IGF-1 and myostatin-mediated co-regulation in skeletal muscle and bone of Daurian ground squirrels (Spermophilus dauricus) during different hibernation stages.

Muscle and bone are cooperatively preserved in Daurian ground squirrels (Spermophilus dauricus) during hibernation. As such, we hypothesized that IGF-1 and myostatin may contribute to musculoskeletal maintenance during this period. Thus, we systematically assessed changes in the protein expression levels of IGF-1 and myostatin, as well as their corresponding downstream targets, in the vastus medialis (VM) muscle and femur in Daurian ground squirrels during different stages. Group differences were determined using one-way analysis of variance (ANOVA). Results indicated that the co-localization levels of IGF-1 and its receptor (IGF-1R) increased by 50% during the pre-hibernation period (PRE) and by 35% during re-entry into torpor (RET) compared to the summer active period (SA). The phosphorylation level of FOXO1 in the VM muscle increased by 50% in the torpor (TOR) group and by 82% in the inter-bout arousal (IBA) group compared to the PRE group. The phosphorylation level of SGK-1 increased by 54% in the IBA group and by 62% in the RET group compared to the SA group. In contrast, the protein expression of IGF-1 and phosphorylation levels of PI3K, Akt, mTOR, and GSK3ß in the VM muscle showed no obvious differences among the different groups. ß-catenin protein expression was up-regulated by 84% in the RET group compared to the SA group, while the content of IGF-1 protein, correlation coefficients of IGF-1 and IGF-1R, and phosphorylation levels of PI3K, Akt, and GSK3ß in the femur showed no significant differences among groups. Regarding myostatin and its downstream targets, myostatin protein expression decreased by 70% in the RET group compared to the SA group, whereas ActRIIB protein expression and Smad2/3 phosphorylation in the VM muscle showed no obvious differences among groups. Furthermore, Smad2/3 phosphorylation decreased by 58% in the TOR group and 53% in the RET group compared to the SA group, whereas ActRIIB protein expression in the femur showed no obvious differences among groups. Overall, the observed changes in IGF-1 and myostatin expression and their downstream targets may be involved in musculoskeletal preservation during hibernation in Daurian ground squirrels.

Integrated Redox-Metabolic Orchestration Sustains Life in Hibernating Ground Squirrels.

Significance: The ultimate manifestations of life, birth, survival under various environmental pressures and death are based on bioenergetics. Hibernation is a unique survival strategy for many small mammals that is characterised by severe metabolic depression and transition from euthermia to hypothermia (torpor) at body temperatures close to 0°C. These manifestations of life were made possible by the remarkable "social" behavior of biomolecules during billions of years of evolution: the evolution of life with oxygen. Oxygen was necessary for energy production and the evolutionary explosion of aerobic organisms. Recent Advances: Nevertheless, reactive oxygen species, formed through oxidative metabolism, are dangerous-they can kill a cell and, on the other hand, play a plethora of fundamentally valuable roles. Therefore, the evolution of life depended on energy metabolism and redox-metabolic adaptations. The more extreme the conditions for survival are, the more sophisticated the adaptive responses of organisms become. Hibernation is a beautiful illustration of this principle. Hibernating animals use evolutionarily conserved molecular mechanisms to survive adverse environmental conditions, including reducing body temperature to ambient levels (often to ∼0°C) and severe metabolic depression. This long-built secret of life lies at the intersection of oxygen, metabolism, and bioenergetics, and hibernating organisms have learned to exploit all the underlying capacities of molecular pathways to survive. Critical Issues: Despite such drastic changes in phenotype, tissues and organs of hibernators sustain no metabolic or histological damage during hibernation or upon awakening from hibernation. This was made possible by the fascinating integration of redox-metabolic regulatory networks whose molecular mechanisms remain undisclosed to this day. Future Directions: Discovering these molecular mechanisms is not warranted only to understand hibernation in itself but to help explain complex medical conditions (hypoxia/reoxygenation, organ transplantation, diabetes, and cancer) and to even help overcome limitations associated with space travel. This is a review of integrated redox-metabolic orchestration in hibernation. Antioxid. Redox Signal. 40, 345-368.

