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Hibernation induction in non-hibernating species

Hibernation induction in non-hibernating species Abstract Hibernation is a natural process in many mammals to maximise survival during extreme environmental conditions. It is characterised by profound physiological changes such as reductions in metabolic rates and core body temperature for prolonged periods of time of up to 9 months. The ability of hibernating animals to recover from hibernation with no obvious signs of organ injury makes them excellent models for the clinical application of this natural protective mechanism, which has generated great interest in medical research. Recent studies have shed more light on the genetic and molecular basis of hibernation and several chemical compounds have been demonstrated to play significant roles in the regulation of hibernation, with an increasing focus on investigating their potential clinical applications in organ preservation and even development of cryonics techniques. In this review, I will present recent progress made in understanding the mechanism of hibernation and highlight evidence for hibernation induction in non-hibernating mammals using chemical compounds; finally, I will discuss the potential for combining genetic techniques to induce metabolic suppression for therapeutic purposes. induction of hibernation, regulation and physiological change, hydrogen sulphide, 5′-AMP, DADLE, genetic basis Introduction Hibernation is an adaptation mainly found in mammals and birds that occurs at physiological, behavioural and molecular levels to sustain life during winter months when resources are insufficient or unpredictable. It is generally associated with a profound reduction in core body temperature to the level of ambient temperature and a reduction in metabolic rate to a fraction of basal metabolic rate (BMR), paralleled with bradypnea and reduced heart rate (Heldmaier, Ortmann and Elvert, 2004). Similar to hibernation, torpor is a survival tactic involving a reduction in body temperature and metabolic rate. The physiological definitions of torpor and hibernation remain unclear but they are used in the scientific literature to express different durations and amplitudes of hypometabolic state. Hibernation generally has a longer duration of up to 9 months whereas torpor lasts <24 h and is commonly referred to as daily torpor. The mean minimum metabolic rate was observed to reach 6% of BMR during hibernation but 35% of BMR during torpor (Ruf and Geiser, 2015). By entering hibernation, hibernators can produce a remarkable saving in energy consumption. In winter, yellow-bellied marmots are able to save up to 85% of their energy, which will greatly enhance winter survival (Armitage, Blumstein and Woods, 2003). Human hibernation, if possible, would have significant clinical applications in three major areas. The induction of a hypometabolic state could better treat critical illness because organ damage could be reduced by inducing hyperthermia in patients who suffer from hypoxia-induced injury such as brain injury and intestinal I/R injury (Aslami and Juffermans, 2010). Another aspect is related to cryonics—a technology that involves lowering the body temperature of patients who cannot be saved with current technologies and preserving them until future technologies can revive and restore them to full health. Cryonics is thought to be an unrealistic practice by most people as simply inducing hypothermia in the human body would lead to ischaemia and reperfusion injury, which result in profound damage in brain and tissues (Best, 2012). Despite being a currently unattainable idea, induction of hibernation in astronauts could significantly reduce resource requirements and associated medical challenges during interstellar space travel. As a Phase I study, NASA placed patients in a mild hypometabolic state for up 14 days with no sign of detrimental effects, suggesting the safety of therapeutic hypothermia in humans (Bradford, Schaffer and Talk, 2014). Hibernation and torpor, processes that occur naturally in many mammals, might offer better alternatives to achieve the aim of long-term preservation of patients and astronauts that currently only exists in science fiction. In this article, I will: (i) provide background information on hibernation including a brief introduction of regulatory mechanisms and detailed physiological changes associated with entrance and arousal from hibernation; (ii) review recent evidence for mechanisms of organ preservation and hibernation induction in non-hibernating mammals using chemical compounds and (iii) discuss the potential to genetically translate the hibernation phenotype to humans with the aim of inducing metabolic suppression or suspended animation for therapeutic purposes, and highlight directions for future research on inducing hibernation in humans. Regulation and physiological changes in hibernation It is thought that hibernation is controlled both by the central nervous system (CNS) and endocrine system. Previous studies identified regions in the brain that are involved in entrance to and arousal from hibernation. Entrance to hibernation involves: (1) activation of hypothalamic regions, and (2) inhibition of cortical regions. In contrast, the hypothalamic nuclei, in particular, are important in arousal from hibernation (Kilduff et al., 1990). 3-Iodothyronamine (T1AM) is an endogenous thyroid that binds to Trace amine-associated receptor 1 (TAAR1), and injection of T1AM into mice effectively reduces cardiac output and heart rate (Chiellini et al., 2007). More generally, thyroid hormones seem to be key modulators in lipid and carbohydrate metabolism and they can regulate basal metabolism at different stages of the life cycle. It was found that plasma total triiodothyronine (tT3) was negatively correlated with its activity prior to hibernation and the level of thyroxine (tT4) increased shortly after right after hibernation termination in female arctic ground squirrels (Wilsterman et al., 2015). There are a number of remarkable physiological changes associated with hibernation. During hibernation or torpor, animals enter a hypometabolic and hypothermic state. The metabolic rate of the Alpine marmot reaches 1/25 of its euthermic BMR and its body temperature drops to 1.5–3°C above ambient temperature (Heldmaier, Ortmann and Elvert, 2004). At the same time, the heart rate is also reduced to a critical level and the inadequate amount of oxygen could lead to hypoxia in non-hibernating animals. Hibernators, however, are able to survive a long period of time without signs of organ damage and there is a generally increased resistance to hypoxia and reperfusion in hibernating animals (Sandovici et al., 2004; Talaei et al., 2011). On a cellular level, programmed cell death (apoptosis) could be triggered by physiological changes associated with entry to and exit from a state of metabolic depression. These include changes in blood flow, oxidative stress and nutrient deprivation. Natural hibernators are able to suppress the apoptotic response as suggested by the constant level of p-p53 S46 in the brain and heart of thirteen-lined ground squirrels during torpor (Rouble et al., 2013). Suppression of apoptosis is done via various mechanisms and one of the ways is to regulate the level of anti-apoptotic proteins. In the same study, an elevation in levels of anti-apoptotic targets (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) was observed in the brain. In addition, a sudden increase in oxygen uptake during arousal from hibernation could result in rapid generation of reactive oxidative species (ROS), which would result in oxidative stress (Storey, 2010). Oxidative stress is a key deleterious factor in brain ischaemia as it could contribute to apoptosis through a number of pathways. However, it has been found that there is a correlation in time between the most rapid fall of ascorbate and peak of oxygen consumption in Artic ground squirrels during arousal from hibernation (Tøien et al., 2001). This suggests that ascorbate may function as an anti-oxidation agent that removes the increased amount of ROS during arousal. Similarly, glutathione peroxidase-3 is an enzyme responsible for hydrogen peroxide (H2O2) detoxification in plasma. Its gene expression level increased 8-fold in the brain of hibernating horseshoe bat compared to non-hibernating animals (Lei et al., 2014). Meanwhile, hibernators will enter an immunosuppressed state during hibernation to prevent general inflammation in the body. It was observed that the number of leucocytes and thrombocytes in blood of European ground squirrels decreased by 90% within 24 h of torpor (Tøien et al., 2001; Bouma et al., 2010). The drop in leucocytes in Syrian hamster and Djungarian hamster was found to be mediated by the level of sphingosine-1-phosphate (S1P) in a temperature-dependent manner (Bouma et al., 2011). When hibernators enter deep torpor, the body temperature drops and so does the S1P level; when animals are aroused from torpor, S1P is restored to the pre-torpor level (Nelson, Otis and Carey, 2010). Apart from suppression of apoptosis, oxidative stress and inflammatory response, hibernators also have to avoid blood clotting as reduced blood flow could lead to deep vein thrombosis, stroke or pulmonary embolism. During torpor, clotting factor FVIII and FIX in 13-lined ground squirrels showed reduced activities by 5-fold and 2-fold, respectively, compared with levels of non-hibernating species (Lechler and Penick, 1963). Von Willebrand factor (VWF), a glycoprotein required for the formation of platelet clot, was observed to be reduced both quantitatively and qualitatively compared to non-hibernating animals (Cooper et al., 2016). Recently some chemical compounds have been suggested to induce hibernation or torpor state in non-hibernating animals, with functions of anti-apoptosis, anti-oxidation, anti-inflammation or anti-coagulation. Now I will discuss evidence regarding the mechanisms of these candidate compounds and point out their limitations in the research on hibernation induction. Hydrogen sulphide Hydrogen sulphide (H2S) is endogenously produced in mammalian tissues and functions as a neuromodulator in the brain (Abe and Kimura, 1996). It is generally known as an inhibitor of oxidative phosphorylation complex IV in mitochondria, via reaction with the copper centre, leading to decreased cellular oxygen consumption and metabolic rate (Szabó, 2007). It is suggested to have an anti-apoptotic, anti-oxidative and anti-inflammatory effect. H2S inhibits apoptosis through activation of the reperfusion injury salvage kinase (RISK) pathway, which is a signalling cascade that involves pro-survival protein kinase (Lambert et al., 2014). Although the specific downstream effects of the RISK pathway have not yet been fully elucidated, the extracellular regulated kinase 1/2 (Erk1/2) arm of the RISK pathway has been shown to activate p90sk, thus phosphorylating and inhibiting Bcl-2-associated death promoter (BAD) protein, which initiates apoptosis (Meng et al., 2013). H2S inhibits oxidation via activation of nuclear factor 2 (Nrf2), which is a regulator of the anti-oxidation response. Keap1 is a negative regulator of Nrf2, and NaHS induces disassociation of Nrf2 from Keap1, enhancing the transcription of genes of detoxifying enzymes that Nrf2 targets, such as glutathione (GSH) reductase and glutamate-cysteine ligase (Yang et al., 2013). H2S is also an anti-inflammatory molecule. Inhibition of the inflammatory response is achieved by inhibition of leucocyte rolling and adhesion, which is further done by triggering the activation of NO synthase and the p38 MAPK-dependent mechanism (Yusof et al., 2009). A summary of pathways in which H2S participates is illustrated in Fig. 1. Meanwhile, hydrogen sulphide was experimentally shown to induce suspended animation by Blackstone et al. in 2005, with the body temperature of house mice falling to approximately 2°C above ambient temperature after exposure to 80 ppm H2S and returning to normal temperature after 6 h of treatment (Blackstone, Morrison and Roth, 2005). Volpato et al. (2008) repeated the experiment in mice and similar results were obtained. Figure 1. Open in new tabDownload slide Pathways of H2S-induced anti-apoptotic, anti-oxidative and anti-inflammatory response. Diagram produced based on Sen, Paul and Gadalla et al. (2012),,Meng et al. (2013), Yang et al. (2013), Lambert et al. (2014) and Yusof et al. (2009). In larger mammals, the effect of H2S is less consistent. Li et al. (2008) failed to induce a suspended animation-like state in anaesthetised piglets with different levels of H2S. This might be due to a dose-response issue as the highest volume of H2S applied was 80 ppm, which might not be sufficient for effective hibernation induction in large mammals. A high concentration of H2S, however, has been reported to be toxic. As described in an earlier study, 500–1000 ppm H2S caused liver damage in humans (Yalamanchili and Smith, 2008). Although studies regarding the ability of H2S to induce a torpor/hibernation-like state in larger mammals appear to be conflicting, it is still a promising candidate and elucidating the mechanisms by which H2S acts is necessary to assess the viability of hibernation induction in humans. 