RESUMO
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
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
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
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
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
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
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
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
Obligate hibernators express circannual patterns of body mass and hibernation, which persist under constant laboratory conditions. Brown adipose tissue (BAT) is important for thermogenesis during arousals from hibernation, whereas white adipose tissue (WAT) serves as energy storage and thermal insulation. The goal of this study was to investigate the effects of environmental temperature on BAT and WAT. We hypothesized that changes to environmental temperature would not influence the pattern of mass gain or BAT and WAT volume in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). To test this, we housed animals at thermoneutral 25°C (warm-housed) or 5°C (cold-housed), with the same photoperiod (12â h light:12â h dark) over an entire year. Throughout the year we measured the volume and water:fat ratio of WAT and BAT using magnetic resonance imaging (MRI). We found no evidence of torpor in the warm-housed animals, indicating that this species might not be an obligate hibernator, as previously assumed. Regardless of ambient temperature, BAT volume increased prior to winter, then decreased in late winter with no change in water:fat ratio. By contrast, both body mass and WAT volume of cold-housed animals declined throughout the winter and recovered after hibernation, but thermoneutral housing produced no circannual pattern in body mass, even though WAT volume declined in late winter. Cold exposure appears to be a primary regulator for WAT but BAT may exhibit an endogenous circannual rhythm in terms of depot volume.
Assuntos
Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Branco/crescimento & desenvolvimento , Temperatura Baixa , Hibernação , Temperatura Alta , Sciuridae/fisiologia , Animais , Masculino , Fotoperíodo , Sciuridae/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Tecido Adiposo Marrom/fisiologia , Lipídeos/fisiologia , Sciuridae/fisiologia , Tecido Adiposo Marrom/irrigação sanguínea , Animais , Hibernação , Lipídeos/análise , Imageamento por Ressonância Magnética , Masculino , Estações do AnoRESUMO
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
We used electrocardiogram (ECG) telemeters to measure the heart rate of hibernating Ictidomys tridecemlineatus (thirteen-lined ground squirrel). An increase in heart rate from 2.2 to 5â beatsâ min-1 accurately identified arousal from torpor before any change in body temperature was detected. Variability in raw heart rate data was significantly reduced by a forward-backward Butterworth low-pass filter, allowing for discrete differential analysis. A decrease in filtered heart rate to 70% of maximum values in interbout euthermia (from approximately 312 to 235â beatsâ min-1) accurately detected entrance into torpor bouts. At this point, body temperature had fallen from 36.1°C to only 34.7°C, much higher than the 30°C typically used to identify entrance. Using these heart rate criteria allowed advanced detection of entrance and arousal (detected 51.9 and 76â min earlier, respectively), compared with traditional body temperature criteria. This method will improve our ability to detect biochemical and molecular markers underlying these transition periods, during which many physiological changes occur.
Assuntos
Nível de Alerta , Eletrocardiografia/métodos , Frequência Cardíaca , Fisiologia/métodos , Sciuridae/fisiologia , Torpor , Animais , Feminino , Hibernação , MasculinoRESUMO
Although seasonal modifications of brown adipose tissue (BAT) in hibernators are well documented, we know little about functional regulation of BAT in different phases of hibernation. In the 13-lined ground squirrel, liver mitochondrial respiration is suppressed by up to 70% during torpor. This suppression is reversed during arousal and interbout euthermia (IBE), and corresponds with patterns of maximal activities of electron transport system (ETS) enzymes. Uncoupling of BAT mitochondria is controlled by free fatty acid release stimulated by sympathetic activation of adipocytes, so we hypothesized that further regulation at the level of the ETS would be of little advantage. As predicted, maximal ETS enzyme activities of isolated BAT mitochondria did not differ between torpor and IBE. In contrast to this pattern, respiration rates of mitochondria isolated from torpid individuals were suppressed by ~60% compared with rates from IBE individuals when measured at 37°C. At 10°C, however, mitochondrial respiration rates tended to be greater in torpor than IBE. As a result, the temperature sensitivity (Q10) of mitochondrial respiration was significantly lower in torpor (~1.4) than IBE (~2.4), perhaps facilitating energy savings during entrance into torpor and thermogenesis at low body temperatures. Despite the observed differences in isolated mitochondria, norepinephrine-stimulated respiration rates of isolated BAT adipocytes did not differ between torpor and IBE, perhaps because the adipocyte isolation requires lengthy incubation at 37°C, potentially reversing any changes that occur in torpor. Such changes may include remodeling of BAT mitochondrial membrane phospholipids, which could change in situ enzyme activities and temperature sensitivities.
Assuntos
Tecido Adiposo Marrom/fisiologia , Temperatura Corporal/fisiologia , Mitocôndrias Hepáticas/fisiologia , Consumo de Oxigênio/fisiologia , Sciuridae/fisiologia , Torpor/fisiologia , Adaptação Fisiológica/fisiologia , Tecido Adiposo Marrom/ultraestrutura , Animais , Nível de Alerta/fisiologia , Hibernação/fisiologia , Fígado/fisiologia , Fígado/ultraestruturaRESUMO
Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.