Integrated metabolomics and proteomics analysis to understand muscle atrophy resistance in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.

Seasonal changes of vitamin D3 and ovarian steroidogenesis in the wild ground squirrels (Citellus dauricus Brandt).

There is mounting evidence that vitamin D3 regulates female reproductive function critically, while little is known about the function of seasonally variable vitamin D3 in regulating ovarian steroidogenesis. This study examined the seasonal expressions of vitamin D receptor (VDR), vitamin D metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes (P450scc, 3ß-HSD, P450c17, and P450arom) in the ovaries of the wild ground squirrels (Citellus dauricus Brandt) during the different breeding seasons. VDR, CYP2R1, CYP27B1, and CYP24A1 were shown to be localized in different types of ovarian cells in the wild ground squirrels during the breeding and non-breeding seasons. Meanwhile, the mRNA levels of VDR, CYP2R1, CYP27B1, CYP11A1, HSD3B1, CYP17A1, and CYP19A1 in the ovaries were remarkably higher in the breeding season. Furthermore, RNA-seq data of ovaries revealed that 6036 genes were differentially expressed genes (DEGs); further analysis revealed that several DEGs known to be involved in ovarian steroidogenesis pathway and cellular response to vitamin D pathway were identified. In addition, during the breeding season, the concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, and 17ß-estradiol were greater in the serum of the wild female ground squirrels. This observation was positively correlated with seasonal changes in the concentration of 25(OH)D3, supporting the fact that the 25(OH)D3 content in the ovaries was significantly higher in the breeding season. These findings suggested that seasonal changes in vitamin D3 might regulate the ovarian steroidogenesis of the wild female ground squirrels.

Differential bone metabolism and protein expression in mice fed a high-fat diet versus Daurian ground squirrels following natural pre-hibernation fattening.

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-|κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated ß|-|catenin (P|-|ß|-|catenin), and glycogen synthase kinase-3ß (GSK-3ß)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P|-|ß|-|catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.

Remarkable Plasticity of Bone Iron Homeostasis in Hibernating Daurian Ground Squirrels (Spermophilus dauricus) May Be Involved in Bone Maintenance.

Iron overload is an independent risk factor for disuse osteoporosis. Hibernating animals are natural models of anti-disuse osteoporosis; however, whether iron metabolism is involved in bone adaptation and maintenance during hibernation is unclear. To investigate this question, Daurian ground squirrels (Spermophilus dauricus) (n = 5-6/group) were used to study changes in bone iron metabolism and its possible role in anti-disuse osteoporosis during hibernation. Iron content in the femur and liver first decreased in the torpor group (vs. summer group, -66.8% and -25.8%, respectively), then recovered in the post-hibernation group, suggesting remarkable plasticity of bone iron content. The expression of ferritin in the femur and hepcidin in the liver also initially decreased in the torpor group (vs. summer group, -28.5% and -38.8%, respectively), then increased in the inter-bout arousal (vs. torpor group, 126.2% and 58.4%, respectively) and post-hibernation groups (vs. torpor group, 153.1% and 27.1%, respectively). In conclusion, bone iron metabolism in hibernating Daurian ground squirrels showed remarkable plasticity, which may be a potential mechanism to avoid disuse bone loss during extended periods of inactivity. However, the specific location of iron during low-iron hibernation and the source of iron in post-hibernation recovery need to be further explored.

Seasonal Change in Adiponectin Associated with Ovarian Morphology and Function in Wild Ground Squirrels (Citellus dauricus Brandt).

