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Representative micrographs of hematoxylin- and eosin-stained liver sections showing congestion and microvesicular steatosis in a spring sham; b spring IR; c hibernating sham; and d hibernating IR squirrels. Shown are higher (×20, insets) and lower (×10) magnification images. Asterisks indicate sinusoidal congestion after IR and arrows indicate microvesicular steatosis in hibernators
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During the hibernation season, livers from 13-lined ground squirrels (Ictidomys tridecemlineatus) are resistant to damage induced by ex vivo, cold ischemia-warm reperfusion (IR) compared with livers from summer squirrels or rats. Here, we tested the hypothesis that hibernation also reduces damage to ground squirrel livers in an in vivo, warm IR mod...
Citations
... In this regard, we sought inspiration from hibernating mammals as they are masters of metabolic adaptation and well known for their tolerance of various types of ischemia-reperfusion stress [35][36][37][38][39][40][41]. From cultured hepatocyte-like cells derived from induced pluripotent stem cells of the 13-lined ground squirrels [7,42,43], we found that the cellular level of 5-ALA increases during cold exposure or OGD, and then decreases during rewarming or reoxygenation. ...
Rationale: Liver resection and transplantation surgeries are accompanied by hepatic ischemia-reperfusion (HIR) injury that hampers the subsequent liver recovery. Given that the liver is the main organ for metabolism and detoxification, ischemia-reperfusion in essence bestows metabolic stress upon the liver and disrupts local metabolic and immune homeostasis. Most of the recent and current research works concerning HIR have been focusing on addressing HIR-induced hepatic injury and inflammation, instead of dealing with the metabolic reprogramming and restoration of redox homeostasis. As our previous work uncovers the importance of 5-aminolevulinate (5-ALA) synthesis during stress adaptation, here we evaluate the effects of supplementing 5-ALA to mitigate HIR injury.
Methods: 5-ALA was supplemented into the mice or cultured cells during the ischemic or oxygen-glucose deprivation (OGD) phase. Following reperfusion or reoxygenation, cellular metabolism and energy homeostasis, mitochondrial production of reactive oxygen species (ROS) and transcriptomic changes were evaluated in HIR mouse models or cultured hepatocytes and macrophages. Liver injury, hepatocytic functional tests, and macrophagic M1/M2 polarization were assessed.
Results: Dynamic changes in the expression of key enzymes in 5-ALA metabolism were first confirmed in donor and mouse liver samples following HIR. Supplemented 5-ALA modulated mouse hepatic lipid metabolism and reduced ATP production in macrophages following HIR, resulting in elevation of anti-inflammatory M2 polarization. Mechanistically, 5-ALA down-regulates macrophagic chemokine receptor CX3CR1 via the repression of RelA following OGD and reoxygenation (OGD/R). Cx3cr1 KO mice demonstrated milder liver injuries and more macrophage M2 polarization after HIR. M2 macrophage-secreted chitinase-like protein 3 (CHIL3; CHI3L1 in human) is an important HIR-induced effector downstream of CX3CR1 deficiency. Addition of CHIL3/CHI3L1 alone improved hepatocellular metabolism and reduced OGD/R-inflicted injuries in cultured mouse and human hepatocytes. Combined treatment with 5-ALA and CHIL3 during the ischemic phase facilitated lipid metabolism and ATP production in the mouse liver following HIR.
Conclusion: Our results reveal that supplementing 5-ALA promotes macrophagic M2 polarization via downregulation of RelA and CX3CR1 in mice following HIR, while M2 macrophage-produced CHIL3/CHI3L1 also manifests beneficial effects to the recovery of hepatic metabolism. 5-ALA and CHIL3/CHI3L1 together mitigate HIR-induced mitochondrial dysfunction and hepatocellular injuries, which may be developed into safe and effective clinical treatments to attenuate HIR injuries.
... Kidney adaptations likewise permit to withstand low T b and organ perfusion as well as reperfusion by the increase of antioxidant enzymes and the downregulation of pro-apoptotic processes [40]. Hepatocytes and the whole liver as such are also protected against damage by torpor-associated conditions and IR, which is mainly attributed to the upregulation of antioxidants and anti-apoptotic proteins [43,50]. ...