5′-Adenosine monophosphate 5′-Adenosine monophosphate (5′-AMP) is a nucleotide produced by hydrolysis of ATP. Zhang et al. (2006) found that a significant dosage of 5′-AMP induces a reversible deep hypometabolic state in non-hibernating mice, in which the metabolic rate was observed to fall to <10% of the euthermic level. By using comparative analysis, a wide suppression of energy generating metabolic pathways was observed in 5′-AMP-induced hypothermia (AIHM; Zhao et al., 2014). In the same study, it was suggested that 5′-AMP has negative effects on haemoglobin’s affinity for oxygen, which further leads to hypometabolism. 5′-AMP also causes glycolysis suppression and results in an increased level of blood 2,3- bisphosphoglycerate (2,3-BPG), an enzyme that binds with greater affinity to deoxygenated haemoglobin, and thus inhibits binding of oxygen to red blood cells. Currently, there are two hypotheses regarding the molecular pathways of AIHM, as shown in Fig. 2. Figure 2. Open in new tabDownload slide Adenosine receptor-mediated and AMPK-mediated pathways to induce AIMP. Diagram produced based on Melvin and Andrews (2009), Iliff and Swoap (2012), Linden and Cekic (2012) and Takedachi et al. (2008). The adenosine receptor-mediated pathway, however, is less convincing. Studies on adenosine receptor-deficient mice demonstrated that mice with deficiency in adenosine receptors showed an ability to enter deep hypometabolism at a similar rate to wild type mice (Daniels et al., 2010). On the other hand, a more recent study suggested that activation of adenosine A1 receptors (A1ARs) in the CNS is essential in induction of hibernation in hibernating species (Jinka, Toien and Drew, 2011). A1ARs actually induced a hypothermic, hibernation-like state in mice, in which a remarkable fall in body temperature was observed, along with a reduction in electroencephalogram amplitude and heart rate (Tupone, Madden and Morrison, 2013). Similar to hydrogen sulphide, 5′-AMP also exhibits anti-inflammatory ability. More specifically, 5′-AMP activates the ERK1/2, p38, JNK and NF-κB signalling pathways, which attenuate LPS-induced inflammation in Wistar rats (Wang et al., 2014). At the same time, the numbers of T- and B-lymphocytes that circulate in the blood were reduced following injection of 5′-AMP into congenic mice (Bouma et al., 2013). The capability of 5′-AMP to induce a significant, safe and reversible hypometabolism was proposed in a previous review (Lee, 2008). Further investigation is needed on molecular mechanisms of AIHM and its clinical potential. DADLE [d-Ala2, d-Leu5]-enkephalin (DADLE) is a synthetic peptide that binds and activates the δ-opioid receptor (DOR). In 2015, a study on rat intestinal epithelial cells demonstrated that DADLE has a protective effect in ischaemia/reperfusion (I/R) injury, via inhibition of the protein kinase kinase 7 (MKK7)-c-Jun N-terminal kinase (JNK) pathway (Wang et al., 2015). JNK pathways are closely associated with I/R injury and MKK7 is an intermediate of JNK involved in activation of JNK (Haeusgen, Herdegen and Waetzig, 2011). Similarly, DADLE has an important role in preventing necrotic and apoptotic processes resulting from I/R injury by inhibiting early apoptotic events, such as serum and glucose deprivation (DEPV)-induced mitochondrial membrane damage (Yao et al., 2007). In the same study, it was shown that the production of reactive oxygen species (ROS) was negatively correlated with the level of DADLE, suggesting that DADLE could prevent ROS-induced apoptosis under the condition of DEPV. DADLE also has neuroprotective effects in both the central and peripheral nervous system (Borlongan, Wang and Su, 2004). It was demonstrated that DADLE protected the terminal membranes of dopaminergic neurons from methamphetamine (METH)-induced neurotoxicity in mice (Tsao et al., 1998). Meanwhile, more evidence of the underlying molecular mechanism in which DADLE works has started to emerge. It was proposed that DADLE is able to reduce the rate of transcription in opioid receptor-deficient LNCaP epithelial cells (Baldelli et al., 2006). The same study also suggested that the inhibitory action of DADLE is independent of opioid receptors, which sheds more light on an alternative mechanism for the compound. In some cases, DADLE is considered to be pharmacodynamically homologous to hibernation induction trigger (HIT), a molecule present in the blood that is capable of inducing hibernation in non-hibernating squirrels by blood transfusion (Dawe and Spurrier, 1969). Similarly to DADLE, HIT has a profound function in organ preservation, as suggested by a study on mongrel dogs (Chien et al., 1991). The preservation of multiple organs prolonged the survival time by up to 48 h and lung function was maintained after transplantation to host animals. Even though the detailed association between HIT and DADLE remains unclear, we must not neglect the role of opioids in torpor and hibernation. The DOR, in particular, is associated with neuronal tolerance to hypoxic/ischaemia stress and anti-oxidative ability. Also, DOR signal is able to stabilise homoeostasis and to increase pro-survival signalling such as protein kinase C, which is involved in cardioprotection (Feng et al., 2012). Despite the significance of DADLE in protecting against damaging effects from I/R injury, there is currently no experimental evidence that DADLE is able to induce torpor or a hibernation-like state in non-hibernating animals. However, a recent study on human neuroblastoma SH-SY5Y cells and primary cortical neurons showed that DADLE is able to reduce the cellular transcription rate to a significant level without causing cell damage or injury (Tian et al., 2014), suggesting that DADLE could be a promising candidate as a therapeutic drug for neuroprotection. Maintenance of hibernation When inducing hibernation, it is also critical to consider ways to maintain and terminate the hibernation state and there are several compounds that play important roles in maintenance and arousal from hibernation. In a study on ground squirrels, mu and kappa opioids were suggested to affect the arousal state of hibernation (Oeltgen et al., 1988). Opioid peptides are involved in thermal regulation during the maintenance phase of hibernation. The injection of two opioid receptor antagonists, naloxone and naloxonazine, effectively elevated body temperature and terminated hibernation in Syrian hamster (Tamura et al., 2005). Glutamate is an amino acid that serves as a metabolic fuel and thus regulates metabolic rate. It acts via NMDA-type glutamate receptors (NMDAR) and the activation of NMDAR was observed to maintain the torpor state outside the CNS of arctic ground squirrel (Jinka, Rasley and Drew, 2012). Meanwhile, it has been found that the interaction between serotonergic and noradrenergic systems (NA) maintains the state of hibernation and that the ratio of these two systems determines entrance or arousal from hibernation in ground squirrels (Semenova and Zakharova, 2015). A higher concentration of serotonin was observed in ground squirrel in autumn prior to hibernation and the NA system is more related to behaviours in spring. In most natural hibernators, the food they consume during summer is converted into white adipose tissue for subsequent consumption in winter (Boyer and Barnes, 1999). If we intend to induce hibernation in humans or other non-hibernating animals, it is necessary to consider the energy resource required for a long period with no intake of food. Consider a healthy adult man aged 50 years old, whose height is 170 cm and weight is 70 kg. Based on the Mifflin St Jeor Equation (Mifflin et al., 1990), which estimates BMR: P=(10m1.0kg+6.25h1.0cm−5.0a1.0year+5)kcalday where P is BMR, m is mass, h is height and a is age, the BMR would be 1517 kcal/day. On average, hibernators are able to reduce energy requirements by up to 90% (Heldmaier, Ortmann and Elvert, 2004), and thus the energy needed during hibernation would be 1517 × 10% = 151.7 kcal/day. It is possible to supply energy using lipid, which has an energy content of 9 kcal/g, and glucose, which has an energy content of 3.75 kcal/g (Food and Agriculture Organization of the United Nations, 2003). To provide sufficient amounts of energy for a man, 16.9 g (151.7/9) of lipid and 40.4 g (151.7/3.75) of glucose would be needed on a daily basis. During natural hibernation, there seems to be a switch from glucose metabolism to lipid metabolism as genes involved in glycolysis are under-expressed while genes for fatty acid catabolism are over-expressed (Yan et al., 2008). It would be more sensible to supply lipid by means of lipid emulsion during hibernation of humans. Genetic and epigenetic basis of hibernation The genes associated with hibernation seem to be present in the genomes of most mammals, including humans (Srere and Martin, 1992). For instance, mitochondrial uncoupling proteins (UCPs) are responsible for non-shivering heat generation in brown adipocyte tissue during hibernation. Its homologous protein, UCP2, was detected in a greatly increased amount in multiple tissues in hibernating ground squirrels and it was found to be present in the human genome (Fleury et al., 1997; Boyer et al., 1998). More examples of genes that function in hibernation and have highly homologous genes in humans are listed in Table 1. Table 1. Comparison of genes associated with hibernation in thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and homologous genes in human Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Open in new tab Table 1. Comparison of genes associated with hibernation in thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and homologous genes in human Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Open in new tab Meanwhile, the evolution of hibernation has been suggested to occur at a molecular level with changes in patterns of gene expression rather than evolution of de novo genes. A model of gene regulation in hibernation has been proposed, as shown in Fig. 3. Figure 3. Open in new tabDownload slide Schematic representation of the central role of differential gene expression during hibernation. Single arrows indicate direct effects, and multiple arrows indicate an effect that requires multiple intermediate events. Reprinted from Srere, H. K. & Martin, S. L. (1992) Central role for differential gene expression in mammalian hibernation. Proceedings of the National Academy of Sciences of the United States of America. 