RESUMO
Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system.NEW & NOTEWORTHY During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs.
Assuntos
Hibernação , MicroRNAs , Sciuridae , Animais , Sciuridae/genética , Hibernação/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Estações do Ano , Torpor/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/genéticaRESUMO
Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.
Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 48oC); whereas Eurasian brown bears see milder temperature drops (down to 2325oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin a protein found in muscles that helps muscles to contract was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 48oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 48oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 48oC. This suggest that resting myosin generates heat at cool temperatures a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.
Assuntos
Hibernação , Animais , Hibernação/fisiologia , Metabolismo Energético , Miosinas de Músculo Esquelético/metabolismo , Ursidae/metabolismo , Ursidae/fisiologia , Trifosfato de Adenosina/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fibras Musculares Esqueléticas/metabolismo , ProteômicaRESUMO
Many research groups explore the regulation of hibernation or compare the physiology of heterothermic mammals between the torpid and aroused, euthermic states. Current methods for monitoring torpor (for example, infrared cameras, body temperature or heart-rate telemetry, and motion sensing) are costly, require specialized techniques, and can be invasive. Here we present an alternate method for determining torpor-bout duration that is cost-effective, noninvasive and accurate: paper towel shredding. In the winter, euthermic thirteen-lined ground squirrels will shred paper towels placed in the cage, but torpid animals will not. The presence of a shredded paper towel, indicating an arousal from torpor, is easily evaluated during routine daily monitoring. In 12 animals over 52 days, this simple technique detected 59 arousals with 100% accuracy when compared with the body temperature telemetry of the same animals. Moreover, this novel method avoids some of the drawbacks of other cheap monitoring systems such as the sawdust technique.
Assuntos
Hibernação , Sciuridae , Animais , Sciuridae/fisiologia , Hibernação/fisiologia , Nível de Alerta/fisiologia , Telemetria/métodos , Telemetria/veterinária , Temperatura Corporal , Masculino , Papel , Abrigo para AnimaisRESUMO
Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000â km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.
Assuntos
Migração Animal , Espécies Reativas de Oxigênio , Aves Canoras , Animais , Aves Canoras/metabolismo , Aves Canoras/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Migração Animal/fisiologia , Citrato (si)-Sintase/metabolismo , Mitocôndrias Musculares/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Voo Animal/fisiologiaRESUMO
Mitochondria serve several important roles in maintaining cellular homeostasis, including adenosine triphosphate (ATP) synthesis, apoptotic signalling, and regulation of both reactive oxygen species (ROS) and calcium. Therefore, mitochondrial studies may reveal insights into metabolism at higher levels of physiological organization. The apparent complexity of mitochondrial function may be daunting to researchers new to mitochondrial physiology. This review is aimed, therefore, at such researchers to provide a brief, yet approachable overview of common techniques used to assess mitochondrial function. Here we discuss the use of high-resolution respirometry in mitochondrial experiments and common analytical platforms used for this technique. Next, we compare the use of common mitochondrial preparation techniques, including adherent cells, tissue homogenate, permeabilized fibers and isolated mitochondria. Finally, we outline additional techniques that can be used in tandem with high-resolution respirometry to assess additional aspects of mitochondrial metabolism, including ATP synthesis, calcium uptake, membrane potential and reactive oxygen species emission. We also include limitations to each of these techniques and outline recommendations for experimental design and interpretation. With a general understanding of methodologies commonly used to study mitochondrial physiology, experimenters may begin contributing to our understanding of this organelle, and how it affects other physiological phenotypes.
Assuntos
Cálcio , Mitocôndrias , Animais , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , HumanosRESUMO
Across many taxa, the complexes of the electron transport system associate with each other within the inner mitochondrial membrane to form supercomplexes (SCs). These SCs are thought to confer some selective advantage, such as increasing cellular respiratory capacity or decreasing the production of damaging reactive oxygen species (ROS). In this study, we investigate the relationship between supercomplex abundance and performance of liver mitochondria isolated from rats that do not hibernate and hibernating ground squirrels in which metabolism fluctuates substantially. We quantified the abundance of SCs (respirasomes (SCs containing CI, CIII, and CIV) or SCs containing CIII and CIV) and examined the relationship with state 3 (OXPHOS) and state 4 (LEAK) respiration rate, as well as net ROS production. We found that, in rats, state 3 and 4 respiration rate correlated negatively with respirasome abundance, but positively with CIII/CIV SC abundance. Despite the greater range of respiration rates in different hibernation stages, these relationships were similar in ground squirrels. This is, to our knowledge, the first report of differential effects of supercomplex types on mitochondrial respiration and ROS production.