The goal of this study is to explore the relationship between altered circulating adiponectin concentration, ovarian tissue morphology, ovarian steroidogenesis, and sex hormone production in ovaries of wild ground squirrels. The ovarian mass differed significantly during the breeding and non-breeding seasons, and the circulating estradiol and progesterone concentrations were significantly higher in the breeding season, while the circulating adiponectin level was significantly lower. The expression levels of gonadotropin receptors (FSHR and LHR) and steroidogenic enzymes (StAR, P450scc, P450arom, and 3ß-HSD) were significantly higher during the breeding season. Comparing the ovarian transcriptome data of wild ground squirrels between the two periods, we found that some differentially expressed genes were enriched for ovarian steroidogenesis and the adipocytokine signaling pathway, which correlated with our present results. Notably, the MAPK signaling pathway was also enriched and its related genes (Erk1, p38 Mapk, Jnk) were up-regulated by qPCR during the non-breeding season. These findings suggested that adiponectin may be involved in the regulation of seasonal changes in the ovarian function of wild ground squirrels, possibly by acting on the MAPK signaling pathway to regulate sex steroidogenesis in the ovaries.

The Protective Effects on Ischemia-Reperfusion Injury Mechanisms of the Thoracic Aorta in Daurian Ground Squirrels (Spermophilus dauricus) over the Torpor-Arousal Cycle of Hibernation.

Hibernators are a natural model of vascular ischemia-reperfusion injury; however, the protective mechanisms involved in dealing with such an injury over the torpor-arousal cycle are unclear. The present study aimed to clarify the changes in the thoracic aorta and serum in summer-active (SA), late-torpor (LT) and interbout-arousal (IBA) Daurian ground squirrels (Spermophilus dauricus). The results show that total antioxidant capacity (TAC) was unchanged, but malondialdehyde (MDA), hydrogen peroxide (H2O2), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) were significantly increased for the LT group, whereas the levels of superoxide dismutase (SOD) and interleukin-10 (IL-10) were significantly reduced in the LT group as compared with the SA group. Moreover, the levels of MDA and IL-1ß were significantly reduced, whereas SOD and IL-10 were significantly increased in the IBA group as compared with the SA group. In addition, the lumen area of the thoracic aorta and the expression of the smooth muscle cells (SMCs) contractile marker protein 22α (SM22α) were significantly reduced, whereas the protein expression of the synthetic marker proteins osteopontin (OPN), vimentin (VIM) and proliferating cell nuclear antigen (PCNA) were significantly increased in the LT group as compared with the SA group. Furthermore, the smooth muscle layer of the thoracic aorta was significantly thickened, and PCNA protein expression was significantly reduced in the IBA group as compared with the SA group. The contractile marker proteins SM22α and synthetic marker protein VIM underwent significant localization changes in both LT and IBA groups, with localization of the contractile marker protein α-smooth muscle actin (αSMA) changing only in the IBA group as compared with the SA group. In tunica intima, the serum levels of heparin sulfate (HS) and syndecan-1 (Sy-1) in the LT group were significantly reduced, but the serum level of HS in the IBA group increased significantly as compared with the SA group. Protein expression and localization of endothelial nitric oxide synthase (eNOS) was unchanged in the three groups. In summary, the decrease in reactive oxygen species (ROS) and pro-inflammatory factors and increase in SOD and anti-inflammatory factors during the IBA period induced controlled phenotypic switching of thoracic aortic SMCs and restoration of endothelial permeability to resist ischemic and hypoxic injury during torpor of Daurian ground squirrels.

Estrogen signaling regulates seasonal changes of the prostate in wild ground squirrels (Spermophilus dauricus).

Previous studies found that testosterone was converted to dihydrotestosterone under the catalysis of 5α-reductase in the prostate of the wild ground squirrels. As a result, this study explored further whether testosterone could be converted to estrogen to affect the prostate gland function in wild ground squirrels. Histological observation showed that the area of epithelial cells and the prostatic secretory lumen were enlarged significantly during the breeding period. Transcriptome analysis revealed that the differentially expressed genes in the prostate were concentrated in the estrogen signaling pathway. Immunohistochemical analysis showed that the immunoreactivities of P450arom were detected in the stromal cells during the breeding and non-breeding periods, indicating the possible conversion of androgen into estrogen locally. Moreover, the immunolocalizations of ERα and ERß were detected mainly in the epithelial or stromal cells. Additionally, qPCR analysis displayed that the mRNA expression level of P450arom in the prostate was significantly higher during the breeding period than that in the non-breeding period. Consistently, the concentration of 17ß-estradiol (E2) was higher in the prostate during the breeding period than the non-breeding period, which is positively correlated with the seasonal changes of prostatic weight. In conclusion, the present results indicated that estrogen produced by P450arom presented in stromal cells might regulate the growth and function of the prostate gland via the locally expressed estrogen receptors in wild ground squirrels. The results of this study were momentous for further uncovering the mechanism of the seasonal regulated by signal pathways in the prostate of wild ground squirrels.

