social environment are associated with maternal fecal glucocorticoid metabolite (FGM) concentrations. Since changes in maternal baseline glucocorticoids may have positive or negative consequences for offspring fitness, we were also interested in estimating their relationship with measures of reproductive success. We fitted generalized linear mixed effects models to a dataset including maternal FGM measurements, social network metrics, maternal/alloparental care, and pup FGM and survival. Agonistic interactions were positively associated with maternal FGM levels, while mothers that engaged in relatively more affiliative interactions had reduced FGM levels when living in environments with low predator pressure. Pups associated with mothers exhibiting high FGM levels had low annual survival rates, received less maternal/alloparental care and had higher FGM levels. Interestingly, offspring from mothers with high FGM levels were more likely to survive the summer when born in small litters. In sum, social interactions likely influence and are influenced by glucocorticoid levels of facultatively social females. Potential benefits of social bonds may be context-specific, and agonistic interactions may be tightly correlated with fitness. Female marmots exhibiting high FGM levels had overall low reproductive success, which is predicted by the cort-fitness hypothesis. However, under adverse conditions, offspring summer survival can be maximized if pups are born in small litters.

Woodchuck VEGF (wVEGF) characteristics: Model for angiogenesis and human hepatocellular carcinoma directed therapies.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis. Human hepatocellular carcinoma (HCC) is a VEGF-driven tumor often associated with chronic hepatitis B or C virus infection. The woodchuck is a well-characterized model of hepatitis B virus related HCC and a valuable tool for translational studies of novel VEGF targeted agents. We cloned the cDNA encoding woodchuck VEGF (wVEGF), transiently expressed it in COS cells and functionally characterized the recombinant protein. The open reading frame of wVEGF contained 645 nucleotides encoding a protein of 214 amino acids. Two protein bands (17 and 25 kDa) were detected in conditioned media of wVEGF expressing COS-1 cells and a single band of 25 kDa was identified in cell lysates. Addition of recombinant wVEGF to COS cells enhanced cell proliferation and stimulated VEGFR2, Akt, ERK1/2, and FAK phosphorylation. Sunitinib, a tyrosine kinase inhibitor, inhibited wVEGF- induced VEGFR2 phosphorylation in a dose-dependent manner. Finally, development of HCC in woodchucks was accompanied by increased laminin and PECAM1 expressing vessels, VEGFR2 expression, increased ligation of VEGF to VEGFR2, and a decrease in collagen IV-positive blood vessels. Our results suggest that woodchuck model can be used further to study angiogenesis and the effect of VEGF directed therapies in human HCC.

Antioxidant defense and protection against cardiac arrhythmias: lessons from a mammalian hibernator (the woodchuck).

Hibernating animals show resistance to hypothermia-induced cardiac arrhythmias. However, it is not clear whether and how mammalian hibernators are resistant to ischemia-induced arrhythmias. The goal of this investigation was to determine the susceptibility of woodchucks ( Marmota monax) to arrhythmias and their mechanisms after coronary artery occlusion at the same room temperature in both winter, the time for hibernation, and summer, when they do not hibernate. By monitoring telemetric electrocardiograms, we found significantly higher arrhythmia scores, calculated as the severity of arrhythmias, with incidence of ventricular tachycardia, ventricular fibrillation, and thus sudden cardiac death (SCD) in woodchucks in summer than they had in winter. The level of catalase expression in woodchuck hearts was significantly higher, whereas the level of oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII) was lower in winter than it was in summer. Ventricular myocytes isolated from woodchucks in winter were more resistant to H2O2-induced early afterdepolarizations (EADs) compared with myocytes isolated from woodchucks in summer. The EADs were eliminated by inhibiting CaMKII (with KN-93), l-type Ca current (with nifedipine), or late Na+ current (with ranolazine). In woodchucks, in the summer, the arrhythmia score was significantly reduced by overexpression of catalase ( via adenoviral vectors) or the inhibition of CaMKII (with KN-93) in the heart. This study suggests that the heart of the mammalian hibernator is more resistant to ischemia-induced arrhythmias and SCD in winter. Increased antioxidative capacity and reduced CaMKII activity may confer resistance in woodchuck hearts against EADs and arrhythmias during winter. The profound protection conferred by catalase overexpression or CaMKII inhibition in this novel natural animal model may provide insights into clinical directions for therapy of arrhythmias.-Zhao, Z., Kudej, R. K., Wen, H., Fefelova, N., Yan, L., Vatner, D. E., Vatner, S. F., Xie, L.-H. Antioxidant defense and protection against cardiac arrhythmias: lessons from a mammalian hibernator (the woodchuck).