Assuntos
Respiração , Sciuridae , Ratos , Animais , Transporte de Elétrons , Espécies Reativas de Oxigênio/metabolismo , Sciuridae/metabolismo , OxigênioRESUMO
Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.
RESUMO
Hibernation confers resistance to ischemia-reperfusion injury in tissue, but the underlying mechanisms remain unclear. Suppression of mitochondrial respiration during torpor may contribute to this tolerance. To explore this concept, we subjected isolated liver mitochondria from torpid, interbout euthermic (IBE) and summer 13-lined ground squirrels (Ictidomys tridecemlineatus) to 5 min of anoxia, followed by reoxygenation (A/R). We also included rat liver mitochondria as a non-hibernating comparison group. Maximum respiration rates of mitochondria from torpid ground squirrels were not affected by A/R, but in IBE and summer, these rates decreased by 50% following A/R and in rats they decreased by 80%. Comparing net ROS production rates among groups, revealed seasonal differences; mitochondria from IBE and torpor produced 75% less ROS than summer ground squirrels and rats. Measurements of oxidative damage to these mitochondria, both freshly isolated, as well as pre- and post-A/R, demonstrated elevated damage to protein, but not lipids, in all groups. Hibernation likely generates oxidative stress, as freshly isolated mitochondria had greater protein damage in torpor and IBE than in summer and rats. When comparing markers of damage pre- and post-A/R, we found that when RET was active, rat macromolecules were more damaged than when RET is inhibited, but in TLGS markers of damage were similar. This result suggests that suppression of RET during hibernation, both in torpor and IBE, lessens oxidative stress produced during arousal. Taken together our study suggests that ischemia-reperfusion tolerance at the mitochondrial level is associated with metabolically suppressed oxidative phosphorylation during hibernation.
Assuntos
Hibernação , Mitocôndrias Hepáticas , Animais , Ratos , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Hibernação/fisiologia , Hipóxia/metabolismo , Sciuridae/fisiologiaRESUMO
Metabolic depression and dormancy (i.e., stopping/greatly reducing activity and feeding) are strategies used by many animals to survive winter conditions characterized by food shortages and cold temperatures. However, controversy exists on whether the reduced metabolism of some fishes at cold temperatures is due to dormancy alone, or also involves active metabolic depression. Thus, we acclimated winter-dormant cunner [Tautogolabrus adspersus, a north temperate wrasse which in Newfoundland is at the northern limit of its distribution] and winter-active Atlantic salmon (Salmo salar) to winter (0°C; 8h light: 16h dark) and summer (10°C; 16h light: 8 h dark) conditions, and measured the thermal sensitivity of ATP-producing and O2-consuming processes in isolated liver mitochondria and hepatocytes when exposed in vitro to temperatures from 20 to 0°C and 10 to 0°C, respectively. We found that: 1) liver mitochondrial State 3 respiration and hepatocyte O2 consumption in cunner were only ~ one-third and two-thirds of that measured in salmon, respectively, at all measurement temperatures; 2) cunner mitochondria also have proton conductance and leak respiration (State 4) values that are only approximately one-third of those in salmon; 3) the mitochondria of cunner show a dramatic reduction in respiratory control ratio (from ~ 8 to 3), and a much greater drop in State 3 respiration, between 10 and 5°C (Q10 values in 10- and 0°C-acclimated fish of 14.5 and 141.2, respectively), as compared with salmon (3.9 and 9.6, respectively); and 4) lowering temperature from 5 to 0°C resulted in ~ 40 and 30% reductions in hepatocyte O2 consumption due to non-mitochondrial respiration and Na+-K+-ATPase activity, respectively, in cunner, but not in salmon. Collectively, these results highlight the intrinsic capacity for metabolic depression in hepatocytes and mitochondria of cunner, and clearly suggest that several cellular processes play a role in the reduced metabolic rates exhibited by some fishes at cold temperatures.