Stress associated ovarian dysfunctions in a seasonal breeder Funambulus pennanti: Role of glucocorticoids and possible amelioration by melatonin.

Studies have shown that stress caused by lack of physical activity disrupts the normal pattern of glucocorticoid secretion which adversely affects the reproductive axis. We studied the effect of chronic movement restriction on ovarian responses in the Indian Palm Squirrel Funambulus pennanti, a highly active diurnal rodent. Physical restraint of squirrels induced stress that led to a significant increase in plasma cortisol, corticosterone and decreased 17ß-estradiol level leading to follicular atresia. Ovarian Reactive Oxygen Species (ROS) content, lipid peroxidation (LPO), activities of superoxide dismutase (SOD) and catalase (CAT) enzymes increased in restrained squirrels. Elevated ROS increased the oxidative load that led to ovarian cell death as evidenced by increased Bax and decreased Bcl2 expression causing further decline in Aromatase and ERα proteins. To elaborate the mechanism(s) involved in stress induced glucocorticoid mediated oxidative damages to the ovary we extended our study by exposing ovaries in vitro to the synthetic glucocorticoid dexamethasone (200 µM). We observed that glucocorticoid receptor (GR) expression was significantly increased in dexamethasone treated ovaries in vitro with a decrease in expression of Nrf2 and HO-1 proteins. Melatonin supplementation (10 nM) along with dexamethasone significantly decreased ovarian ROS production, lipid peroxidation and increased antioxidant enzyme activities by improving the expression of Nrf2 and HO-1, reinstating the cellular redox homeostasis. Therefore, it can be suggested that physical restraint induced glucocorticoid and its receptor activation interfered with the ovarian antioxidant defense mechanism. Melatonin via its receptor MT1 significantly alleviated ovarian damages acting as a cytoprotective agent.

5'-Adenosine monophosphate deaminase regulation in ground squirrels during hibernation.

Hibernation is an important winter survival strategy for many small mammals. By sinking into a deep torpor where metabolic rate can be as low as 1-5% of the resting rate in euthermia, animals accrue huge energy savings that allow survival, typically without eating, for many months. Hibernating ground squirrels show a net reduction in the total adenylate pool of skeletal muscle during torpor, but the ATP/ADP ratio and adenylate energy charge remain stable. A key enzyme involved in managing adenylate pool size is 5'-adenosine monophosphate deaminase (AMPD). Assessing skeletal muscle AMPD from both Richardson's ground squirrels (Urocitellus richardsonii) (RGS) and 13-lined ground squirrels (Ictidomys tridecemlineatus) (TLGS), the present study shows that muscle AMPD of euthermic versus hibernating animals displays markedly different kinetic properties, differential responses to temperature and to effectors, and is regulated by reversible protein phosphorylation. AMPD activity decreased during hibernation in both TLGS and RGS skeletal muscle, by 70 and 84%, respectively. Stimulation of total protein phosphatases, total serine/threonine protein phosphatases, PP1, PP2B or PP2C, all reduced AMPD activity between 54 and 92% in extracts of euthermic RGS muscle. The same incubation did not change the activity of AMPD from muscle of hibernating animals. Oppositely, both euthermic and hibernating AMPD showed a strong increase in activity when incubated under conditions that promoted the enzyme phosphorylation by PKA, PKC or PKG. Overall, the data indicate that both low activity of AMPD and low affinity of the enzyme for AMP during torpor reduce the rate of adenylate degradation, the primary driver of these changes being covalent phosphorylation of AMPD.

Multi-tissue profile of NFκB pathway regulation during mammalian hibernation.

