-
[show abstract]
[hide abstract]
ABSTRACT: Mammalian hibernation involves periods of substantial suppression of metabolic rate (torpor) allowing energy conservation during winter. In thirteen-lined ground squirrels (Ictidomys tridecemlineatus), suppression of liver mitochondrial respiration during entrance into torpor occurs rapidly (within 2 h) before core body temperature falls below 30°C, whereas reversal of this suppression occurs slowly during arousal from torpor. We hypothesized that this pattern of rapid suppression in entrance and slow reversal during arousal was related to changes in the phosphorylation state of mitochondrial enzymes during torpor catalyzed by temperature-dependent kinases and phosphatases. We compared mitochondrial protein phosphorylation among hibernation metabolic states using immunoblot analyses and assessed how phosphorylation related to mitochondrial respiration rates. No proteins showed torpor-specific changes in phosphorylation, nor did phosphorylation state correlate with mitochondrial respiration. However, several proteins showed seasonal (summer vs. winter) differences in phosphorylation of threonine or serine residues. Using MALDI-TOF/TOF MS, we identified three of these proteins: F1-ATPase α-chain, long-chain specific acyl-CoA dehydrogenase, and ornithine transcarbamylase. Therefore, we conclude that protein phosphorylation is likely a mechanism involved in bringing about seasonal changes in mitochondrial metabolism in hibernating ground squirrels, but it seems unlikely to play any role in acute suppression of mitochondrial metabolism during torpor.
Physiological Genomics 04/2013; · 2.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hibernation is a crucial strategy of winter survival used by many mammals. During hibernation, thirteen-lined ground squirrels, Ictidomys tridecemlineatus, cycle through a series of torpor bouts, each lasting more than a week, during which the animals are largely immobile. Previous hibernation studies have demonstrated that such natural models of skeletal muscle disuse cause limited or no changes in either skeletal muscle size or mechanical performance. However, work loop analysis of skeletal muscle, which provides a realistic assessment of in vivo power output, has not previously been undertaken in mammals that undergo prolonged torpor during hibernation. In the present study, our aim was to assess the effects of 3 months of hibernation on mechanical performance (using the work loop technique) and several biochemical properties that may affect performance. There was no significant difference in soleus muscle power output-cycle frequency curves between winter (torpid) and summer animals. Total antioxidant capacity of gastrocnemius muscle was 156% higher in torpid than summer animals, suggesting one potential mechanism for maintenance of acute muscle performance. Soleus muscle fatigue resistance was significantly lower in torpid than summer animals. Gastrocnemius muscle glycogen content was unchanged. However, state 3 and state 4 mitochondrial respiration rates were significantly suppressed, by 59% and 44% respectively, in mixed hind-limb skeletal muscle from torpid animals compared with summer controls. These findings in hind-limb skeletal muscles suggest that, although maximal mechanical power output is maintained in torpor, there is both suppression of ATP production capacity and reduced fatigue resistance.
Journal of Experimental Biology 03/2013; · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hibernating ground squirrels (Ictidomys tridecemlineatus) alternate between two distinct metabolic states throughout winter: torpor, during which metabolic rate (MR) and body temperature (T(b)) are considerably suppressed, and interbout euthermia (IBE), during which MR and T(b) briefly return to euthermic levels. Previous studies showed suppression of succinate-fueled respiration during torpor in liver and skeletal muscle mitochondria; however, these studies used only a single, saturating succinate concentration. Therefore, they could not address whether mitochondrial metabolic suppression occurs under physiological substrate concentrations or whether differences in the kinetics of mitochondrial responses to changing substrate concentration might also contribute to mitochondrial metabolic regulation during torpor. The present study confirmed that succinate oxidation is reduced during torpor in liver and skeletal muscle at 37°C and 10°C over a 100-fold range of succinate concentrations. At 37°C, this suppression resulted from inhibition of succinate dehydrogenase (SDH), which had a greater affinity for oxaloacetate (an SDH inhibitor) during torpor. At 10°C, SDH was not inhibited, suggesting that SDH inhibition initiates but does not maintain mitochondrial suppression during torpor. Moreover, in both liver and skeletal muscle, mitochondria from torpid animals maintained relatively higher respiration rates at low succinate concentrations, which reduces the extent of energy savings that can be achieved during torpor but may also maintain mitochondrial oxidative capacity above some lower critical threshold, thereby preventing cellular and/or mitochondrial injury during torpor and facilitating rapid recruitment of oxidative capacity during arousal.
Journal of Experimental Biology 01/2013; · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.