Assuntos
Temperatura Baixa , Perciformes , Aclimatação , Animais , Depressão , Peixes/metabolismo , TemperaturaRESUMO
Many insects enter a state of dormancy (diapause) during winter in which they lower their metabolism to save energy. Metabolic suppression is a hallmark of diapause, yet we know little about the mechanisms underpinning metabolic suppression in winter or how it is reversed in the spring. Here, we show that metabolic suppression in dormant Colorado potato beetles results from the breakdown of flight muscle mitochondria via mitophagy. Diapausing Colorado potato beetles suppress their metabolism by 90%, and this lowered metabolic rate coincides with a similar reduction in flight muscle mitochondrial function and density. During early diapause, beetles increase the expression of mitophagy-related transcripts (Parkin and ATG5) in their flight muscle coincident with an increase in mitophagy-related structures in the flight muscle. Knocking down Parkin expression with RNA interference in diapausing beetles prevented some mitochondrial breakdown and partially restored the whole animal metabolic rate, suggesting that metabolic suppression in diapausing beetles is driven by mitophagy. In other animals and in models of disease, such large-scale mitochondrial degradation is irreversible. However, we show that as diapause ends, beetles reverse mitophagy and increase the expression of PGC1α and NRF1 to replenish flight muscle mitochondrial pools. This mitochondrial biogenesis is activated in anticipation of diapause termination and in the absence of external stimuli. Our study provides a mechanistic link between mitochondrial degradation in insect tissues over the winter and whole-animal metabolic suppression.
Assuntos
Besouros , Diapausa de Inseto , Mitofagia , Animais , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Besouros/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hibernators rapidly and reversibly suppress mitochondrial respiration and whole animal metabolism. Posttranslational modifications likely regulate these mitochondrial changes, which may help conserve energy in winter. These modifications are affected by reactive oxygen species (ROS), so suppressing mitochondrial ROS production may also be important for hibernators, just as it is important for surviving ischemia-reperfusion injury.
Assuntos
Hibernação , Animais , Metabolismo Energético/fisiologia , Hibernação/fisiologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sciuridae/metabolismoRESUMO
AbstractDuring hibernation, especially during arousal from torpor to interbout euthermia (IBE), blood flow changes drastically. In nonhibernating mammals, similar changes during ischemia/reperfusion lead to oxidative damage. We hypothesized that suppression of mitochondrial metabolism during hibernation protects against such damage. We compared markers of oxidative damage and total antioxidant capacity in eight tissues among summer, torpid, and IBE thirteen-lined ground squirrels. Overall, summer tissue had less lipid and protein oxidative damage than tissue from the hibernation season, but DNA damage (in four tissues) and total antioxidant capacity (in all eight tissues) were similar among all groups. During torpor, when mitochondrial metabolism is suppressed, lipid damage in heart, brown adipose tissue, and small intestine was lower than IBE by as much as fivefold. By contrast, oxidative damage to protein was at least twofold higher in liver and skeletal muscle in torpor compared with IBE. Our findings suggest that arousal from torpor creates oxidative damage similar to ischemia/reperfusion injury but that this damage is repaired during IBE. These differences cannot be explained by changes in antioxidant capacity, so they are likely due to differences is reactive oxygen species production among hibernation states that may relate to the well-characterized reversible suppression of mitochondrial metabolism during torpor.
Assuntos
Hibernação , Torpor , Animais , Antioxidantes/metabolismo , Nível de Alerta/fisiologia , Hibernação/fisiologia , Lipídeos , Estresse Oxidativo , Sciuridae/fisiologia , Torpor/fisiologiaRESUMO
Complexes of the electron transport system can associate with each other to form supercomplexes (SCs) within mitochondrial membranes, perhaps increasing respiratory capacity or reducing reactive oxygen species production. In this study, we determined the abundance, composition, and stability of SCs in a mammalian hibernator, in which both whole animal and mitochondrial metabolism change greatly throughout winter. We isolated mitochondria from thirteen-lined ground squirrels (Ictidomys tridecemlineatus) in different hibernation states, as well as from rats (Rattus norvegicus). We extracted mitochondrial proteins using two nonionic detergents of different strengths and quantified SC abundance using two-dimensional gel electrophoresis and immunoblotting. Rat heart and liver had fewer SCs than ground squirrels. Within ground squirrels, SCs are dynamic, changing among hibernation states within a matter of hours. In brown adipose tissue, Complex III composition in different SCs differed between the torpid and interbout euthermic phase of a hibernation bout. In heart and liver, complex III composition changed between winter and summer. We also evaluated the stability of liver SCs using a stronger detergent and found that the stability of SCs differed; torpor SCs were more stable than the SCs of ground squirrels in other states and rats. This study is the first report of SC changes during hibernation and the first to demonstrate their dynamics on a short timescale.