89 (15), 7119–7123. In concert with the hypothesis that most physiological changes are associated with gene regulation, it has been found that gene regulation of 5′-AMP-induced hypometabolism occurs at a post-translational level. In the study by Zhang et al. (2011), >99.9% of mRNA analysed did not significantly change in level in the liver of mice. Global depression of transcription has been observed in golden-mantled ground squirrels during hibernation, which might be due to the combined effects of mild suppression of initiation and severe inhibition of elongation during transcription (Van Breukelen and Martin, 2002). Epigenetics refers to changes in gene expression that are not the result of alteration in DNA sequence. It also plays a major role in transcriptional and translational suppression in hibernation, which includes histone tail modification and DNA methylation. Morin and Storey (2006) found that the relative content of acetylation and phosphorylation on H3 fell by 25% and 40%, respectively, and that the amount of histone deacetylase protein rose during hibernation in skeletal muscle of thirteen-lined ground squirrels, which led to elevated chromatin packing and transcriptional silencing. A similar result was obtained in another study on brown adipose tissue from ground squirrels (Biggar and Storey, 2014). Changes in levels of methylation of DNA occurred in response to environmental stress, as demonstrated in a recent study on mouse neuronal cells (Hartley et al., 2013). When neurons in hippocampuswere exposed to hypoxia, both hyper- and hypo-methylation on CpG islands or promoter regions of different genes were observed to produce transcriptional modulation. Similarly, DNA methylation contributes to the transition into the hibernation phenotype in the liver and skeletal muscle of thirteen-lined ground squirrels (Alvarado et al., 2015). They successfully identified a gene, myocyte enhancer factor 2C (mef2c), in skeletal muscle whose promoter is methylated in response to hibernation initiation. Given that most of the genes associated with hibernation are present in the human genome, it could be possible to temporarily induce hibernation using molecular biology techniques. We should be immediately aware that modification should not be done at the genome or germ-line level because of genetic inheritance and ethical concerns, implying that most of the changes would be post-transcriptional modifications. Therefore, we can (i) deliver genes that are upregulated in hibernation into humans using viral or non-viral vectors to increase gene expression; (ii) exploit the mechanism of RNAi and deliver small RNA molecules to perform gene silencing and achieve downregulation at the transcriptional level and (iii) modify histone or DNA methylation to up- or downregulate gene expression. Genes that are upregulated in hibernation can be delivered into humans using vectors. However, this is less practical when the number of genes is enormous. It has been reported that 225 genes are upregulated in response to hypoxia exposure (Hartley et al., 2013), which will make the design and delivery of genes a tremendous amount of work. DNA pieces of the genes that we would like to downregulate could be introduced into non-hibernating animals. Introduced DNA will yield dsRNA or short hairpin RNA (shRNA) after transcription, which will be targeted by the host RNA interference system (RNAi) to produce short interfering RNA (siRNA). The RNAi system will cleave host mRNA that is complementary to siRNA and silence the expression of the gene. However, siRNA would lead to miRNA-like off-target effects as first recognised in 2003 (Aimee et al., 2003). This is because siRNA can target mRNA sequences whose 3′UTRs are complementary to the 5′UTR of siRNA. Synthetic siRNA has been reported to stimulate the production of interferons and inflammatory cytokines both in vivo and in vitro (Adam et al., 2005). Therefore the immunogenic effect of siRNA remains under investigation. Conclusion and future directions Hibernation is a strategy adopted by animals to conserve energy during periods with insufficient resources. Some studies have successfully induced torpor or hibernation-like state in non-hibernating animals using chemical compounds such as H2S and 5′-AMP, but there has been limited success in larger mammals. More research on hibernation induction needs to be done on primates, which are good models for human medicine owning to their genetic and physiological similarities. Nevertheless, these molecules have great potential in clinical applications such as trauma, heart attack, stroke and many other serious medical emergencies. Meanwhile, methods to maintain and terminate hibernation remain to be determined despite increased research on molecules involved in the maintenance phase of hibernation. At the genetic level, hibernation appears to be largely associated with the regulation of gene expression. The fact that most hibernation genes are also present in the human genome may allow us to explore ways to induce hibernation/torpor using current molecular technologies such as the CRISPR-Cas system in the future. Author biography Mingke is studying a BSc(Hons) in Biology with One Year in Industry at Imperial College London. She did an industrial placement at GlaxoSmithKline and worked on a hybridomas optimisation project. During her studies and placement, she was particularly attracted to research studies on natural adaptations in animals and how they could be utilised in future medicine. Meanwhile, she is equally interested developing biotechnologies that could make a difference to society. Mingke hopes to further pursue a PhD degree in relevant fields or start a biotechnology company in the near future. References Abe , K. and Kimura , H. 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Email: n.franks@imperial.ac.uk Biography: Mingke is studying a BSc(Hons) in Biology with One Year in Industry at Imperial College London. She did an industrial placement at GlaxoSmithKline and worked on a hybridomas optimisation project. During her studies and placement, she was particularly attracted to research studies on natural adaptations in animals and how they could be utilised in future medicine. Meanwhile, she is equally interested developing biotechnologies that could make a difference to society. Mingke hopes to further pursue a PhD degree in relevant fields or start a biotechnology company in the near future. © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BioScience Horizons Oxford University Press

Hibernation induction in non-hibernating species

BioScience Horizons , Volume 11 – Jan 1, 2018

Hibernation induction in non-hibernating species

BioScience Horizons , Volume 11 – Jan 1, 2018

Abstract

Abstract Hibernation is a natural process in many mammals to maximise survival during extreme environmental conditions. It is characterised by profound physiological changes such as reductions in metabolic rates and core body temperature for prolonged periods of time of up to 9 months. The ability of hibernating animals to recover from hibernation with no obvious signs of organ injury makes them excellent models for the clinical application of this natural protective mechanism, which has generated great interest in medical research. Recent studies have shed more light on the genetic and molecular basis of hibernation and several chemical compounds have been demonstrated to play significant roles in the regulation of hibernation, with an increasing focus on investigating their potential clinical applications in organ preservation and even development of cryonics techniques. In this review, I will present recent progress made in understanding the mechanism of hibernation and highlight evidence for hibernation induction in non-hibernating mammals using chemical compounds; finally, I will discuss the potential for combining genetic techniques to induce metabolic suppression for therapeutic purposes. induction of hibernation, regulation and physiological change, hydrogen sulphide, 5′-AMP, DADLE, genetic basis Introduction Hibernation is an adaptation mainly found in mammals and birds that occurs at physiological, behavioural and molecular levels to sustain life during winter months when resources are insufficient or unpredictable. It is generally associated with a profound reduction in core body temperature to the level of ambient temperature and a reduction in metabolic rate to a fraction of basal metabolic rate (BMR), paralleled with bradypnea and reduced heart rate (Heldmaier, Ortmann and Elvert, 2004). Similar to hibernation, torpor is a survival tactic involving a reduction in body temperature and metabolic rate. The physiological definitions of torpor and hibernation remain unclear but they are used in the scientific literature to express different durations and amplitudes of hypometabolic state. Hibernation generally has a longer duration of up to 9 months whereas torpor lasts <24 h and is commonly referred to as daily torpor. The mean minimum metabolic rate was observed to reach 6% of BMR during hibernation but 35% of BMR during torpor (Ruf and Geiser, 2015). By entering hibernation, hibernators can produce a remarkable saving in energy consumption. In winter, yellow-bellied marmots are able to save up to 85% of their energy, which will greatly enhance winter survival (Armitage, Blumstein and Woods, 2003). Human hibernation, if possible, would have significant clinical applications in three major areas. The induction of a hypometabolic state could better treat critical illness because organ damage could be reduced by inducing hyperthermia in patients who suffer from hypoxia-induced injury such as brain injury and intestinal I/R injury (Aslami and Juffermans, 2010). Another aspect is related to cryonics—a technology that involves lowering the body temperature of patients who cannot be saved with current technologies and preserving them until future technologies can revive and restore them to full health. Cryonics is thought to be an unrealistic practice by most people as simply inducing hypothermia in the human body would lead to ischaemia and reperfusion injury, which result in profound damage in brain and tissues (Best, 2012). Despite being a currently unattainable idea, induction of hibernation in astronauts could significantly reduce resource requirements and associated medical challenges during interstellar space travel. As a Phase I study, NASA placed patients in a mild hypometabolic state for up 14 days with no sign of detrimental effects, suggesting the safety of therapeutic hypothermia in humans (Bradford, Schaffer and Talk, 2014). Hibernation and torpor, processes that occur naturally in many mammals, might offer better alternatives to achieve the aim of long-term preservation of patients and astronauts that currently only exists in science fiction. In this article, I will: (i) provide background information on hibernation including a brief introduction of regulatory mechanisms and detailed physiological changes associated with entrance and arousal from hibernation; (ii) review recent evidence for mechanisms of organ preservation and hibernation induction in non-hibernating mammals using chemical compounds and (iii) discuss the potential to genetically translate the hibernation phenotype to humans with the aim of inducing metabolic suppression or suspended animation for therapeutic purposes, and highlight directions for future research on inducing hibernation in humans. Regulation and physiological changes in hibernation It is thought that hibernation is controlled both by the central nervous system (CNS) and endocrine system. Previous studies identified regions in the brain that are involved in entrance to and arousal from hibernation. Entrance to hibernation involves: (1) activation of hypothalamic regions, and (2) inhibition of cortical regions. In contrast, the hypothalamic nuclei, in particular, are important in arousal from hibernation (Kilduff et al., 1990). 