Hibernation enables many species of the mammalian kingdom to overcome periods of harsh environmental conditions. During this physically inactive state metabolic rate and body temperature are drastically downregulated, thereby reducing energy requirements (torpor) also over shorter time periods. Since blood cells reflect the organism´s current condition, it was suggested that transcriptomic alterations in blood cells mirror the torpor-associated physiological state. Transcriptomics on blood cells of torpid and non-torpid Djungarian hamsters and QIAGEN Ingenuity Pathway Analysis (IPA) revealed key target molecules (TMIPA), which were subjected to a comparative literature analysis on transcriptomic alterations during torpor/hibernation in other mammals. Gene expression similarities were identified in 148 TMIPA during torpor nadir among various organs and phylogenetically different mammalian species. Based on TMIPA, IPA network analyses corresponded with described inhibitions of basic cellular mechanisms and immune system-associated processes in torpid mammals. Moreover, protection against damage to the heart, kidney, and liver was deduced from this gene expression pattern in blood cells. This study shows that blood cell transcriptomics can reflect the general physiological state during torpor nadir. Furthermore, the understanding of molecular processes for torpor initiation and organ preservation may have beneficial implications for humans in extremely challenging environments, such as in medical intensive care units and in space.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00424-023-02842-8.
... During early arousal, it is suggested that restoring the expression of MAPK members leads to activation of TFs, such as MYC and CREB1, which is facilitated by hypomethylation. Furthermore, torpid animals tolerate hepatic ischaemia following profound reductions of blood flow, while maintaining mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lowering vascular resistance and Kupffer cell phagocytosis [56,57]. Our data provide further indications that administration of MAPK inhibitors might protect from cell damage by arresting cell cycle during ischaemia [58] and that MAPK-regulated TFs may be interesting targets to avert damage by regulating cell cycle progression during organ reperfusion. ...
Hibernation consists of alternating torpor-arousal phases, during which animals cope with repetitive hypothermia and ischaemia-reperfusion. Due to limited transcriptomic and methylomic information for facultative hibernators, we here conducted RNA and whole-genome bisulfide sequencing in liver of hibernating Syrian hamster (Mesocricetus auratus). Gene ontology analysis was performed on 844 differentially expressed genes and confirmed the shift in metabolic fuel utilization, inhibition of RNA transcription and cell cycle regulation as found in seasonal hibernators. Additionally, we showed a so far unreported suppression of mitogen-activated protein kinase (MAPK) and protein phosphatase 1 pathways during torpor. Notably, hibernating hamsters showed upregulation of MAPK inhibitors (dual-specificity phosphatases and sproutys) and reduced levels of MAPK-induced transcription factors (TFs). Promoter methylation was found to modulate the expression of genes targeted by these TFs. In conclusion, we document gene regulation between hibernation phases, which may aid the identification of pathways and targets to prevent organ damage in transplantation or ischaemia-reperfusion.
... Hibernating mammals, which build up tremendous stores of fat reserves prior to hibernation, drastically decrease their metabolic rate and rely on catabolic programs associated with the metabolism of fatty acids (Florant 1998). Hibernating mammals have elevated resistance to multiple stressors, including oxidative damage (Otis et al. 2017), traumatic injury (Iaizzo et al. 2012), and hypothermia (Matos-Cruz et al. 2017). Dormancy programs in lower organisms, such as dauer in Caenorhabditis elegans (Fielenbach & Antebi 2008) and extreme abiotic states in tardigrades (Hashimoto et al. 2016), also show pronounced resilience to environmental stressors. ...
... These small mammals maintain their body temperature at 37 °C in non-hibernating seasons; during hibernation, they live through cycles of deep torpor that lasts for days with a body temperature typically below 5 °C, and short interbout arousal in which the animals quickly raise their body temperature to 37 °C for a few hours while enduring IR-like physiology [16][17][18]. It has been shown that livers from hibernating thirteen-lined ground squirrels (GSs; Ictidomys tridecemlineatus) are more resistant to both cold and warm IR injuries [19,20]. Neuroprotective effects have also been extensively studied in hibernating ground squirrel species, piquing interests in translating these spectacular feats into effective treatments of stroke and other IR-type of injuries to the brain [21][22][23][24]. ...
