When faced with adverse environmental conditions, the marsupial Dromiciops gliroides uses either daily or seasonal torpor to support survival and is the only known hibernating mammal in South America. As the sole living representative of the ancient Order Microbiotheria, this species can provide crucial information about the evolutionary origins and biochemical mechanisms of hibernation. Hibernation is a complex energy-saving strategy that involves changes in gene expression that are elicited in part by microRNAs. To better elucidate the role of microRNAs in orchestrating hypometabolism, a modified stem-loop technique and quantitative PCR were used to characterize the relative expression levels of 85 microRNAs in liver and skeletal muscle of control and torpid D. gliroides. Thirty-nine microRNAs were differentially regulated during torpor; of these, 35 were downregulated in liver and 11 were differentially expressed in skeletal muscle. Bioinformatic analysis predicted that the downregulated liver microRNAs were associated with activation of MAPK, PI3K-Akt and mTOR pathways, suggesting their importance in facilitating marsupial torpor. In skeletal muscle, hibernation-responsive microRNAs were predicted to regulate focal adhesion, ErbB, and mTOR pathways, indicating a promotion of muscle maintenance mechanisms. These tissue-specific responses suggest that microRNAs regulate key molecular pathways that facilitate hibernation, thermoregulation, and prevention of muscle disuse atrophy.
The North American wood frog, Rana sylvatica, is one of just a few anuran species that tolerates whole body freezing during the winter and has been intensely studied to identify the biochemical adaptations that support freeze tolerance. Among these adaptations is the altered expression of many genes, making freeze-responsive changes to gene regulatory mechanisms a topic of interest. The present study focuses on the potential involvement of microRNAs as one such regulatory mechanism and aims to better understand freeze/thaw stress-induced microRNA responses in the freeze-tolerant wood frog. Using quantitative PCR, relative levels of 53 microRNAs were measured in heart and skeletal muscle of control, 24 h frozen, and 8 h thawed frogs. MicroRNAs showed tissue specific expression patterns: 21 microRNAs decreased in the heart during thawing, whereas 16 microRNAs increased during freezing stress in skeletal muscle. These findings suggest that select genes may be activated and suppressed in heart and skeletal muscle, respectively, in response to freezing. Bioinformatics analysis using the DIANA miRPath program (v.2.0) predicted that the differentially expressed microRNAs may collectively regulate tissue-specific cellular pathways to promote survival of wood frogs undergoing freezing and thawing.
Hibernation is a highly regulated stress response that is utilized by some mammals to survive harsh winter conditions and involves a complex metabolic reprogramming at the cellular level to maintain tissue protections at low temperature. In this study, we profiled the expression of 117 conserved microRNAs in the heart, muscle, and liver of the 13-lined ground squirrel (Ictidomys tridecemlineatus) across four stages of the torpor-arousal cycle (euthermia, early torpor, late torpor, and interbout arousal) by real-time PCR. We found significant differential regulation of numerous microRNAs that were both tissue specific and torpor stage specific. Among the most significant regulated microRNAs was miR-208b, a positive regulator of muscle development that was found to be upregulated by fivefold in the heart during late torpor (13-fold during arousal), while decreased by 3.7-fold in the skeletal muscle, implicating a potential regulatory role in the development of cardiac hypertrophy and skeletal muscle atrophy in the ground squirrels during torpor. In addition, the insulin resistance marker miR-181a was upregulated by 5.7-fold in the liver during early torpor, which supports previous suggestions of hyperinsulinemia in hibernators during the early stages of the hibernation cycle. Although microRNA expression profiles were largely unique between the three tissues, GO annotation analysis revealed that the putative targets of upregulated microRNAs tend to enrich toward suppression of progrowth-related processes in all three tissues. These findings implicate microRNAs in the regulation of both tissue-specific processes and general suppression of cell growth during hibernation.
All authors contributed to the conception and design of the project and to the editing of the manuscript. EL, SG, JES, BC, GG, JMA, ALE, and FB conducted the brown bear experiments and provided the tissue samples. BEL and KBS conducted biochemical assays. Data analysis and assembly of the manuscript was carried out by BEL, FB, and KBS. All authors read and approved the final manuscript.
During hibernation, the metabolic rate of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) can drop to <5 % of normal resting rate at 37 °C, core body temperature can decrease to as low as 1-5 °C, and heart rate can fall from 350-400 to 5-10 bpm. Energy saved by hibernating allows squirrels to survive the winter when food is scarce, and living off lipid reserves in white adipose tissue (WAT) is crucial. While hibernating, some energy must be used to cope with conditions that would normally be damaging for mammals (e.g., low core body temperatures, ischemia) and could induce cell death via apoptosis. Cell survival is largely dependent on the relative amounts and activities of pro- and anti-apoptotic Bcl-2 family proteins. The present study analyzed how anti-apoptotic proteins respond to protect WAT cells during hibernation. Relative levels of several anti-apoptotic proteins were quantified in WAT via immunoblotting over six time points of the torpor-arousal cycle. These included anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, and Mcl-l, as well as caspase inhibitors x-IAP and c-IAP. Changes in the relative protein levels and/or phosphorylation levels were also observed for various regulators of apoptosis (p-JAKs, p-STATs, SOCS, and PIAS). Mcl-1 and x-IAP protein levels increased whereas Bcl-xL, Bcl-2, and c-IAP protein/phosphorylation levels decreased signifying important roles for certain Bcl-2 family members in cell survival over the torpor-arousal cycle. Importantly, the relative phosphorylation of selected STAT proteins increased, suggesting a mechanism for Bcl-2 family activation. These results suggest that an increase in WAT cytoprotective mechanisms supports survival efforts during hibernation.
Phthalate administration to male rats has been shown to negatively impact neural development while development of the female rat brain is less affected. Because a number of exogenous agents have been shown to interfere with dopamine function, we evaluated post-adolescent behavioral (operant conditioning for food reward and locomotor activity), histological (tyrosine hydroxylase; TH), and genetic (mRNA levels) outcomes of preadolescent (postnatal days [PND] 16-22) phthalate exposure. Male and female Long-Evans rats were administered 4 doses (0, 1, 10, or 20 mg/kg) of di-(2-ethylhexyl)phthalate (DEHP) i.p. from PND16 to 22. Rats were trained on an operant task to bar press for chocolate-flavored pellets from PND55-63 then euthanized on PND78. The 10 mg/kg DEHP dose was associated with elevated bar pressing for food reward during acquisition and extinction while the 20 mg/kg dose was associated with elevated locomotor activity in both males and females. Stereological analysis revealed reduced TH+ densities in the SNc in DEHP- (10 and 20 mg/kg) treated male and female rats. In the VTA, TH+ staining was reduced in male rats treated with 10 or 20 mg/kg DEHP while in females, the TH: CV ratio was higher at the 10 mg/kg dose compared with controls. An examination of Th mRNA showed a main effect of sex with females showing increased Th expression at all DEHP doses. The present results show that preadolescent phthalate exposure results in detrimental dopaminergic system development impacting neurobehavioral function in post-adolescent rats.
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