Hibernators have evolved effective mechanisms to overcome the challenges of torpor-arousal cycling. This study focuses on the antioxidant and inflammatory defenses under the control of the redox-sensitive and inflammatory-centered NFκB transcription factor in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), a well-established model of mammalian hibernation. While hibernators significantly depress oxygen consumption and overall metabolic rate during torpor, arousal brings with it a rapid increase in respiration that is associated with an influx of reactive oxygen species. As such, hibernators employ a variety of antioxidant defenses to combat oxidative damage. Herein, we used Luminex multiplex technology to examine the expression of key proteins in the NFκB transcriptional network, including NFκB, super-repressor IκBα, upstream activators TNFR1 and FADD, and downstream target c-Myc. Transcription factor DNA-binding ELISAs were also used to measure the relative degree of NFκB binding to DNA during hibernation. Analyses were performed across eight different tissues, cerebral cortex, brainstem, white and brown adipose tissue, heart, liver, kidney, and spleen, during euthermic control and late torpor to highlight tissue-specific NFκB mediated cytoprotective responses against oxidative stress experienced during torpor-arousal. Our findings demonstrated brain-specific NFκB activation during torpor, with elevated levels of upstream activators, inactive-phosphorylated IκBα, active-phosphorylated NFκB, and enhanced NFκB-DNA binding. Protein levels of downstream protein, c-Myc, also increased in the brain and adipose tissues during late torpor. The results show that NFκB regulation might serve a critical neuroprotective and cytoprotective role in hibernating brains and selective peripheral tissue.

Autophagy and Akt-mTOR signaling display periodic oscillations during torpor-arousal cycles in oxidative skeletal muscle of Daurian ground squirrels (Spermophilus dauricus).

Whether hibernation accelerates or suppresses autophagy is still unknown. In the current study, we examined changes in autophagy in oxidative soleus (SOL) muscle in summer active (SA), pre-hibernation (PRE), torpor (TOR), interbout arousal (IBA), and post-hibernation groups of Daurian ground squirrels (Spermophilus dauricus). Here, the SOL muscle showed no significant atrophy during hibernation in regard to muscle wet weight, fiber cross-sectional area, or MuRF1 protein level. Autophagy-related proteins beclin1 and Atg7 increased significantly, whereas LC3-II decreased significantly in the PRE group compared with the SA group. However, neither the expression nor activity of cathepsin L showed any differences between the SA and PRE groups. In addition, beclin1, LC3-II, and the LC3-II/LC3-I ratio increased, p62 decreased, LC3 puncta increased, p62 puncta decreased, and cathepsin L activity increased in the TOR group compared with the PRE group. In contrast, beclin1, LC3-II, and the LC3-II/LC3-I ratio decreased, p62 increased, LC3 puncta decreased, p62 puncta increased, and cathepsin L activity declined in the IBA group compared with the TOR group. Moreover, the phosphorylation of Akt (Ser473) and mTOR (Ser2448) changed significantly during hibernation and showed an inverse relationship with autophagy changes. In conclusion, autophagy proteins displayed periodic oscillation in the torpor-arousal cycle, which may be advantageous in maintaining SOL muscle mass during the entire hibernation period. Furthermore, the Akt-mTOR signaling was decreased in TOR and increased in IBA group in the SOL muscle of Daurian ground squirrels during hibernation.

Changes in copper, zinc and cadmium distributions in the liver of Formosan squirrels with characteristic high copper accumulation.

We discovered previously that Formosan squirrels (Callosciurus erythraeus) accumulate copper (Cu) in their livers at levels averaging 1700 µg per dry g (approximately 420 µg per wet g). In the current study, we investigated the relationship between Cu accumulation and hepatic injury, and we determined the distribution and chemical form of Cu in the liver supernatant. In particular, we explored the role of metallothionein in the liver supernatant. We observed no significant differences in hepatic Cu concentration between squirrels that showed pathological changes in the liver and those that did not. Serum alanine aminotransferase activity did not increase with increasing hepatic Cu concentration. These results suggest that abnormal Cu accumulation in the livers of Formosan squirrels does not induce severe hepatic injury. We found that 26.7% of the Cu in the liver was distributed to the supernatant, and only 11.0% of the Cu in the liver was bound to metallothionein, suggesting that metallothionein in the hepatic supernatant does not contribute to detoxification of excess Cu in Formosan squirrels.