3-Iodothyronamine (T1AM) is an endogenous thyroid that binds to Trace amine-associated receptor 1 (TAAR1), and injection of T1AM into mice effectively reduces cardiac output and heart rate (Chiellini et al., 2007). More generally, thyroid hormones seem to be key modulators in lipid and carbohydrate metabolism and they can regulate basal metabolism at different stages of the life cycle. It was found that plasma total triiodothyronine (tT3) was negatively correlated with its activity prior to hibernation and the level of thyroxine (tT4) increased shortly after right after hibernation termination in female arctic ground squirrels (Wilsterman et al., 2015). There are a number of remarkable physiological changes associated with hibernation. During hibernation or torpor, animals enter a hypometabolic and hypothermic state. The metabolic rate of the Alpine marmot reaches 1/25 of its euthermic BMR and its body temperature drops to 1.5–3°C above ambient temperature (Heldmaier, Ortmann and Elvert, 2004). At the same time, the heart rate is also reduced to a critical level and the inadequate amount of oxygen could lead to hypoxia in non-hibernating animals. Hibernators, however, are able to survive a long period of time without signs of organ damage and there is a generally increased resistance to hypoxia and reperfusion in hibernating animals (Sandovici et al., 2004; Talaei et al., 2011). On a cellular level, programmed cell death (apoptosis) could be triggered by physiological changes associated with entry to and exit from a state of metabolic depression. These include changes in blood flow, oxidative stress and nutrient deprivation. Natural hibernators are able to suppress the apoptotic response as suggested by the constant level of p-p53 S46 in the brain and heart of thirteen-lined ground squirrels during torpor (Rouble et al., 2013). Suppression of apoptosis is done via various mechanisms and one of the ways is to regulate the level of anti-apoptotic proteins. In the same study, an elevation in levels of anti-apoptotic targets (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) was observed in the brain. In addition, a sudden increase in oxygen uptake during arousal from hibernation could result in rapid generation of reactive oxidative species (ROS), which would result in oxidative stress (Storey, 2010). Oxidative stress is a key deleterious factor in brain ischaemia as it could contribute to apoptosis through a number of pathways. However, it has been found that there is a correlation in time between the most rapid fall of ascorbate and peak of oxygen consumption in Artic ground squirrels during arousal from hibernation (Tøien et al., 2001). This suggests that ascorbate may function as an anti-oxidation agent that removes the increased amount of ROS during arousal. Similarly, glutathione peroxidase-3 is an enzyme responsible for hydrogen peroxide (H2O2) detoxification in plasma. Its gene expression level increased 8-fold in the brain of hibernating horseshoe bat compared to non-hibernating animals (Lei et al., 2014). Meanwhile, hibernators will enter an immunosuppressed state during hibernation to prevent general inflammation in the body. It was observed that the number of leucocytes and thrombocytes in blood of European ground squirrels decreased by 90% within 24 h of torpor (Tøien et al., 2001; Bouma et al., 2010). The drop in leucocytes in Syrian hamster and Djungarian hamster was found to be mediated by the level of sphingosine-1-phosphate (S1P) in a temperature-dependent manner (Bouma et al., 2011). When hibernators enter deep torpor, the body temperature drops and so does the S1P level; when animals are aroused from torpor, S1P is restored to the pre-torpor level (Nelson, Otis and Carey, 2010). Apart from suppression of apoptosis, oxidative stress and inflammatory response, hibernators also have to avoid blood clotting as reduced blood flow could lead to deep vein thrombosis, stroke or pulmonary embolism. During torpor, clotting factor FVIII and FIX in 13-lined ground squirrels showed reduced activities by 5-fold and 2-fold, respectively, compared with levels of non-hibernating species (Lechler and Penick, 1963). Von Willebrand factor (VWF), a glycoprotein required for the formation of platelet clot, was observed to be reduced both quantitatively and qualitatively compared to non-hibernating animals (Cooper et al., 2016). Recently some chemical compounds have been suggested to induce hibernation or torpor state in non-hibernating animals, with functions of anti-apoptosis, anti-oxidation, anti-inflammation or anti-coagulation. Now I will discuss evidence regarding the mechanisms of these candidate compounds and point out their limitations in the research on hibernation induction. Hydrogen sulphide Hydrogen sulphide (H2S) is endogenously produced in mammalian tissues and functions as a neuromodulator in the brain (Abe and Kimura, 1996). It is generally known as an inhibitor of oxidative phosphorylation complex IV in mitochondria, via reaction with the copper centre, leading to decreased cellular oxygen consumption and metabolic rate (Szabó, 2007). It is suggested to have an anti-apoptotic, anti-oxidative and anti-inflammatory effect. H2S inhibits apoptosis through activation of the reperfusion injury salvage kinase (RISK) pathway, which is a signalling cascade that involves pro-survival protein kinase (Lambert et al., 2014). Although the specific downstream effects of the RISK pathway have not yet been fully elucidated, the extracellular regulated kinase 1/2 (Erk1/2) arm of the RISK pathway has been shown to activate p90sk, thus phosphorylating and inhibiting Bcl-2-associated death promoter (BAD) protein, which initiates apoptosis (Meng et al., 2013). H2S inhibits oxidation via activation of nuclear factor 2 (Nrf2), which is a regulator of the anti-oxidation response. Keap1 is a negative regulator of Nrf2, and NaHS induces disassociation of Nrf2 from Keap1, enhancing the transcription of genes of detoxifying enzymes that Nrf2 targets, such as glutathione (GSH) reductase and glutamate-cysteine ligase (Yang et al., 2013). H2S is also an anti-inflammatory molecule. Inhibition of the inflammatory response is achieved by inhibition of leucocyte rolling and adhesion, which is further done by triggering the activation of NO synthase and the p38 MAPK-dependent mechanism (Yusof et al., 2009). A summary of pathways in which H2S participates is illustrated in Fig. 1. Meanwhile, hydrogen sulphide was experimentally shown to induce suspended animation by Blackstone et al. in 2005, with the body temperature of house mice falling to approximately 2°C above ambient temperature after exposure to 80 ppm H2S and returning to normal temperature after 6 h of treatment (Blackstone, Morrison and Roth, 2005). Volpato et al. (2008) repeated the experiment in mice and similar results were obtained. Figure 1. Open in new tabDownload slide Pathways of H2S-induced anti-apoptotic, anti-oxidative and anti-inflammatory response. Diagram produced based on Sen, Paul and Gadalla et al. (2012),,Meng et al. (2013), Yang et al. (2013), Lambert et al. (2014) and Yusof et al. (2009). In larger mammals, the effect of H2S is less consistent. Li et al. (2008) failed to induce a suspended animation-like state in anaesthetised piglets with different levels of H2S. This might be due to a dose-response issue as the highest volume of H2S applied was 80 ppm, which might not be sufficient for effective hibernation induction in large mammals. A high concentration of H2S, however, has been reported to be toxic. As described in an earlier study, 500–1000 ppm H2S caused liver damage in humans (Yalamanchili and Smith, 2008). Although studies regarding the ability of H2S to induce a torpor/hibernation-like state in larger mammals appear to be conflicting, it is still a promising candidate and elucidating the mechanisms by which H2S acts is necessary to assess the viability of hibernation induction in humans. 5′-Adenosine monophosphate 5′-Adenosine monophosphate (5′-AMP) is a nucleotide produced by hydrolysis of ATP. Zhang et al. (2006) found that a significant dosage of 5′-AMP induces a reversible deep hypometabolic state in non-hibernating mice, in which the metabolic rate was observed to fall to <10% of the euthermic level. By using comparative analysis, a wide suppression of energy generating metabolic pathways was observed in 5′-AMP-induced hypothermia (AIHM; Zhao et al., 2014). In the same study, it was suggested that 5′-AMP has negative effects on haemoglobin’s affinity for oxygen, which further leads to hypometabolism. 5′-AMP also causes glycolysis suppression and results in an increased level of blood 2,3- bisphosphoglycerate (2,3-BPG), an enzyme that binds with greater affinity to deoxygenated haemoglobin, and thus inhibits binding of oxygen to red blood cells. Currently, there are two hypotheses regarding the molecular pathways of AIHM, as shown in Fig. 2. Figure 2. Open in new tabDownload slide Adenosine receptor-mediated and AMPK-mediated pathways to induce AIMP. Diagram produced based on Melvin and Andrews (2009), Iliff and Swoap (2012), Linden and Cekic (2012) and Takedachi et al. (2008). The adenosine receptor-mediated pathway, however, is less convincing. Studies on adenosine receptor-deficient mice demonstrated that mice with deficiency in adenosine receptors showed an ability to enter deep hypometabolism at a similar rate to wild type mice (Daniels et al., 2010). On the other hand, a more recent study suggested that activation of adenosine A1 receptors (A1ARs) in the CNS is essential in induction of hibernation in hibernating species (Jinka, Toien and Drew, 2011). A1ARs actually induced a hypothermic, hibernation-like state in mice, in which a remarkable fall in body temperature was observed, along with a reduction in electroencephalogram amplitude and heart rate (Tupone, Madden and Morrison, 2013). Similar to hydrogen sulphide, 5′-AMP also exhibits anti-inflammatory ability. More specifically, 5′-AMP activates the ERK1/2, p38, JNK and NF-κB signalling pathways, which attenuate LPS-induced inflammation in Wistar rats (Wang et al., 2014). At the same time, the numbers of T- and B-lymphocytes that circulate in the blood were reduced following injection of 5′-AMP into congenic mice (Bouma et al., 2013). The capability of 5′-AMP to induce a significant, safe and reversible hypometabolism was proposed in a previous review (Lee, 2008). Further investigation is needed on molecular mechanisms of AIHM and its clinical potential. DADLE [d-Ala2, d-Leu5]-enkephalin (DADLE) is a synthetic peptide that binds and activates the δ-opioid receptor (DOR). In 2015, a study on rat intestinal epithelial cells demonstrated that DADLE has a protective effect in ischaemia/reperfusion (I/R) injury, via inhibition of the protein kinase kinase 7 (MKK7)-c-Jun N-terminal kinase (JNK) pathway (Wang et al., 2015). JNK pathways are closely associated with I/R injury and MKK7 is an intermediate of JNK involved in activation of JNK (Haeusgen, Herdegen and Waetzig, 2011). Similarly, DADLE has an important role in preventing necrotic and apoptotic processes resulting from I/R injury by inhibiting early apoptotic events, such as serum and glucose deprivation (DEPV)-induced mitochondrial membrane damage (Yao et al., 2007). In the same study, it was shown that the production of reactive oxygen species (ROS) was negatively correlated with the level of DADLE, suggesting that DADLE could prevent ROS-induced apoptosis under the condition of DEPV. DADLE also has neuroprotective effects in both the central and peripheral nervous system (Borlongan, Wang and Su, 2004). It was demonstrated that DADLE protected the terminal membranes of dopaminergic neurons from methamphetamine (METH)-induced neurotoxicity in mice (Tsao et al., 1998). Meanwhile, more evidence of the underlying molecular mechanism in which DADLE works has started to emerge. It was proposed that DADLE is able to reduce the rate of transcription in opioid receptor-deficient LNCaP epithelial cells (Baldelli et al., 2006). The same study also suggested that the inhibitory action of DADLE is independent of opioid receptors, which sheds more light on an alternative mechanism for the compound. In some cases, DADLE is considered to be pharmacodynamically homologous to hibernation induction trigger (HIT), a molecule present in the blood that is capable of inducing hibernation in non-hibernating squirrels by blood transfusion (Dawe and Spurrier, 1969). Similarly to DADLE, HIT has a profound function in organ preservation, as suggested by a study on mongrel dogs (Chien et al., 1991). The preservation of multiple organs prolonged the survival time by up to 48 h and lung function was maintained after transplantation to host animals. Even though the detailed association