The effect of maternal glucocorticoid levels on juvenile docility in yellow-bellied marmots.

Maternal effects can have significant and long-term consequences on offspring behavior and survival, while consistent individual differences (i.e., personality) can have profound impacts on individual fitness. Thus, both can influence population dynamics. However, the underlying mechanisms that determine variation in personality traits are poorly understood. Maternal effects are one potential mechanism that may explain personality variation. We capitalized on a long-term study of yellow-bellied marmots (Marmota flaviventris) to identify maternal effects on juvenile docility. To do so, we partitioned the variance in juvenile docility using a quantitative genetic modeling approach to isolate potential maternal effects. We also directly tested whether maternal stress, measured through fecal glucocorticoid metabolite levels during lactation of 82 mothers, was associated with offspring docility. Docility scores were estimated for 645 juveniles trapped between 2002 and 2012. We found an interaction between maternal glucocorticoid levels and dam age on juvenile docility. We also found significant maternal, litter, permanent environment, and year effects. These results suggest that a mother's life history stage interacts with stress to influence offspring personality. This early life influence can have long lasting effects on an individual's docility throughout life.

Molecular cloning, characterization and expression analysis of TGF-ß and receptor genes in the woodchuck model.

Transforming growth factor beta (TGF-ß) is an important cytokine with pleiotropic regulatory functions in the immune system and in the responses against viral infections. TGF-ß acts on a variety of immune cells through the cell surface TGF-ß receptor (University of Duisburg-EssenTGFBR). The woodchuck has been used as a biomedical model for studies of obesity and energy balance, endocrine and metabolic function, cardiovascular, cerebrovascular and neoplastic disease. Woodchucks infected with woodchuck hepatitis virus (WHV) represent an informative animal model to study hepatitis B virus (HBV) infection. In this study, the cDNA sequences of woodchuck TGF-ß1, TGF-ß2, TGFBR1 and TGFBR2 were cloned, sequenced and characterized. The full-length TGFBR1 cDNA sequence consisted of 1305bp coding sequence (CDS) that encoded 434 amino acids with a molecular weight of 48.9kDa. The phylogenetic tree analysis revealed that the woodchuck TGF-ß family genes had a closer genetic relationship with Ictidomys tridecemlineatus. One antibody with cross-reactivity to woodchuck TGFBR1 was identified by flow cytometry. Moreover, the expression of these genes were analyzed at the transcriptional level. The quantitative PCR analysis showed that the TGF-ß family transcripts were constitutively expressed in many tissues tested. Altered expression levels of the TGF-ß family transcripts in the liver of WHV infected woodchucks were observed. These results serve as a foundation for further insight into the role of the TGF-ß family in viral hepatitis in woodchuck model. Our work also possesses the potential value for characterizing the TGF-ß family in other related diseases, such as obesity-related diseases, metabolic disorder, cardiovascular disease and cancer.

Molecular characterization of woodchuck IFI16 and AIM2 and their expression in woodchucks infected with woodchuck hepatitis virus (WHV).

IFI16 and AIM2 are important DNA sensors in antiviral immunity. To characterize these two molecules in a woodchuck model, which is widely used to study hepatitis B virus (HBV) infection, we cloned and analyzed the complete coding sequences (CDSs) of woodchuck IFI16 and AIM2, and found that AIM2 was highly conserved in mammals, whereas the degree of sequence identity between woodchuck IFI16 and its mammalian orthologues was low. IFI16 and IFN-ß were upregulated following VACV ds 70 mer transfection, while AIM2 and IL-1ß were upregulated following poly (dA:dT) transfection, both in vitro and in vivo; IFI16-targeted siRNA decreased the transcription of IFI16 and IFN-ß stimulated by VACV ds 70 mer, and AIM2 siRNA interference downregulated AIM2 and IL-1ß transcripts stimulated by poly (dA:dT), in vitro, suggesting that woodchuck IFI16 and AIM2 may play pivotal roles in the DNA-mediated induction of IFN-ß and IL-1ß, respectively. IFI16 and AIM2 transcripts were upregulated in the liver and spleen following acute WHV infection, while IFI16 was downregulated in the liver following chronic infection, implying that IFI16 and AIM2 may be involved in WHV infection. These data provide the basis for the study of IFI16- and AIM2-mediated innate immunity using the woodchuck model.