Agmatine modulates calcium handling in cardiomyocytes of hibernating ground squirrels through calcium-sensing receptor signaling.

True hibernators are remarkable group of mammals whose hearts are resistant to such stressors as deep hypothermia, ischemia, arrhythmia. Capability of cardiac cells from hibernating species to effectively rule Ca2+ homeostasis during torpor is poorly studied. Better understanding of these mechanisms could allow to introduce new strategies for improvement the cardiac performance and may be useful for cardiovascular medicine. Here for the first time we have shown that the regulation of Ca2+ handling and thereby cardiomyocyte contractility by endogenous neurotransmitter agmatine occurs through the modulation of calcium-sensing receptor (CaSR). In isolated cardiocytes of hibernating ground squirrels generating stationary Ca2+ transients in the absence of actual myocellular excitation, low doses of this polyamine (up to 500 µM) induce the Gßγ-dependent activation of PI3-kinase with subsequent stimulation of Akt-kinase and nitric oxide (NO) production by endothelial NO-synthase (eNOS). NO production abolishes Ca2+ oscillations in virtue of the enhancement of Ca2+ reuptake by sarco(endo)plasmic Ca2+ ATPase (SERCA). Simultaneously, the activation of phospholipase A2 (PLA2) and arachidonic-acid dependent Ca2+ entry occur providing replenishment of Ca2+ store. High concentrations of agmatine (> 2 mM) induce other CaSR-mediated pathways involving phospholipase C (PLC) pathway, the formation of inositoltriphosphate (IP3) and diacylglicerol (DAG) followed by induction of their targets: IP3 receptors and protein kinase C isoforms (PKC), respectively. Furthermore, it is also responsible for the stimulation of PLA2 and elevation of intracellular calcium caused by arachidonic acid-regulated Ca2+-permeable (ARC) channels. Additionally, there is a potent store-operated Ca2+ entry (SOC) in cardiomyocyte. Negative (NPS 2143) and positive (R 568) allosteric modulators of CaSR recapitulate effects of low and high agmatine doses on Ca2+ handling and NO synthesis. These facts and the alteration of agmatine influence in response to an increase of extracellular Ca2+, which is the direct agonist of CaSR, may confirm the participation of CaSR in regulation of Ca2+ handling and excitability of cardiomyocytes by agmatine.

FECAL PROGESTERONE METABOLITES IN POSTPARTUM SIBERIAN FLYING SQUIRRELS.

The Siberian flying squirrel ( Pteromys volans) produces up to two litters a year. To deliver second litters in breeding season, P. volans may have a postpartum estrus similarly to that of a variety of small mammals. If this were the case, females would have periods of elevated progesterone levels because of the formation of corpora lutea (CL) after postpartum ovulation. To test this hypothesis, fecal progesterone metabolite dynamics was investigated during lactation in this species using an enzyme immunoassay. In five of the six lactating females, periods of high fecal progesterone metabolite concentration were observed, and, furthermore, progesterone secretion patterns were periodic. Therefore, the source of progesterone during lactation could be arising CL from postpartum ovulation, indicating that ovarian activity was reinitiated after parturition and the CL that formed began secreting progesterone. This study thus showed that P. volans likely has the physiologic potential to mate during lactation.

Integrated transcriptomic and metabolomic analysis reveals adaptive changes of hibernating retinas.

Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities.

Remarkable plasticity of Na+, K+-ATPase, Ca2+-ATPase and SERCA contributes to muscle disuse atrophy resistance in hibernating Daurian ground squirrels.