Assuntos
Crassostrea/enzimologia , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/enzimologia , Animais , Crassostrea/genética , Transporte de Elétrons , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Doenças Mitocondriais/terapia , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
Low temperature tolerance is the main predictor of variation in the global distribution and performance of insects, yet the molecular mechanisms underlying cold tolerance variation are poorly known, and it is unclear whether the mechanisms that improve cold tolerance within the lifetime of an individual insect are similar to those that underlie evolved differences among species. The accumulation of cold-induced injuries by hemimetabolous insects is associated with loss of Na(+) and K(+) homeostasis. Here we show that this model holds true for Drosophila; cold exposure increases haemolymph [K(+)] in D. melanogaster, and cold-acclimated flies maintain low haemolymph [Na(+)] and [K(+)], both at rest and during a cold exposure. This pattern holds across 24 species of the Drosophila phylogeny, where improvements in cold tolerance have been consistently paired with reductions in haemolymph [Na(+)] and [K(+)]. Cold-acclimated D. melanogaster have low activity of Na(+)/K(+)-ATPase, which may contribute to the maintenance of low haemolymph [Na(+)] and underlie improvements in cold tolerance. Modifications to ion balance are associated with both phenotypic plasticity within D. melanogaster and evolutionary differences in cold tolerance across the Drosophila phylogeny, which suggests that adaptation and acclimation of cold tolerance in insects may occur through similar mechanisms. Cold-tolerant flies maintain haemolymph osmolality despite low haemolymph [Na(+)] and [K(+)], possibly through modest accumulations of organic osmolytes. We propose that this could have served as an evolutionary route by which chill-susceptible insects developed more extreme cold tolerance strategies.
Assuntos
Drosophila melanogaster/fisiologia , Aclimatação , Adaptação Fisiológica , Animais , Evolução Biológica , Temperatura Baixa , Hemolinfa/metabolismo , Filogenia , ATPase Trocadora de Sódio-Potássio/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
The time required to recover from cold-induced paralysis (chill-coma) is a common measure of insect cold tolerance used to test central questions in thermal biology and predict the effects of climate change on insect populations. The onset of chill-coma in the fall field cricket (Gryllus pennsylvanicus, Orthoptera: Gryllidae) is accompanied by a progressive drift of Na(+) and water from the hemolymph to the gut, but the physiological mechanisms underlying recovery from chill-coma are not understood for any insect. Using a combination of gravimetric methods and atomic absorption spectroscopy, we demonstrate that recovery from chill-coma involves a reestablishment of hemolymph ion content and volume driven by removal of Na(+) and water from the gut. Recovery is associated with a transient elevation of metabolic rate, the time span of which increases with increasing cold exposure duration and closely matches the duration of complete osmotic recovery. Thus, complete recovery from chill-coma is metabolically costly and encompasses a longer period than is required for the recovery of muscle potentials and movement. These findings provide evidence that physiological mechanisms of hemolymph ion content and volume regulation, such as ion-motive ATPase activity, are instrumental in chill-coma recovery and may underlie natural variation in insect cold tolerance.
Assuntos
Gryllidae/fisiologia , Aclimatação/fisiologia , Animais , Clima Frio , Fenômenos Fisiológicos do Sistema Digestório , Feminino , Hemolinfa/fisiologia , Homeostase , Transporte de Íons , Modelos Biológicos , Contração Muscular/fisiologia , Potássio/metabolismo , Sódio/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
Pulmonary surfactant lines the entire alveolar surface, serving primarily to reduce the surface tension at the air-liquid interface. Surfactant films adsorb as a monolayer interspersed with multilayers with surfactant lipids segregating into different phases or domains. Temperature variation, which influences lipid physical properties, affects both the lipid phase segregation and the surface activity of surfactants. In hibernating animals, such as 13-lined ground squirrels, which vary their body temperature, surfactant must be functional over a wide range of temperatures. We hypothesised that surfactant from the 13-lined ground squirrel, Ictidomys tridecemlineatus, would undergo appropriate lipid structural re-arrangements at air-water interfaces to generate phase separation, sufficient to attain the low surface tensions required to remain stable at both low and high body temperatures. Here, we examined pressure-area isotherms at 10, 25 and 37°C and found that surfactant films from both hibernating and summer-active squirrels reached their highest surface pressure on the Wilhelmy-Langmuir balance at 10°C. Epifluorescence microscopy demonstrated that films of hibernating squirrel surfactant display different lipid micro-domain organisation characteristics than surfactant from summer-active squirrels. These differences were also reflected at the nanoscale as determined by atomic force microscopy. Such re-arrangement of lipid domains in the relatively more fluid surfactant films of hibernating squirrels may contribute to overcoming collapse pressures and support low surface tension during the normal breathing cycle at low body temperatures.
Assuntos
Adaptação Fisiológica , Hibernação/fisiologia , Lipídeos/química , Surfactantes Pulmonares/química , Animais , Microscopia de Força Atômica , Sciuridae , Propriedades de Superfície , Tensão Superficial , TemperaturaRESUMO
Hibernation evolved in some small mammals that live in cold environments, presumably to conserve energy when food supplies are low. Throughout the winter, hibernators cycle spontaneously between torpor, with low metabolism and near-freezing body temperatures, and euthermia, with high metabolism and body temperatures near 37°C. Understanding the mechanisms underlying this natural model of extreme metabolic plasticity is important for fundamental and applied science. During entrance into torpor, reductions in metabolic rate begin before body temperatures fall, even when thermogenesis is not active, suggesting active mechanisms of metabolic suppression, rather than passive thermal effects. Mitochondrial respiration is suppressed during torpor, especially when measured in liver mitochondria fuelled with succinate at 37°C in vitro. This suppression of mitochondrial metabolism appears to be invoked quickly during entrance into torpor when body temperature is high, but is reversed slowly during arousal when body temperature is low. This pattern may reflect body temperature-sensitive, enzyme-mediated post-translational modifications of oxidative phosphorylation complexes, for instance by phosphorylation or acetylation.
Assuntos
Hibernação , Mamíferos/fisiologia , Mitocôndrias/metabolismo , Animais , Respiração CelularRESUMO
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.