Molecular characterization of woodchuck CD4 (wCD4) and production of a depletion monoclonal antibody against wCD4.

CD4 T cells play an important role in the immune response against hepatitis B virus (HBV) infection. Woodchucks represent an excellent animal model to study HBV infection. In this study, we characterized the cDNA sequence of woodchuck CD4 (wCD4). The deduced wCD4 protein has four extracellular immunoglobulin-like domains comparable to the other mammalian CD4 molecules. The important extracellular cysteine residues and the intracellular tyrosine protein kinase-binding site of wCD4 are also conserved. The deduced wCD4 protein shows 53-63% identity with the counterparts of other mammalians. Phylogenetic analysis indicates that wCD4 is closely related with the counterparts of primates. Two polyclonal antibodies (pAbs) and four monoclonal Abs (mAbs) against wCD4 were produced. Two pAbs and one mAbs (G2) were found to effectively suppress ConA induced proliferation in vitro. Anti-wCD4 mAb G2 depleted 60% of CD4 cells from healthy woodchucks, while the remaining CD4 cells responded well to ConA stimulation. This work provides a basis for studying CD4 T cell mediated immune responses against HBV infection in the woodchuck model.

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.

Hibernation does not reduce cortical bone density, area or second moments of inertia in woodchucks (Marmota monax).

Long periods of inactivity in most mammals result in bone loss that may not be completely recoverable during an individual's lifetime regardless of future activity. Prolonged inactivity is normal during hibernation, but it remains uncertain whether hibernating mammals suffer decreased bone properties after hibernation that affects survival. We test the hypothesis that relative cortical area (C(A) ), apparent density, bone area fraction (B.Ar/T.Ar), and moments of inertia do not differ between museum samples of woodchucks (Marmota monax) collected before and after hibernation. We used peripheral quantitative computed tomography to examine bone geometry in the femur, tibia, humerus and mandible. We see little evidence for changes in bone measures with hibernation supporting our hypothesis. In fact, when including subadults to increase sample sizes and controlling age statistically, we observed a trend toward increased bone properties following hibernation. Diaphyses were significantly denser in the humerus, femur, and tibia after hibernation, and relative mandibular cortical area was significantly larger. Similarly, relative mechanical indices were significantly larger in the mandible after hibernation. Although tests of individual measures in many cases were not significantly different prehibernation versus posthibernation, the overall pattern of average increase posthibernation was significant for relative C(A) and densities as well as relative diaphyseal mechanical indices when examining outcomes collectively. The exception to this pattern was a reduction in metaphyseal trabecular bone following hibernation. Individually, only humeral B.Ar/T.Ar was significantly reduced, but the average reduction in trabecular measures post-hibernation was significant when examined collectively. Because the sample included subadults, we suggest that much of the increased bone relates to their continued growth during hibernation. Our results indicate that woodchucks are more similar to large hibernators that maintain skeletal integrity compared to smaller-bodied hibernators that may lose bone. This result suggests a potential size-related trend in bone response to hibernation across mammals.

The reproductive endocrinology and behavior of Vancouver Island marmot (Marmota vancouverensis).

The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America's most endangered species with fewer than 150 individuals remaining in the wild. A captive breeding program was established across four facilities in Canada as an insurance population and source of animals for reintroduction to the wild. The purpose of this study was to gather information about the basic reproductive biology and behavior of this species, which is essential to improve captive breeding programs. Regular fecal samples were obtained from adult female (n = 14) and male (n = 10) marmots, 2 years of age and older, over 1-3 breeding seasons (2-3 months duration posthibernation) for steroid hormone analysis. Enzyme immunoassays were validated for quantifying fecal testosterone metabolite concentrations for males, and fecal estrogen and progesterone metabolite concentrations for females. Results indicated that fecal progesterone metabolite concentrations can be used to monitor ovulation and pregnancy. Behavioral monitoring through infrared video surveillance was conducted in four breeding pairs over a 2-year period (n = 7 behavioral profiles). Breeding behaviors correlated strongly with changes in reproductive endocrine profiles. A high frequency of play behavior or "wrestling" was observed in conjunction with breeding activity before an elevation in progesterone metabolite concentrations. Impending parturition was associated with increased aggression and exclusion of the male from the maternal nestbox as well as an increase in nesting activity. Observational data combined with hormonal analysis suggest that female VIMs are induced ovulators and that multiple breeding attempts may be required for ovulation and conception. Gestation appears to be approximately 34 days from peak breeding activity (32 days from estimated ovulation). Fecal testosterone concentrations suggest that testicular activity is seasonal with the reproductive activity occurring immediately posthibernation. Monitoring breeding behavior is a useful means of indicating estrus, conception and pregnancy, which can also be supported by the hormonal analysis of daily fecal samples of individual animals.