Rationale: Hibernating thirteen-lined ground squirrels (GS; Ictidomys tridecemlineatus) are naturally adapted to prolonged periods of ultraprofound hypothermia (body temperature < 5 ºC) during torpor, and drastic oscillations of body temperature and ischemia/reperfusion-like stress during their short euthermic interbout arousals. Thus, their superior adaptability may hold tremendous promise for the advancement of donor organ cold preservation and subsequent organ transplantation. However, bridging hibernation research and translational medicine has been impeded by a dearth of in vitro research tools, till the recent establishment of the GS induced pluripotent stem cells (iPSCs). In this study, we reported the generation of functional hepatocyte-like cells (HLCs) from GS iPSCs. As temperature and oxygen supply affect cellular metabolism, we hypothesized that the GS HLCs can metabolically counter drastic temperature and oxygen supply changes. Differentially regulated metabolites can be evaluated and included into the preservation solution to mitigate temperature and ischemia/reperfusion-associated damage to donor livers.
Methods: A protocol has been developed to produce GS iPSCs-derived HLCs. Comparative metabolomic analysis on GS HLCs and human donor liver samples revealed changes in metabolites caused by cold storage and rewarming. Human embryonic stem cell (ESC)-derived HLCs and ex vivo cold preservation and reperfusion of isolated rat livers were used to assess candidate metabolites that may have protective effects against preservation-related injuries.
Results: GS iPSCs were efficiently differentiated into expandable, cryopreservation-compatible and functional HLCs. Metabolomic analysis unveiled distinct changes of mitochondrial metabolites between GS and human cells following cold storage and rewarming. GS and human HLC-based experiments indicated that the metabolism of 5-aminolevulinate (5-ALA) is key to restricting free radical production during rewarming. Survival of human HLCs was significantly increased following cold exposure and rewarming, as supplemented 5-ALA enhanced Complex III activity and improved mitochondrial respiration. Further, 5-ALA mitigated damage in rat livers following 48-h cold preservation and ex vivo reperfusion. Metabolomic and transcriptomic analyses revealed that supplemented 5-ALA promoted both anabolic and catabolic activities while alleviating cell death, inflammation, hypoxia and other stress responses in isolated perfused rat livers.
Conclusion: In the liver, rewarming from ultraprofound hypothermia imposes complex metabolic challenges and stresses on the mitochondria. Metabolites such as 5-ALA can help alleviate mitochondrial stress. Supplementing 5-ALA to the liver preservation solution can substantially improve the functional recovery of rat livers following prolonged cold preservation, rewarming and reperfusion.
... In addition, seasonal remodeling of lipid composition in tissues occurs in a diet-independent manner, while there are hypotheses that dietary fatty acids, in particular, the ratio of dietary omega-3/omega-6 fatty acids, may affect the expression and quality of torpor [8][9][10] . In contrast, resistance to ischemia/ reperfusion stresses also develops during the transition from summer to winter in 13-lined ground squirrels [11][12][13] . Arctic ground squirrels also undergo body remodeling, such as pre-HIB fattening and global gene expression changes 14,15 , but also exhibit intrinsic resistance to global ischemia independently of seasons 16,17 . ...
Mammalian hibernators endure severe and prolonged hypothermia that is lethal to non-hibernators, including humans and mice. The mechanisms responsible for the cold resistance remain poorly understood. Here, we found that hepatocytes from a mammalian hibernator, the Syrian hamster, exhibited remarkable resistance to prolonged cold culture, whereas murine hepatocytes underwent cold-induced cell death that fulfills the hallmarks of ferroptosis such as necrotic morphology, lipid peroxidation and prevention by an iron chelator. Unexpectedly, hepatocytes from Syrian hamsters exerted resistance to cold- and drug-induced ferroptosis in a diet-dependent manner, with the aid of their superior ability to retain dietary α-tocopherol (αT), a vitamin E analog, in the liver and blood compared with those of mice. The liver phospholipid composition is less susceptible to peroxidation in Syrian hamsters than in mice. Altogether, the cold resistance of the hibernator’s liver is established by the ability to utilize αT effectively to prevent lipid peroxidation and ferroptosis. Daisuke Anegawa et al. investigated the mechanisms responsible for cold resistance in the Syrian hamster’s hepatocytes, which exhibited remarkable resistance to prolonged cold culture. Their results suggest that hepatocytes exhibit diet-dependent resistance to cold, which is linked to the retention of α-tocopherol in the liver.