Assuntos
Hibernação , Torpor , Animais , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hibernação/fisiologia , Ratos , Sciuridae/fisiologia , Torpor/fisiologiaRESUMO
Hibernating mammals may suppress their basal metabolic rate during torpor by up to 95% to reduce energy expenditure during winter, but the underlying mechanisms remain poorly understood. Here we show that hydrogen sulfide (H2S), a ubiquitous signaling molecule, is a powerful inhibitor of respiration of liver mitochondria isolated from torpid 13-lined ground squirrels, but has a weak effect on mitochondria isolated during summer and hibernation arousals, where metabolic rate is normal. Consistent with these in vitro effects, we find strong seasonal variations of in vivo levels of H2S in plasma and increases of H2S levels in the liver of squirrels during torpor compared to levels during arousal and summer. The in vivo changes of liver H2S levels correspond with low activity of the mitochondrial H2S oxidizing enzyme sulfide:quinone oxidoreductase (SQR) during torpor. Taken together, these results suggest that during torpor, H2S accumulates in the liver due to a low SQR activity and contributes to inhibition of mitochondrial respiration, while during arousals and summer these effects are reversed, H2S is degraded by active SQR and mitochondrial respiration rates increase. This study provides novel insights into mechanisms underlying mammalian hibernation, pointing to SQR as a key enzyme involved in the control of mitochondrial function.
Assuntos
Hibernação , Sulfeto de Hidrogênio , Animais , Mitocôndrias , Respiração , SciuridaeRESUMO
The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is assumed to be an obligate hibernator - commencing and terminating hibernation on a circannual rhythm, regardless of environmental conditions - but, until now, this assumption had never been fully tested. We housed three groups of captive-born ground squirrels from Aug. 2017 to Aug. 2018 under constant photoperiod (12 h L:12 h D) at 5, 16 or 25 °C, and monitored hibernation using body temperature loggers. At 5 and 16 °C all animals hibernated from autumn to spring with no differences in date of first/last torpor or duration of interbout euthermic periods (IBE), but torpor bout duration was 25% shorter at 16 °C. One of 4 animals housed at 25 °C did not hibernate. For the other three 25 °C animals, the first torpor date did not differ from the other groups, but the last torpor bout (5 Feb.) occurred almost 8 weeks earlier. These animals aroused from torpor more frequently and IBE lasted significantly longer, so the total time spent torpid was less than 50% of the other groups. Unlike the 5 or 16 °C animals, 25 °C animals re-entered torpor in late spring 2018. Taken together these data suggest that this species is an obligate hibernator, but that high ambient temperatures can accelerate the endogenous circannual hibernation rhythm.
Assuntos
Temperatura Alta , Sciuridae/fisiologia , Torpor , Animais , Temperatura Corporal , Feminino , Masculino , Estações do AnoRESUMO
Post-translational glycosylation of proteins with O-linked ß-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.