between HIT and DADLE remains unclear, we must not neglect the role of opioids in torpor and hibernation. The DOR, in particular, is associated with neuronal tolerance to hypoxic/ischaemia stress and anti-oxidative ability. Also, DOR signal is able to stabilise homoeostasis and to increase pro-survival signalling such as protein kinase C, which is involved in cardioprotection (Feng et al., 2012). Despite the significance of DADLE in protecting against damaging effects from I/R injury, there is currently no experimental evidence that DADLE is able to induce torpor or a hibernation-like state in non-hibernating animals. However, a recent study on human neuroblastoma SH-SY5Y cells and primary cortical neurons showed that DADLE is able to reduce the cellular transcription rate to a significant level without causing cell damage or injury (Tian et al., 2014), suggesting that DADLE could be a promising candidate as a therapeutic drug for neuroprotection. Maintenance of hibernation When inducing hibernation, it is also critical to consider ways to maintain and terminate the hibernation state and there are several compounds that play important roles in maintenance and arousal from hibernation. In a study on ground squirrels, mu and kappa opioids were suggested to affect the arousal state of hibernation (Oeltgen et al., 1988). Opioid peptides are involved in thermal regulation during the maintenance phase of hibernation. The injection of two opioid receptor antagonists, naloxone and naloxonazine, effectively elevated body temperature and terminated hibernation in Syrian hamster (Tamura et al., 2005). Glutamate is an amino acid that serves as a metabolic fuel and thus regulates metabolic rate. It acts via NMDA-type glutamate receptors (NMDAR) and the activation of NMDAR was observed to maintain the torpor state outside the CNS of arctic ground squirrel (Jinka, Rasley and Drew, 2012). Meanwhile, it has been found that the interaction between serotonergic and noradrenergic systems (NA) maintains the state of hibernation and that the ratio of these two systems determines entrance or arousal from hibernation in ground squirrels (Semenova and Zakharova, 2015). A higher concentration of serotonin was observed in ground squirrel in autumn prior to hibernation and the NA system is more related to behaviours in spring. In most natural hibernators, the food they consume during summer is converted into white adipose tissue for subsequent consumption in winter (Boyer and Barnes, 1999). If we intend to induce hibernation in humans or other non-hibernating animals, it is necessary to consider the energy resource required for a long period with no intake of food. Consider a healthy adult man aged 50 years old, whose height is 170 cm and weight is 70 kg. Based on the Mifflin St Jeor Equation (Mifflin et al., 1990), which estimates BMR: P=(10m1.0kg+6.25h1.0cm−5.0a1.0year+5)kcalday where P is BMR, m is mass, h is height and a is age, the BMR would be 1517 kcal/day. On average, hibernators are able to reduce energy requirements by up to 90% (Heldmaier, Ortmann and Elvert, 2004), and thus the energy needed during hibernation would be 1517 × 10% = 151.7 kcal/day. It is possible to supply energy using lipid, which has an energy content of 9 kcal/g, and glucose, which has an energy content of 3.75 kcal/g (Food and Agriculture Organization of the United Nations, 2003). To provide sufficient amounts of energy for a man, 16.9 g (151.7/9) of lipid and 40.4 g (151.7/3.75) of glucose would be needed on a daily basis. During natural hibernation, there seems to be a switch from glucose metabolism to lipid metabolism as genes involved in glycolysis are under-expressed while genes for fatty acid catabolism are over-expressed (Yan et al., 2008). It would be more sensible to supply lipid by means of lipid emulsion during hibernation of humans. Genetic and epigenetic basis of hibernation The genes associated with hibernation seem to be present in the genomes of most mammals, including humans (Srere and Martin, 1992). For instance, mitochondrial uncoupling proteins (UCPs) are responsible for non-shivering heat generation in brown adipocyte tissue during hibernation. Its homologous protein, UCP2, was detected in a greatly increased amount in multiple tissues in hibernating ground squirrels and it was found to be present in the human genome (Fleury et al., 1997; Boyer et al., 1998). More examples of genes that function in hibernation and have highly homologous genes in humans are listed in Table 1. Table 1. Comparison of genes associated with hibernation in thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and homologous genes in human Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Open in new tab Table 1. Comparison of genes associated with hibernation in thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and homologous genes in human Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Open in new tab Meanwhile, the evolution of hibernation has been suggested to occur at a molecular level with changes in patterns of gene expression rather than evolution of de novo genes. A model of gene regulation in hibernation has been proposed, as shown in Fig. 3. Figure 3. Open in new tabDownload slide Schematic representation of the central role of differential gene expression during hibernation. Single arrows indicate direct effects, and multiple arrows indicate an effect that requires multiple intermediate events. Reprinted from Srere, H. K. & Martin, S. L. (1992) Central role for differential gene expression in mammalian hibernation. Proceedings of the National Academy of Sciences of the United States of America. 89 (15), 7119–7123. In concert with the hypothesis that most physiological changes are associated with gene regulation, it has been found that gene regulation of 5′-AMP-induced hypometabolism occurs at a post-translational level. In the study by Zhang et al. (2011), >99.9% of mRNA analysed did not significantly change in level in the liver of mice. Global depression of transcription has been observed in golden-mantled ground squirrels during hibernation, which might be due to the combined effects of mild suppression of initiation and severe inhibition of elongation during transcription (Van Breukelen and Martin, 2002). Epigenetics refers to changes in gene expression that are not the result of alteration in DNA sequence. It also plays a major role in transcriptional and translational suppression in hibernation, which includes histone tail modification and DNA methylation. Morin and Storey (2006) found that the relative content of acetylation and phosphorylation on H3 fell by 25% and 40%, respectively, and that the amount of histone deacetylase protein rose during hibernation in skeletal muscle of thirteen-lined ground squirrels, which led to elevated chromatin packing and transcriptional silencing. A similar result was obtained in another study on brown adipose tissue from ground squirrels (Biggar and Storey, 2014). Changes in levels of methylation of DNA occurred in response to environmental stress, as demonstrated in a recent study on mouse neuronal cells (Hartley et al., 2013). When neurons in hippocampuswere exposed to hypoxia, both hyper- and hypo-methylation on CpG islands or promoter regions of different genes were observed to produce transcriptional modulation. Similarly, DNA methylation contributes to the transition into the hibernation phenotype in the liver and skeletal muscle of thirteen-lined ground squirrels (Alvarado et al., 2015). They successfully identified a gene, myocyte enhancer factor 2C (mef2c), in skeletal muscle whose promoter is methylated in response to hibernation initiation. Given that most of the genes associated with hibernation are present in the human genome, it could be possible to temporarily induce hibernation using molecular biology techniques. We should be immediately aware that modification should not be done at the genome or germ-line level because of genetic inheritance and ethical concerns, implying that most of the changes would be post-transcriptional modifications. Therefore, we can (i) deliver genes that are upregulated in hibernation into humans using viral or non-viral vectors to increase gene expression; (ii) exploit the mechanism of RNAi and deliver small RNA molecules to perform gene silencing and achieve downregulation at the transcriptional level and (iii) modify histone or DNA methylation to up- or downregulate gene expression. Genes that are upregulated in hibernation can be delivered into humans using vectors. However, this is less practical when the number of genes is enormous. It has been reported that 225 genes are upregulated in response to hypoxia exposure (Hartley et al., 2013), which will make the design and delivery of genes a tremendous amount of work. DNA pieces of the genes that we would like to downregulate could be introduced into non-hibernating animals. Introduced DNA will yield dsRNA or short hairpin RNA (shRNA) after transcription, which will be targeted by the host RNA interference system (RNAi) to produce short interfering RNA (siRNA). The RNAi system will cleave host mRNA that is complementary to siRNA and silence the expression of the gene. However, siRNA would lead to miRNA-like off-target effects as first recognised in 2003 (Aimee et al., 2003). This is because siRNA can target mRNA sequences whose 3′UTRs are complementary to the 5′UTR of siRNA. Synthetic siRNA has been reported to stimulate the production of interferons and inflammatory cytokines both in vivo and in vitro (Adam et al., 2005). Therefore the immunogenic effect of siRNA remains under investigation. Conclusion and future directions Hibernation is a strategy adopted by animals to conserve energy during periods with insufficient resources. Some studies have successfully induced torpor or hibernation-like state in non-hibernating animals using chemical compounds such as H2S and 5′-AMP, but there has been limited success in larger mammals. More research on hibernation induction needs to be done on primates, which are good models for human medicine owning to their genetic and physiological similarities. Nevertheless, these molecules have great potential in clinical applications such as trauma, heart attack, stroke and many other serious medical emergencies. Meanwhile, methods to maintain and terminate hibernation remain to be determined despite increased research on molecules involved in the maintenance phase of hibernation. At the genetic level, hibernation appears to be largely associated with the regulation of gene expression. The fact that most hibernation genes are also present in the human genome may allow us to explore ways to induce hibernation/torpor using current molecular technologies such as the CRISPR-Cas system in the future. Author biography Mingke is studying a BSc(Hons) in Biology with One Year in Industry at Imperial College London. She did an industrial placement at GlaxoSmithKline and worked on a hybridomas optimisation project. During her studies and placement, she was particularly attracted to research studies on natural adaptations in animals and how they could be utilised in future medicine. Meanwhile, she is equally interested developing biotechnologies that could make a difference to society. Mingke hopes to further pursue a PhD degree in relevant fields or start a biotechnology company in the near future. References Abe , K. and Kimura , H. 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Email: n.franks@imperial.ac.uk Biography: Mingke is studying a BSc(Hons) in Biology with One Year in Industry at Imperial College London. She did an industrial placement at GlaxoSmithKline and worked on a hybridomas optimisation project. During her studies and placement, she was particularly attracted to research studies on natural adaptations in animals and how they could be utilised in future medicine. Meanwhile, she is equally interested developing biotechnologies that could make a difference to society. Mingke hopes to further pursue a PhD degree in relevant fields or start a biotechnology company in the near future. © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press.