... However, hibernator tissues resist ischemia and reperfusion better than non-hibernators and this resistance improves in the winter hibernation season (Lindell et al. 2005). In fact, damage to hepatocytes induced by ischemia and reperfusion is reduced by up to 50% in winter hibernating ground squirrels compared with non-hibernating animals sampled in the spring (Otis et al. 2017). Mechanisms underlying the metabolic suppression associated with hibernation and how it may contribute to resist such stressful conditions remain poorly understood. ...
... O-GlcNAcylation can modulate the activity of multiple regulatory proteins and transcription factors that change gene expression profiles in effector cells thereby aiding in the response to environmental stress or preserving specific cell functions (Bond and Hanover 2015;Martinez et al. 2017;Yang and Qian 2017). Therefore, the second part of our study focused on profiling galectin genes and protein expression in the liver of active and hibernating thirteen-lined ground squirrels, a tissue that exhibits improved resistance to the stress of ischemia/reperfusion in winter (Lindell et al. 2005;Otis et al. 2017). Galectins are soluble glycan-binding proteins with multiple modes of pro-apoptotic and pro-survival actions depending on their localization in cells or secretion (Garner and Baum 2008;Compagno et al. 2014;Vladoiu et al. 2014;Nabi et al. 2015;Timoshenko 2015). ...
Post-translational glycosylation of proteins with O-linked β-N-acetylglucosamine (O-GlcNAcylation) and changes of galectin expression profiles are essential in many cellular stress responses. We examine this regulation in the liver tissue of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) representing a biological model of hypometabolism and physiological stress resistance. The tissue levels of O-GlcNAcylated proteins as well as galectin-1 and galectin-3 proteins detected by immunodot blot assay were significantly lower by 4.6–5.4-, 2.2–2.3- and 2.5–2.9-fold, respectively, in the non-hibernating summer squirrels compared with those in winter, whether hibernating or aroused. However, there were no differences in the expression of genes encoding enzymes involved in O-GlcNAc cycle (O-GlcNAc transferase and O-GlcNAcase) and such galectins as LGALS1, LGALS2, LGALS3, LGALS4 and LGALS9. Only the expression of LGALS8 gene in the liver tissue was significantly decreased by 37.6 ± 0.1% in hibernating ground squirrels relative to summer animals. Considering that the expression of a proven genetic biomarker ELOVL6 encoding ELOVL fatty acid elongase 6 was readily upregulated in non-hibernating animals by 11.3–32.9-fold, marginal differential changes in the expression of galectin genes cannot be classified as biomarkers of hibernation. Thus, this study provides evidence that hibernation in Ictidomys tridecemlineatus is associated with increasing O-GlcNAcylation of liver proteins and suggests that the contribution of galectins deserves further studies at the protein level.
... Indeed, during and after natural hibernation, when core temperature drops dramatically to~4 • C, no organ damage was found [6,7]. Even more interesting is that outside the hibernation season, hibernators withstand iatrogenic damage, such as ischemia/reperfusion injury (IRI) and energy deprivation [8][9][10][11], whereas IRI in humans leads to organ failure as found, for example, in organ transplantation [12] and myocardial infarction [13]. Because of these features, hibernation is a highly interesting model to define new preservation techniques in conditions such as organ transplantation [14,15] and cardiac arrest [16]. ...