Scared and less noisy: glucocorticoids are associated with alarm call entropy.

The nonlinearity and arousal hypothesis predicts that highly aroused mammals will produce nonlinear, noisy vocalizations. We tested this prediction by measuring faecal glucocorticoid metabolites (GCMs) in adult yellow-bellied marmots (Marmota flaviventris), and asking if variation in GCMs was positively correlated with Wiener entropy-a measure of noise. Contrary to our prediction, we found a significant negative relationship: marmots with more faecal GCMs produced calls with less noise than those with lower levels of GCMs. A previous study suggested that glucocorticoids modulate the probability that a marmot will emit a call. This study suggests that, like some other species, calls emitted from highly aroused individuals are less noisy. Glucocorticoids thus play an important, yet underappreciated role, in alarm call production.

Older mothers follow conservative strategies under predator pressure: the adaptive role of maternal glucocorticoids in yellow-bellied marmots.

When the maternal environment is a good predictor of the offspring environment, maternal glucocorticoid (GC) levels might serve to pre-program offspring to express certain phenotypes or life-history characteristics that will increase their fitness. We conducted a field study to assess the effects of naturally occurring maternal GC levels on their offspring in yellow-bellied marmots (Marmota flaviventris) subjected to different predator pressures. Maternal fecal corticosteroid metabolites (FCM) were positively correlated with predator pressure. Predators had both direct and indirect effects on pups. We found that older mothers with higher FCM levels had smaller and female-biased litters. Moreover, sons from older mothers with high FCM levels dispersed significantly more than those from older mothers with low FCM levels, whereas the opposite pattern was found in pups from younger mothers. These age-related effects may permit females to make adaptive decisions that increase their pups' fitness according to their current situation.

Characterization of woodchuck apolipoprotein A-I: a new tool for drug delivery and identification of altered isoforms in the woodchuck chronic hepatitis model.

Apolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum, and participates in the reverse transport of cholesterol from tissues to the liver for excretion. The natural HDL tropism to the liver and cancer cells has been used extensively to target encapsulated drugs. The alteration of the plasmatic isoforms of ApoA-I is a hallmark of chronic hepatitis and hepatocarcinoma in mice and humans. Woodchucks infected with the woodchuck hepatitis virus (WHV) represent the best animal model for the study of chronic viral hepatitis B and viral induced hepatocarcinoma (HCC). WHV-infected woodchuck represents a clinically relevant animal model under which new treatment strategies can be evaluated and optimized. Therapeutic efficacy in this model is likely to be translated into a successful therapy for patients infected with HBV. The present study describes, for the first time, the cloning and characterization of woodchuck ApoA-I. The open reading frame (ORF) of the woodchuck ApoA-I is 795 bp long, coding for 264 amino acids. Unexpectedly, phylogenetic analysis revealed that the closest sequences are those of human and macaque. Woodchuck HDLs were isolated successfully from sera by density gradient ultracentrifugation. A commercial antibody that recognized the woodchuck ApoA-I was also identified. Finally, taking advantage of the techniques and tools developed in this study, two potential applications of woodchuck HDLs are illustrated: drug delivery to a woodchuck hepatocarcinoma cell line and the use of isoelectrofocusing to identify ApoA-I isoforms.

Latitudinal differences in the hibernation characteristics of woodchucks (Marmota monax).