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Abstract

Abstract Hibernation is a natural process in many mammals to maximise survival during extreme environmental conditions. It is characterised by profound physiological changes such as reductions in metabolic rates and core body temperature for prolonged periods of time of up to 9 months. The ability of hibernating animals to recover from hibernation with no obvious signs of organ injury makes them excellent models for the clinical application of this natural protective mechanism, which has generated great interest in medical research. Recent studies have shed more light on the genetic and molecular basis of hibernation and several chemical compounds have been demonstrated to play significant roles in the regulation of hibernation, with an increasing focus on investigating their potential clinical applications in organ preservation and even development of cryonics techniques. In this review, I will present recent progress made in understanding the mechanism of hibernation and highlight evidence for hibernation induction in non-hibernating mammals using chemical compounds; finally, I will discuss the potential for combining genetic techniques to induce metabolic suppression for therapeutic purposes. induction of hibernation, regulation and physiological change, hydrogen sulphide, 5′-AMP, DADLE, genetic basis Introduction Hibernation is an adaptation mainly found in mammals and birds that occurs at physiological, behavioural and molecular levels to sustain life during winter months when resources are insufficient or unpredictable. It is generally associated with a profound reduction in core body temperature to the level of ambient temperature and a reduction in metabolic rate to a fraction of basal metabolic rate (BMR), paralleled with bradypnea and reduced heart rate (Heldmaier, Ortmann and Elvert, 2004). Similar to hibernation, torpor is a survival tactic involving a reduction in body temperature and metabolic rate. The physiological definitions of torpor and hibernation remain unclear but they are used in the scientific literature to express different durations and amplitudes of hypometabolic state. Hibernation generally has a longer duration of up to 9 months whereas torpor lasts <24 h and is commonly referred to as daily torpor. The mean minimum metabolic rate was observed to reach 6% of BMR during hibernation but 35% of BMR during torpor (Ruf and Geiser, 2015). By entering hibernation, hibernators can produce a remarkable saving in energy consumption. In winter, yellow-bellied marmots are able to save up to 85% of their energy, which will greatly enhance winter survival (Armitage, Blumstein and Woods, 2003). Human hibernation, if possible, would have significant clinical applications in three major areas. The induction of a hypometabolic state could better treat critical illness because organ damage could be reduced by inducing hyperthermia in patients who suffer from hypoxia-induced injury such as brain injury and intestinal I/R injury (Aslami and Juffermans, 2010). Another aspect is related to cryonics—a technology that involves lowering the body temperature of patients who cannot be saved with current technologies and preserving them until future technologies can revive and restore them to full health. Cryonics is thought to be an unrealistic practice by most people as simply inducing hypothermia in the human body would lead to ischaemia and reperfusion injury, which result in profound damage in brain and tissues (Best, 2012). Despite being a currently unattainable idea, induction of hibernation in astronauts could significantly reduce resource requirements and associated medical challenges during interstellar space travel. As a Phase I study, NASA placed patients in a mild hypometabolic state for up 14 days with no sign of detrimental effects, suggesting the safety of therapeutic hypothermia in humans (Bradford, Schaffer and Talk, 2014). Hibernation and torpor, processes that occur naturally in many mammals, might offer better alternatives to achieve the aim of long-term preservation of patients and astronauts that currently only exists in science fiction. In this article, I will: (i) provide background information on hibernation including a brief introduction of regulatory mechanisms and detailed physiological changes associated with entrance and arousal from hibernation; (ii) review recent evidence for mechanisms of organ preservation and hibernation induction in non-hibernating mammals using chemical compounds and (iii) discuss the potential to genetically translate the hibernation phenotype to humans with the aim of inducing metabolic suppression or suspended animation for therapeutic purposes, and highlight directions for future research on inducing hibernation in humans. Regulation and physiological changes in hibernation It is thought that hibernation is controlled both by the central nervous system (CNS) and endocrine system. Previous studies identified regions in the brain that are involved in entrance to and arousal from hibernation. Entrance to hibernation involves: (1) activation of hypothalamic regions, and (2) inhibition of cortical regions. In contrast, the hypothalamic nuclei, in particular, are important in arousal from hibernation (Kilduff et al., 1990). 3-Iodothyronamine (T1AM) is an endogenous thyroid that binds to Trace amine-associated receptor 1 (TAAR1), and injection of T1AM into mice effectively reduces cardiac output and heart rate (Chiellini et al., 2007). More generally, thyroid hormones seem to be key modulators in lipid and carbohydrate metabolism and they can regulate basal metabolism at different stages of the life cycle. It was found that plasma total triiodothyronine (tT3) was negatively correlated with its activity prior to hibernation and the level of thyroxine (tT4) increased shortly after right after hibernation termination in female arctic ground squirrels (Wilsterman et al., 2015). There are a number of remarkable physiological changes associated with hibernation. During hibernation or torpor, animals enter a hypometabolic and hypothermic state. The metabolic rate of the Alpine marmot reaches 1/25 of its euthermic BMR and its body temperature drops to 1.5–3°C above ambient temperature (Heldmaier, Ortmann and Elvert, 2004). At the same time, the heart rate is also reduced to a critical level and the inadequate amount of oxygen could lead to hypoxia in non-hibernating animals. Hibernators, however, are able to survive a long period of time without signs of organ damage and there is a generally increased resistance to hypoxia and reperfusion in hibernating animals (Sandovici et al., 2004; Talaei et al., 2011). On a cellular level, programmed cell death (apoptosis) could be triggered by physiological changes associated with entry to and exit from a state of metabolic depression. These include changes in blood flow, oxidative stress and nutrient deprivation. Natural hibernators are able to suppress the apoptotic response as suggested by the constant level of p-p53 S46 in the brain and heart of thirteen-lined ground squirrels during torpor (Rouble et al., 2013). Suppression of apoptosis is done via various mechanisms and one of the ways is to regulate the level of anti-apoptotic proteins. In the same study, an elevation in levels of anti-apoptotic targets (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) was observed in the brain. In addition, a sudden increase in oxygen uptake during arousal from hibernation could result in rapid generation of reactive oxidative species (ROS), which would result in oxidative stress (Storey, 2010). Oxidative stress is a key deleterious factor in brain ischaemia as it could contribute to apoptosis through a number of pathways. However, it has been found that there is a correlation in time between the most rapid fall of ascorbate and peak of oxygen consumption in Artic ground squirrels during arousal from hibernation (Tøien et al., 2001). This suggests that ascorbate may function as an anti-oxidation agent that removes the increased amount of ROS during arousal. Similarly, glutathione peroxidase-3 is an enzyme responsible for hydrogen peroxide (H2O2) detoxification in plasma. Its gene expression level increased 8-fold in the brain of hibernating horseshoe bat compared to non-hibernating animals (Lei et al., 2014). Meanwhile, hibernators will enter an immunosuppressed state during hibernation to prevent general inflammation in the body. It was observed that the number of leucocytes and thrombocytes in blood of European ground squirrels decreased by 90% within 24 h of torpor (Tøien et al., 2001; Bouma et al., 2010). The drop in leucocytes in Syrian hamster and Djungarian hamster was found to be mediated by the level of sphingosine-1-phosphate (S1P) in a temperature-dependent manner (Bouma et al., 2011). When hibernators enter deep torpor, the body temperature drops and so does the S1P level; when animals are aroused from torpor, S1P is restored to the pre-torpor level (Nelson, Otis and Carey, 2010). Apart from suppression of apoptosis, oxidative stress and inflammatory response, hibernators also have to avoid blood clotting as reduced blood flow could lead to deep vein thrombosis, stroke or pulmonary embolism. During torpor, clotting factor FVIII and FIX in 13-lined ground squirrels showed reduced activities by 5-fold and 2-fold, respectively, compared with levels of non-hibernating species (Lechler and Penick, 1963). Von Willebrand factor (VWF), a glycoprotein required for the formation of platelet clot, was observed to be reduced both quantitatively and qualitatively compared to non-hibernating animals (Cooper et al., 2016). Recently some chemical compounds have been suggested to induce hibernation or torpor state in non-hibernating animals, with functions of anti-apoptosis, anti-oxidation, anti-inflammation or anti-coagulation. Now I will discuss evidence regarding the mechanisms of these candidate compounds and point out their limitations in the research on hibernation induction. Hydrogen sulphide Hydrogen sulphide (H2S) is endogenously produced in mammalian tissues and functions as a neuromodulator in the brain (Abe and Kimura, 1996). It is generally known as an inhibitor of oxidative phosphorylation complex IV in mitochondria, via reaction with the copper centre, leading to decreased cellular oxygen consumption and metabolic rate (Szabó, 2007). It is suggested to have an anti-apoptotic, anti-oxidative and anti-inflammatory effect. H2S inhibits apoptosis through activation of the reperfusion injury salvage kinase (RISK) pathway, which is a signalling cascade that involves pro-survival protein kinase (Lambert et al., 2014). Although the specific downstream