Mitochondrial failure is recognized to play an important role in a variety of diseases. We previously showed hibernating species to have cell-autonomous protective mechanisms to resist cellular stress and sustain mitochondrial function. Here, we set out to detail these mitochondrial features of hibernators. We compared two hibernator-derived cell lines (HaK and DDT1MF2) with two non-hibernating cell lines (HEK293 and NRK) during hypothermia (4 °C) and rewarming (37 °C). Although all cell lines showed a strong decrease in oxygen consumption upon cooling, hibernator cells maintained functional mitochondria during hypothermia, without mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential decline or decreased adenosine triphosphate (ATP) levels, which were all observed in both non-hibernator cell lines. In addition, hibernator cells survived hypothermia in the absence of extracellular energy sources, suggesting their use of an endogenous substrate to maintain ATP levels. Moreover, hibernator-derived cells did not accumulate reactive oxygen species (ROS) damage and showed normal cell viability even after 48 h of cold-exposure. In contrast, non-hibernator cells accumulated ROS and showed extensive cell death through ferroptosis. Understanding the mechanisms that hibernators use to sustain mitochondrial activity and counteract damage in hypothermic circumstances may help to define novel preservation techniques with relevance to a variety of fields, such as organ transplantation and cardiac arrest.
... Hibernation seems to protect against cellular damage and promote wound healing, but the mechanisms underlying this cytoprotective effect are largely unknown (Otis et al., 2017). We observed increased expression of cytoprotective HO-1 in animals during pre-hibernation ( Figure 1C). ...
Heme oxygenase (HO)-1 plays an important role during hibernation by catalyzing the degradation of heme to biliverdin/bilirubin, ferrous iron, and carbon monoxide, which activates the protective mechanisms against stress. In this context, it was important to analyze the metabolic processes of heme. Nevertheless, to date, no study has approached on biosynthesis of heme. Therefore, our study aims to understand the process of heme biosynthesis, which regulates cell survival in conditions of hypothermia and calorie restriction (CR). During hibernation, the mRNA levels of enzymes responsible for de novo heme biosynthesis were increased in the liver tissue of a Syrian hamster model of hibernation. Moreover, heme trafficking and iron metabolism were found to be more active, as assessed based on the changes in the levels of heme transporter and ferroportin mRNA. The levels of HO-1, a powerful antioxidant, were also upregulated during hibernation. Additionally, increased levels of Sirt-1 mRNA were also observed. These enzymes are known to act as cellular metabolic sensors that activate the cytoprotective mechanisms. These results indicate that HO-1 induction, brought about by the upregulation of heme during the pre-hibernation period, may protect against external stress. Here, we describe heme catabolism during hibernation by analyzing the regulation of the key molecular players involved in heme metabolism. Therefore, this study offers a new strategy for the better regulation of intracellular heme concentrations during hypothermia and other stresses.
... The periodic resumption of normal metabolism and return to normothermia is energetically expensive and causes fluctuations in tissue perfusion [8,9] The organs of hibernating animals experience multiple bouts of ischemia followed by reperfusion during torpor, yet remarkably their organs do not undergo ischemia reperfusion (IR) injury [9][10][11]. Understanding the molecular and cellular mechanisms that allow animals to preserve organ function during extreme shifts in tissue perfusion, as experienced by hibernators, will likely lead to drug targets to prevent IR injury in human organs. ...
... The periodic resumption of normal metabolism and return to normothermia is energetically expensive and causes fluctuations in tissue perfusion [8,9] The organs of hibernating animals experience multiple bouts of ischemia followed by reperfusion during torpor, yet remarkably their organs do not undergo ischemia reperfusion (IR) injury [9][10][11]. Understanding the molecular and cellular mechanisms that allow animals to preserve organ function during extreme shifts in tissue perfusion, as experienced by hibernators, will likely lead to drug targets to prevent IR injury in human organs. ...
... The natural resistance to IR injury that hibernators experience persists outside of the hibernating season, and stable molecular adaptations to cellular stress have been demonstrated in many hibernators [1,3]. Many different cell types of hibernators, when studied ex vivo, demonstrate an ability to preserve mitochondrial membrane potential, regulate ATP production and avoid cell death when exposed to hypoxic stress conditions [3,6,9,12]. The 13-lined ground squirrel, which is an important model for understanding the mechanisms of hibernation in vivo, has shown us that the organs of hibernators are remarkably resistant to either cold or warm IR injury [9]. ...