We investigated cytosolic calcium (Ca2+) and sarcoplasmic reticulum Ca2+ regulation in skeletal muscle fibers of hibernating Daurian ground squirrels (Spermophilus dauricus), non-hibernating hindlimb-unloaded (HLU) squirrels, and HLU rats to clarify the molecular mechanisms involved in preventing muscle atrophy in hibernators. The Na+, K+-ATPase and Ca2+-ATPase activities in the soleus muscle (SOL) of squirrels were maintained in hibernation, decreased during interbout arousal (IB-A), and increased to autumn/pre-hibernation (AUT/Pre-H) levels in torpor after interbout arousal (Post-IBA), whereas activities in the extensor digitorum longus muscle (EDL) were stable during hibernation, but increased during post-hibernation (Post-H). Activities increased in the SOL of HLU rats, but were stable in HLU squirrels. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity in the SOL decreased in IB-A squirrels, but returned to AUT/Pre-H levels in the Post-IBA group; no significant changes were found in the EDL. SERCA activity increased in the EDL of HLU squirrels and SOL of HLU rats. Compared with AUT/Pre-H, SERCA type 2 protein expression increased in the SOL and EDL of IB-A and Post-IBA squirrels, but increased in the SOL only in HLU animals. We also describe the protein kinase A changes in this paper. Thus, hibernating ground squirrels displayed remarkable Na+, K+-ATPase, Ca2+-ATPase, and SERCA plasticity.

Seasonal changes of androgen receptor, estrogen receptors and aromatase expression in the hippocampus of the wild male ground squirrels (Citellus dauricus Brandt).

The wild ground squirrel is a typical seasonal breeder whose annual life cycle can be roughly divided into the breeding season, the post-breeding season and hibernation. Our previous study has reported the seasonal changes in the expressions of androgen receptor (AR), estrogen receptors α and ß (ERα and ERß), and aromatase cytochrome P450 (P450arom) in the hypothalamus of male wild ground squirrels. To further seek evidence of seasonal expression of steroid hormone receptors and steroid hormone synthases in other brain regions, we investigated the protein and mRNA expressions of AR, ERα, ERß and P450arom in the hippocampus of the male wild ground squirrels during these different reproductive periods. Histological observation showed that the number of pyramidal cells in Cornu Ammonis 1 (CA1) increased in the breeding season. Both protein and mRNA of AR, ERα, ERß and P450arom were present in CA1 and CA3 of all seasons. There was significant increment in the immune-signal intensity and mRNA level of AR and ERα during the pre-hibernation, whereas those of ERß and P450arom were higher during the post-breeding season. In addition, the profile of plasma testosterone concentration showed the nadir in the post-breeding season, a marked elevation in the pre-hibernation, and the summit in the breeding season. These findings suggested that the hippocampus may be a direct target of androgen and estrogen; androgen may play important regulatory roles through its receptor and/or the aromatized estrogen in the hippocampus of the wild male ground squirrels.

Short day length enhances physiological resilience of the immune system against 2-deoxy-d-glucose-induced metabolic stress in a tropical seasonal breeder Funambulus pennanti.

Studies demonstrate the importance of metabolic resources in the regulation of reproduction and immune functions in seasonal breeders. In this regard, the restricted energy availability can be considered as an environmental variable that may act as a seasonal stressor and can lead to compromised immune functions. The present study explored the effect of photoperiodic variation in the regulation of immune function under metabolic stress condition. The T-cell-dependent immune response in a tropical seasonal breeder Funambulus pennanti was studied following the inhibition of cellular glucose utilization with 2-deoxy-d-glucose (2-DG). 2-DG treatment resulted in the suppression of general (e.g., proliferative response of lymphocytes) and antigen-specific [anti-keyhole limpet hemocyanin IgG titer and delayed-type hypersensitivity response] T-cell responses with an activation of the hypothalamic-pituitary-adrenal axis, which was evident from the increased levels of plasma corticosterone. 2-DG administration increased the production of inflammatory cytokines [interleukin (IL)-1ß and tumor necrosis factor (TNF)-α] and decreased the autocrine T-cell growth factor IL-2. The immunocompromising effect of 2-DG administration was retarded in animals exposed to short photoperiods compared with the control and long photoperiod-exposed groups. This finding suggested that short photoperiodic conditions enhanced the resilience of the immune system, possibly by diverting metabolic resources from the reproductive organs toward the immune system. In addition, melatonin may have facilitated the energy "trade-off" between reproductive and immune mechanisms, thereby providing an advantage to the seasonal breeders for their survival during stressful environmental conditions.