effects of the RISK pathway have not yet been fully elucidated, the extracellular regulated kinase 1/2 (Erk1/2) arm of the RISK pathway has been shown to activate p90sk, thus phosphorylating and inhibiting Bcl-2-associated death promoter (BAD) protein, which initiates apoptosis (Meng et al., 2013). H2S inhibits oxidation via activation of nuclear factor 2 (Nrf2), which is a regulator of the anti-oxidation response. Keap1 is a negative regulator of Nrf2, and NaHS induces disassociation of Nrf2 from Keap1, enhancing the transcription of genes of detoxifying enzymes that Nrf2 targets, such as glutathione (GSH) reductase and glutamate-cysteine ligase (Yang et al., 2013). H2S is also an anti-inflammatory molecule. Inhibition of the inflammatory response is achieved by inhibition of leucocyte rolling and adhesion, which is further done by triggering the activation of NO synthase and the p38 MAPK-dependent mechanism (Yusof et al., 2009). A summary of pathways in which H2S participates is illustrated in Fig. 1. Meanwhile, hydrogen sulphide was experimentally shown to induce suspended animation by Blackstone et al. in 2005, with the body temperature of house mice falling to approximately 2°C above ambient temperature after exposure to 80 ppm H2S and returning to normal temperature after 6 h of treatment (Blackstone, Morrison and Roth, 2005). Volpato et al. (2008) repeated the experiment in mice and similar results were obtained. Figure 1. Open in new tabDownload slide Pathways of H2S-induced anti-apoptotic, anti-oxidative and anti-inflammatory response. Diagram produced based on Sen, Paul and Gadalla et al. (2012),,Meng et al. (2013), Yang et al. (2013), Lambert et al. (2014) and Yusof et al. (2009). In larger mammals, the effect of H2S is less consistent. Li et al. (2008) failed to induce a suspended animation-like state in anaesthetised piglets with different levels of H2S. This might be due to a dose-response issue as the highest volume of H2S applied was 80 ppm, which might not be sufficient for effective hibernation induction in large mammals. A high concentration of H2S, however, has been reported to be toxic. As described in an earlier study, 500–1000 ppm H2S caused liver damage in humans (Yalamanchili and Smith, 2008). Although studies regarding the ability of H2S to induce a torpor/hibernation-like state in larger mammals appear to be conflicting, it is still a promising candidate and elucidating the mechanisms by which H2S acts is necessary to assess the viability of hibernation induction in humans. 5′-Adenosine monophosphate 5′-Adenosine monophosphate (5′-AMP) is a nucleotide produced by hydrolysis of ATP. Zhang et al. (2006) found that a significant dosage of 5′-AMP induces a reversible deep hypometabolic state in non-hibernating mice, in which the metabolic rate was observed to fall to <10% of the euthermic level. By using comparative analysis, a wide suppression of energy generating metabolic pathways was observed in 5′-AMP-induced hypothermia (AIHM; Zhao et al., 2014). In the same study, it was suggested that 5′-AMP has negative effects on haemoglobin’s affinity for oxygen, which further leads to hypometabolism. 5′-AMP also causes glycolysis suppression and results in an increased level of blood 2,3- bisphosphoglycerate (2,3-BPG), an enzyme that binds with greater affinity to deoxygenated haemoglobin, and thus inhibits binding of oxygen to red blood cells. Currently, there are two hypotheses regarding the molecular pathways of AIHM, as shown in Fig. 2. Figure 2. Open in new tabDownload slide Adenosine receptor-mediated and AMPK-mediated pathways to induce AIMP. Diagram produced based on Melvin and Andrews (2009), Iliff and Swoap (2012), Linden and Cekic (2012) and Takedachi et al. (2008). The adenosine receptor-mediated pathway, however, is less convincing. Studies on adenosine receptor-deficient mice demonstrated that mice with deficiency in adenosine receptors showed an ability to enter deep hypometabolism at a similar rate to wild type mice (Daniels et al., 2010). On the other hand, a more recent study suggested that activation of adenosine A1 receptors (A1ARs) in the CNS is essential in induction of hibernation in hibernating species (Jinka, Toien and Drew, 2011). A1ARs actually induced a hypothermic, hibernation-like state in mice, in which a remarkable fall in body temperature was observed, along with a reduction in electroencephalogram amplitude and heart rate (Tupone, Madden and Morrison, 2013). Similar to hydrogen sulphide, 5′-AMP also exhibits anti-inflammatory ability. More specifically, 5′-AMP activates the ERK1/2, p38, JNK and NF-κB signalling pathways, which attenuate LPS-induced inflammation in Wistar rats (Wang et al., 2014). At the same time, the numbers of T- and B-lymphocytes that circulate in the blood were reduced following injection of 5′-AMP into congenic mice (Bouma et al., 2013). The capability of 5′-AMP to induce a significant, safe and reversible hypometabolism was proposed in a previous review (Lee, 2008). Further investigation is needed on molecular mechanisms of AIHM and its clinical potential. DADLE [d-Ala2, d-Leu5]-enkephalin (DADLE) is a synthetic peptide that binds and activates the δ-opioid receptor (DOR). In 2015, a study on rat intestinal epithelial cells demonstrated that DADLE has a protective effect in ischaemia/reperfusion (I/R) injury, via inhibition of the protein kinase kinase 7 (MKK7)-c-Jun N-terminal kinase (JNK) pathway (Wang et al., 2015). JNK pathways are closely associated with I/R injury and MKK7 is an intermediate of JNK involved in activation of JNK (Haeusgen, Herdegen and Waetzig, 2011). Similarly, DADLE has an important role in preventing necrotic and apoptotic processes resulting from I/R injury by inhibiting early apoptotic events, such as serum and glucose deprivation (DEPV)-induced mitochondrial membrane damage (Yao et al., 2007). In the same study, it was shown that the production of reactive oxygen species (ROS) was negatively correlated with the level of DADLE, suggesting that DADLE could prevent ROS-induced apoptosis under the condition of DEPV. DADLE also has neuroprotective effects in both the central and peripheral nervous system (Borlongan, Wang and Su, 2004). It was demonstrated that DADLE protected the terminal membranes of dopaminergic neurons from methamphetamine (METH)-induced neurotoxicity in mice (Tsao et al., 1998). Meanwhile, more evidence of the underlying molecular mechanism in which DADLE works has started to emerge. It was proposed that DADLE is able to reduce the rate of transcription in opioid receptor-deficient LNCaP epithelial cells (Baldelli et al., 2006). The same study also suggested that the inhibitory action of DADLE is independent of opioid receptors, which sheds more light on an alternative mechanism for the compound. In some cases, DADLE is considered to be pharmacodynamically homologous to hibernation induction trigger (HIT), a molecule present in the blood that is capable of inducing hibernation in non-hibernating squirrels by blood transfusion (Dawe and Spurrier, 1969). Similarly to DADLE, HIT has a profound function in organ preservation, as suggested by a study on mongrel dogs (Chien et al., 1991). The preservation of multiple organs prolonged the survival time by up to 48 h and lung function was maintained after transplantation to host animals. Even though the detailed association between HIT and DADLE remains unclear, we must not neglect the role of opioids in torpor and hibernation. The DOR, in particular, is associated with neuronal tolerance to hypoxic/ischaemia stress and anti-oxidative ability. Also, DOR signal is able to stabilise homoeostasis and to increase pro-survival signalling such as protein kinase C, which is involved in cardioprotection (Feng et al., 2012). Despite the significance of DADLE in protecting against damaging effects from I/R injury, there is currently no experimental evidence that DADLE is able to induce torpor or a hibernation-like state in non-hibernating animals. However, a recent study on human neuroblastoma SH-SY5Y cells and primary cortical neurons showed that DADLE is able to reduce the cellular transcription rate to a significant level without causing cell damage or injury (Tian et al., 2014), suggesting that DADLE could be a promising candidate as a therapeutic drug for neuroprotection. Maintenance of hibernation When inducing hibernation, it is also critical to consider ways to maintain and terminate the hibernation state and there are several compounds that play important roles in maintenance and arousal from hibernation. In a study on ground squirrels, mu and kappa opioids were suggested to affect the arousal state of hibernation (Oeltgen et al., 1988). Opioid peptides are involved in thermal regulation during the maintenance phase of hibernation. The injection of two opioid receptor antagonists, naloxone and naloxonazine, effectively elevated body temperature and terminated hibernation in Syrian hamster (Tamura et al., 2005). Glutamate is an amino acid that serves as a metabolic fuel and thus regulates metabolic rate. It acts via NMDA-type glutamate receptors (NMDAR) and the activation of NMDAR was observed to maintain the torpor state outside the CNS of arctic ground squirrel (Jinka, Rasley and Drew, 2012). Meanwhile, it has been found that the interaction between serotonergic and noradrenergic systems (NA) maintains the state of hibernation and that the ratio of these two systems determines entrance or arousal from hibernation in ground squirrels (Semenova and Zakharova, 2015). A higher concentration of serotonin was observed in ground squirrel in autumn prior to hibernation and the NA system is more related to behaviours in spring. In most natural hibernators, the food they consume during summer is converted into white adipose tissue for subsequent consumption in winter (Boyer and Barnes, 1999). If we intend to induce hibernation in humans or other non-hibernating animals, it is necessary to consider the energy resource required for a long period with no intake of food. Consider a healthy adult man aged 50 years old, whose height is 170 cm and weight is 70 kg. Based on the Mifflin St Jeor Equation (Mifflin et al., 1990), which estimates BMR: P=(10m1.0kg+6.25h1.0cm−5.0a1.0year+5)kcalday where P is BMR, m is mass, h is height and a is age, the BMR would be 1517 kcal/day. On average, hibernators are able to reduce energy requirements by up to 90% (Heldmaier, Ortmann and Elvert, 2004), and thus the energy needed during hibernation would be 1517 × 10% = 151.7 kcal/day. It is possible to supply energy using lipid, which has an energy content of 9 kcal/g, and glucose, which has an energy content of 3.75 kcal/g (Food and Agriculture Organization of the United Nations, 