Assuntos
Acetilglucosamina/metabolismo , Galectinas/metabolismo , Hibernação , Fígado/metabolismo , Sciuridae , Animais , GlicosilaçãoRESUMO
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. METHODS: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. RESULTS: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. CONCLUSIONS: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/metabolismo , Mutação de Sentido Incorreto/genética , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Sarcômeros/metabolismo , Adenosina Trifosfatases , Animais , Cardiomiopatia Hipertrófica/genética , Células Cultivadas , Metabolismo Energético , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Simulação de Dinâmica Molecular , Relaxamento Muscular , Contração Miocárdica , Miócitos Cardíacos/citologia , Conformação Proteica , Sarcômeros/genéticaRESUMO
Hibernators survive challenging winters by entering torpor, which lowers body temperature (Tb) to â¼5⯰C for 12-14 days, followed by spontaneous arousals where Tb increases to â¼37⯰C for 10-12â¯h before entering another torpor bout. This Tb cycle is accompanied by significant fluctuations in metabolic rate. Little is known about the role of the liver in lipid metabolism during hibernation. In this study we measured the effect of ambient temperature on liver volume and lipid content in 13-lined ground squirrels (Ictidomys tridecemlineatus). We housed animals at thermoneutral (25⯰C) or cold (5⯰C) ambient temperatures, with the same photoperiod (12â¯h light:12â¯h dark) for an entire year. We determined volume and water-fat ratio of the liver using magnetic resonance imaging (MRI). Ambient temperature significantly affected both liver volume and fat content. From October to August squirrels housed at 25⯰C had 25% smaller livers compared to the squirrels housed at 5⯰C, but their average lipid content (13.3%) was 37% higher. Because the squirrels housed at 25⯰C appeared to continue feeding throughout the winter but did not enter extended torpor, more carbohydrates may have been diverted to lipid stores. By contrast, animals housed at 5⯰C did not appear to feed, and carbohydrates would likely be preferentially stored in the liver as glycogen to supply glucose for brain metabolism. These results suggest that the fat burden caused by hibernators preparing for winter can lead to symptoms of metabolic syndrome, but that these symptoms are reversible in the spring.
Assuntos
Tecido Adiposo/anatomia & histologia , Hibernação , Fígado/anatomia & histologia , Sciuridae/fisiologia , Tecido Adiposo/metabolismo , Animais , Temperatura Baixa , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Tamanho do Órgão , Sciuridae/metabolismoRESUMO
During hibernation, small mammals, including the 13-lined ground squirrel (Ictidomys tridecemlineatus), cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by >95% and body temperature falls to ~5°C, and interbout euthermia (IBE), where both metabolic rate and body temperature rapidly increase to euthermic levels. Suppression of whole animal metabolism during torpor is paralleled by rapid, reversible suppression of mitochondrial respiration. We hypothesized that these changes in mitochondrial metabolism are regulated by posttranslational modifications to mitochondrial proteins. Differential two-dimensional gel electrophoresis and two-dimensional blue-native PAGE revealed differences in the isoelectric point of several liver mitochondrial proteins between torpor and IBE. Quadrupole time-of-flight LC/MS and matrix-assisted laser desorption/ionization MS identified these as proteins involved in ß-oxidation, the tricarboxylic acid cycle, reactive oxygen species detoxification, and the electron transport system (ETS). Immunoblots revealed that subunit 1 of ETS complex IV was acetylated during torpor but not IBE. Phosphoprotein staining revealed significantly greater phosphorylation of succinyl-CoA ligase and the flavoprotein subunit of ETS complex II in IBE than torpor. In addition, the 75-kDa subunit of ETS complex I was 1.5-fold more phosphorylated in torpor. In vitro treatment with alkaline phosphatase increased the maximal activity of complex I from liver mitochondria isolated from torpid, but not IBE, animals. By contrast, phosphatase treatment decreased complex II activity in IBE but not torpor. These findings suggest that the rapid changes in mitochondrial metabolism in hibernators are mediated by posttranslational modifications of key metabolic enzymes, perhaps by intramitochondrial kinases and deacetylases.
Assuntos
Hibernação/fisiologia , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Animais , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Torpor/fisiologiaRESUMO
We discovered a previously undescribed orbital lipid depot in the thirteen-lined ground squirrel during the first ever magnetic resonance image (MRI) of this common experimental model of mammalian hibernation. In animals housed at constant ambient temperatures (5°C or 25°C, 12â h:12â h light:dark photoperiod), the volume of this depot increased in the autumn and decreased in the spring, suggesting an endogenous circannual pattern. Water-fat MRI revealed that throughout the year this depot is composed of â¼40% lipid, similar to brown adipose tissue (BAT). During arousal from torpor, thermal images showed higher surface temperatures near this depot before the rest of the head warmed, suggesting a thermoregulatory function. This depot, however, does not contain uncoupling protein 1, a BAT biomarker, or uncoupling protein 3. Histology shows blood vessels in close proximity to each other, suggesting it may serve as a vascular rete, perhaps to preferentially warm the eye and brain during arousals.