There is little information on the phenotypic flexibility of hibernation characteristics within species. To address this issue, we observed differences in hibernation characteristics of three free-ranging populations of woodchucks (Marmota monax) distributed along a latitudinal gradient from Maine to South Carolina. Data from free-ranging animals exhibited a direct relationship between latitude and length of the hibernation season. As expected, woodchucks in the northern latitudes hibernated longer than those in the southern latitudes. Also, the length of interbout arousals decreased with increase in latitude, whereas the length of torpor bouts and the number of arousals increased. Thus, we observed phenotypic plasticity in hibernation characteristics based primarily on latitudinal temperature differences in each population. Further analysis revealed a direct relationship between latitude and total time spent in torpor. Maine animals spent 68% more time in torpor than South Carolina animals. However, total time spent euthermic did not differ among the three populations. The "cost-benefit" hypothesis of hibernation may help to explain these results. It assumes that hibernators avoid the physiological stress of torpor by staying euthermic as much as possible. Woodchucks in each population maximized time spent euthermic, utilizing torpor only at the level needed to survive winter hibernation and to commence reproduction in the spring.

The interferon-alpha gene family of Marmota himalayana, a Chinese marmot species with susceptibility to woodchuck hepatitis virus infection.

The interferon-alpha (IFN-alpha) gene family is an important part of the immune system. Recombinant interferon-alpha is widely used to treat viral hepatitis and malignant diseases. Marmota himalayana has been found to be susceptible to woodchuck hepatitis virus, a virus genetically related to hepatitis B virus (HBV), and is suitable as an animal model for studies on HBV infection. Here, the IFN-alpha gene family of M. himalayana (cwIFN-alpha) was characterized. Sequence data indicate that the cwIFN-alpha family consists of at least 8 functional sequences and 6 pseudogenes with high homology within the family and to IFN-alpha of Marmota monax, a related species and well-established animal model. The recombinant cwIFN-alpha subtypes were expressed and tested to be active in viral protection assay and to induce expression of MxA in a species-specific manner. This work provides essential information for future work on testing new therapeutic approaches of HBV infection based on IFN-alpha in M. himalayana.

Cloning, expression and polyclonal antibody preparation of the asialoglycoprotein receptor of Marmota himalayan.

The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota Himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

Faecal glucocorticoid metabolites and alarm calling in free-living yellow-bellied marmots.

When individuals of a variety of species encounter a potential predator, some, but not all, emit alarm calls. To explain the proximate basis of this variation, we compared faecal glucocorticoid metabolite concentrations in live-trapped yellow-bellied marmots (Marmota flaviventris) between occasions when they did and did not emit alarm calls. We found that marmots had significantly higher glucocorticoid levels when they called than when they did not call, suggesting that stress or arousal may play an important role in potentiating alarm calls. Marmots are sensitive to variation in the reliability of callers. The present finding provides one possible mechanism underlying caller variation: physiological arousal influences the propensity to emit alarm calls.

Hexokinase and glucose-6-phosphatase activity in woodchuck model of hepatitis virus-induced hepatocellular carcinoma.

2-Deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) is used for PET imaging of woodchuck (Marmota monax) model of hepatocellular carcinoma (HCC). The usefulness of FDG on this animal model needs to be validated according to the hypothesized mechanisms. In this study, two key enzymes involved in glucose or [(18)F] FDG metabolism, hexokinase (HK) and glucose-6-phophatase (G6Pase), were examined for their enzymatic activities in the woodchuck models of HCC, which has not been studied before. After dynamic PET scans, woodchuck liver tissue samples were harvested and the homogenate was centrifuged. The supernatant was used for HK activity assay and the microsomal pellet was used for G6Pase assay. HK and G6Pase activities were measured by means of colorimetric reactions via kinetic and end-point assays, respectively. Total protein content was measured by the Bradford method and used to normalize all enzyme activities. HK and G6Pase activities in woodchuck HCC will be used to correlate with in vivo PET imaging data. The woodchuck model of HCC had significantly increased levels of HK in the livers compared to the age-matching healthy woodchuck (7.96 +/- 1.27 vs. 2.74 +/- 0.66 mU/mg protein, P < 0.01) and significantly decreased levels of G6Pase compared to healthy woodchuck (40.35 +/- 19.28 vs. 237.01 +/- 17.32 mU/mg protein, P < 0.01), reflecting an increase in glycolysis. In addition, significant differences were found in HK and G6Pase activities between HCC liver region (HK: 7.96 +/- 1.27 mU/mg protein; G6Pase: 40.35 +/- 19.28 mU/mg protein) and surrounding normal liver region (HK: 2.98 +/- 0.92 mU/mg protein; G6Pase: 140.87 +/- 30.62 mU/mg protein) in the same woodchuck model of HCC (P < 0.01). Our study demonstrated an increased HK activity and a decreased G6Pase activity in liver of the woodchuck models of HCC as compared to normal woodchuck liver.