2003). To provide sufficient amounts of energy for a man, 16.9 g (151.7/9) of lipid and 40.4 g (151.7/3.75) of glucose would be needed on a daily basis. During natural hibernation, there seems to be a switch from glucose metabolism to lipid metabolism as genes involved in glycolysis are under-expressed while genes for fatty acid catabolism are over-expressed (Yan et al., 2008). It would be more sensible to supply lipid by means of lipid emulsion during hibernation of humans. Genetic and epigenetic basis of hibernation The genes associated with hibernation seem to be present in the genomes of most mammals, including humans (Srere and Martin, 1992). For instance, mitochondrial uncoupling proteins (UCPs) are responsible for non-shivering heat generation in brown adipocyte tissue during hibernation. Its homologous protein, UCP2, was detected in a greatly increased amount in multiple tissues in hibernating ground squirrels and it was found to be present in the human genome (Fleury et al., 1997; Boyer et al., 1998). More examples of genes that function in hibernation and have highly homologous genes in humans are listed in Table 1. Table 1. Comparison of genes associated with hibernation in thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and homologous genes in human Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Open in new tab Table 1. Comparison of genes associated with hibernation in thirteen-lined ground squirrel (Spermophilus tridecemlineatus) and homologous genes in human Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Protein name . Function . Homologous proteins in human . Identity percentage . References . Trace amine-associated receptor 1 (TAAR1) Binds to 3- iodothyronamine to reduce cardiac output and heart rate Homo sapiens trace amine-associated receptor 1 (TAAR1) 86 Chiellini et al. (2007) Hypoxia inducible factor, alpha subunit (HIF-1a) Regulate genes that enhance hypoxia tolerance Homo sapiens hypoxia inducible factor 1 alpha subunit (HIF-1A), RefSeqGene on chromosome 14 78 Höpfl, Ogunshola and Gassmann (2003) Activating transcription factor (ATF4) Enhance expression of chaperones, which provides stability to cellular protein Homo sapiens activating transcription factor 4 (ATF4), transcript variant 1 86 Mamady and Storey (2008) Nuclear factor E2-related factor 2 (Nrf2) Increase expression of anti-oxidant proteins Homo sapiens nuclear factor (erythroid-derived 2)-like 2, mRNA (cDNA clone MGC:20033 IMAGE:4548874), complete cds 87 Morin et al. (2008) Synthetic construct Homo sapiens clone ccsbBroadEn_01090 NFE2L2 gene, encodes complete protein 89 Glutathione peroxidase-3 Detoxification of H2O2 in plasma Homo sapiens glutathione peroxidase-3 (plasma) (GPX3) gene, complete cds 78 Lei et al. (2014) α2-Macroglobulin Prevent blood clots Homo sapiens alpha-2-macroglobulin (A2M), RefSeqGene on chromosome 12 80 Srere and Martin (1992) Peroxisome proliferator-activated receptor (PPARγ) Regulate expression of genes related to lipid metabolism Homo sapiens peroxisome proliferative activated receptor gamma (PPARG) gene, complete cds 84 Eddy, Morin and Storey (2005), Berger and Moller (2002) H isoform of fatty acid-binding protein (H-FABP) Enhance intracellular transport of fatty acid Human fatty acid-binding protein FABP gene, complete cds 88 Hittel and Storey (2002) Open in new tab Meanwhile, the evolution of hibernation has been suggested to occur at a molecular level with changes in patterns of gene expression rather than evolution of de novo genes. A model of gene regulation in hibernation has been proposed, as shown in Fig. 3. Figure 3. Open in new tabDownload slide Schematic representation of the central role of differential gene expression during hibernation. Single arrows indicate direct effects, and multiple arrows indicate an effect that requires multiple intermediate events. Reprinted from Srere, H. K. & Martin, S. L. (1992) Central role for differential gene expression in mammalian hibernation. Proceedings of the National Academy of Sciences of the United States of America. 89 (15), 7119–7123. In concert with the hypothesis that most physiological changes are associated with gene regulation, it has been found that gene regulation of 5′-AMP-induced hypometabolism occurs at a post-translational level. In the study by Zhang et al. (2011), >99.9% of mRNA analysed did not significantly change in level in the liver of mice. Global depression of transcription has been observed in golden-mantled ground squirrels during hibernation, which might be due to the combined effects of mild suppression of initiation and severe inhibition of elongation during transcription (Van Breukelen and Martin, 2002). Epigenetics refers to changes in gene expression that are not the result of alteration in DNA sequence. It also plays a major role in transcriptional and translational suppression in hibernation, which includes histone tail modification and DNA methylation. Morin and Storey (2006) found that the relative content of acetylation and phosphorylation on H3 fell by 25% and 40%, respectively, and that the amount of histone deacetylase protein rose during hibernation in skeletal muscle of thirteen-lined ground squirrels, which led to elevated chromatin packing and transcriptional silencing. A similar result was obtained in another study on brown adipose tissue from ground squirrels (Biggar and Storey, 2014). Changes in levels of methylation of DNA occurred in response to environmental stress, as demonstrated in a recent study on mouse neuronal cells (Hartley et al., 2013). When neurons in hippocampuswere exposed to hypoxia, both hyper- and hypo-methylation on CpG islands or promoter regions of different genes were observed to produce transcriptional modulation. Similarly, DNA methylation contributes to the transition into the hibernation phenotype in the liver and skeletal muscle of thirteen-lined ground squirrels (Alvarado et al., 2015). They successfully identified a gene, myocyte enhancer factor 2C (mef2c), in skeletal muscle whose promoter is methylated in response to hibernation initiation. Given that most of the genes associated with hibernation are present in the human genome, it could be possible to temporarily induce hibernation using molecular biology techniques. We should be immediately aware that modification should not be done at the genome or germ-line level because of genetic inheritance and ethical concerns, implying that most of the changes would be post-transcriptional modifications. Therefore, we can (i) deliver genes that are upregulated in hibernation into humans using viral or non-viral vectors to increase gene expression; (ii) exploit the mechanism of RNAi and deliver small RNA molecules to perform gene silencing and achieve downregulation at the transcriptional level and (iii) modify histone or DNA methylation to up- or downregulate gene expression. Genes that are upregulated in hibernation can be delivered into humans using vectors. However, this is less practical when the number of genes is enormous. It has been reported that 225 genes are upregulated in response to hypoxia exposure (Hartley et al., 2013), which will make the design and delivery of genes a tremendous amount of work. DNA pieces of the genes that we would like to downregulate could be introduced into non-hibernating animals. Introduced DNA will yield dsRNA or short hairpin RNA (shRNA) after transcription, which will be targeted by the host RNA interference system (RNAi) to produce short interfering RNA (siRNA). The RNAi system will cleave host mRNA that is complementary to siRNA and silence the expression of the gene. However, siRNA would lead to miRNA-like off-target effects as first recognised in 2003 (Aimee et al., 2003). This is because siRNA can target mRNA sequences whose 3′UTRs are complementary to the 5′UTR of siRNA. Synthetic siRNA has been reported to stimulate the production of interferons and inflammatory cytokines both in vivo and in vitro (Adam et al., 2005). Therefore the immunogenic effect of siRNA remains under investigation. Conclusion and future directions Hibernation is a strategy adopted by animals to conserve energy during periods with insufficient resources. Some studies have successfully induced torpor or hibernation-like state in non-hibernating animals using chemical compounds such as H2S and 5′-AMP, but there has been limited success in larger mammals. More research on hibernation induction needs to be done on primates, which are good models for human medicine owning to their genetic and physiological similarities. Nevertheless, these molecules have great potential in clinical applications such as trauma, heart attack, stroke and many other serious medical emergencies. Meanwhile, methods to maintain and terminate hibernation remain to be determined despite increased research on molecules involved in the maintenance phase of hibernation. At the genetic level, hibernation appears to be largely associated with the regulation of gene expression. The fact that most hibernation genes are also present in the human genome may allow us to explore ways to induce hibernation/torpor using current molecular technologies such as the CRISPR-Cas system in the future. Author biography Mingke is studying a BSc(Hons) in Biology with One Year in Industry at Imperial College London. She did an industrial placement at GlaxoSmithKline and worked on a hybridomas optimisation project. During her studies and placement, she was particularly attracted to research studies on natural adaptations in animals and how they could be utilised in future medicine. Meanwhile, she is equally interested developing biotechnologies that could make a difference to society. Mingke hopes to further pursue a PhD degree in relevant fields or start a biotechnology company in the near future. References Abe , K. and Kimura , H. 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Email: n.franks@imperial.ac.uk Biography: Mingke is studying a BSc(Hons) in Biology with One Year in Industry at Imperial College London. She did an industrial placement at GlaxoSmithKline and worked on a hybridomas optimisation project. During her studies and placement, she was particularly attracted to research studies on natural adaptations in animals and how they could be utilised in future medicine. Meanwhile, she is equally interested developing biotechnologies that could make a difference to society. Mingke hopes to further pursue a PhD degree in relevant fields or start a biotechnology company in the near future. © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press.

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BioScience HorizonsOxford University Press

Published: Jan 1, 2018

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