AJP Regulatory Integrative and Comparative Physiology 01/2012; 302(1):R15-28. · 3.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) appears to be due to a mismatch between ATP supply and demand in lamprey, depleting glycogen stores and starving the nervous system of ATP. The cause of this TFM-induced ATP deficit is unclear. One possibility is that TFM uncouples mitochondrial oxidative phosphorylation, thus impairing ATP production. To test this hypothesis, mitochondria were isolated from the livers of sea lamprey and rainbow trout, and O(2) consumption rates were measured in the presence of TFM or 2,4-dinitrophenol (2,4-DNP), a known uncoupler of oxidative phosphorylation. TFM and 2,4-DNP markedly increased State IV respiration in a dose-dependent fashion, but had no effect on State III respiration, which is consistent with uncoupling of oxidative phosphorylation. To determine how TFM uncoupled oxidative phosphorylation, the mitochondrial transmembrane potential (TMP) was recorded using the mitochondria-specific dye rhodamine 123. Mitochondrial TMP decreased by 22% in sea lamprey, and by 28% in trout following treatment with 50μmolL(-1) TFM. These findings suggest that TFM acted as a protonophore, dissipating the proton motive force needed to drive ATP synthesis. We conclude that the mode of TFM toxicity in sea lamprey and rainbow trout is via uncoupling of oxidative phosphorylation, leading to impaired ATP production.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 04/2011; 153(3):342-9. · 2.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: During fasting, mice (Mus musculus) undergo daily bouts of torpor, considerably reducing body temperature (T(b)) and metabolic rate (MR). We examined females of different laboratory strains (Balb/c, C57/6N, and CD1) to determine whether liver mitochondrial metabolism is actively reduced during torpor. In all strains, we found that state 3 (phosphorylating) respiration rate measured at 37 degrees C was reduced up to 35% during torpor for at least one of the substrates (glutamate and succinate) used to fuel respiration. The extent of this suppression varied and was correlated with T(b) at sampling. This suggests that, at the biochemical level, the transition to and from a hypometabolic torpid state is gradual. In fasted non-torpid animals, T(b) and MR still fluctuated greatly: T(b) dropped by as much as 4 degrees C and MR was reduced up to 25% compared to fed controls. Changes in T(b) and MR in fasted, non-torpid animals were correlated with changes in mitochondrial state 3 respiration rate measured at 37 degrees C. This suggests that fasting mice may conserve energy even when not torpid by occasionally reducing T(b) and mitochondrial oxidative capacity to reduce MR. Furthermore, proton conductance was higher in torpid compared to non-torpid animals when measured at 15 degrees C (the lower limit of torpid T(b)). This pattern is similar to that reported previously for daily torpor in Phodopus sungorus.
Biochimica et Biophysica Acta 04/2010; 1797(4):476-86. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Lower ROS release rate in long-lived species is likely caused by decreased reduction of electron transport chain (ETC) complexes, but how this is achieved remains largely unknown. We compared liver mitochondrial H(2)O(2) release rates among endotherms of comparable size and metabolic rate: house sparrow and big brown bat (both long-lived) and house mouse (short-lived). We hypothesized that low ROS release rates in long-lived species result from (i) lower mitochondrial respiration rate, (ii) increased mitochondrial proton conductance ('uncoupling to survive'), and/or (iii) increased ETC oxidative capacity ('spare oxidative capacity'). H(2)O(2) release rate was 70% lower in bats than mice despite similar respiration rates. Consistent with 'uncoupling to survive', proton leakiness was 3-fold higher in bats at membrane potentials above 130mV. Basal H(2)O(2) release rate and respiration rates were 2-fold higher in sparrows than mice. Consistent with 'spare oxidative capacity', subsaturating succinate decreased H(2)O(2) release rate in sparrows but not mice. Moreover, succinate:Cytochrome c oxidoreductase activity was 3-fold higher in sparrows, and ETC inhibitors increased ROS release rate 20-27-fold in sparrows (with glutamate or subsaturating succinate) but only 4-5-fold in mice. Taken together these data suggest that complexes I and III are less reduced under physiological conditions in sparrows. We conclude that different long-lived species may use distinct mechanisms to lower mitochondrial ROS release rate.
Mechanisms of ageing and development 06/2009; 130(8):467-76. · 4.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) were fed one of four isocaloric, isolipemic diets containing 16, 22, 35 or 55 mg linoleic acid (18:2n-6) per gram. Mitochondrial properties were compared between hibernating and summer active states, and between diet groups. As in other studies, state 3 respiration was significantly reduced in hibernation, but only in animals fed the 22 mg g(-1) 18:2 diet. In the other diet groups, there was no difference in state 3 respiration between the hibernating and summer active groups. In the 22 mg g(-1) 18:2 diet group, there was no difference in mitochondrial proton conductance between hibernating and summer active animals, again in agreement with earlier studies. However, for all other diet groups, mitochondrial proton conductance was significantly reduced during hibernation. Mitochondrial phospholipid fatty acids changed significantly with hibernation, including increases in unsaturation indices and n-6/n-3, but no differences were found among diet groups. Mitochondrial proton conductance in hibernation showed a positive correlation with the content of linoleic acid (18:2) and arachidonic acid (20:4) in mitochondrial phospholipids. Lipid peroxidation was higher in mitochondria from hibernating animals, probably due to higher unsaturation, but there was no effect of dietary 18:2 on this pattern. Despite the dietary effects on mitochondrial metabolism, all animals hibernated with no differences in bout durations, body temperatures or whole-animal metabolic rates among the diet groups. The reduced mitochondrial proton leak in the 15, 35 and 55 mg g(-1) 18:2 diet groups might compensate for the inability to suppress respiration, permitting whole-animal energy savings over the hibernation season.
Journal of Experimental Biology 09/2008; 211(Pt 16):2689-99. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: During daily torpor in the dwarf Siberian hamster, Phodopus sungorus, metabolic rate is reduced by 65% compared with the basal rate, but the mechanisms involved are contentious. We examined liver mitochondrial respiration to determine the possible role of active regulated changes and passive thermal effects in the reduction of metabolic rate. When assayed at 37 degrees C, state 3 (phosphorylating) respiration, but not state 4 (nonphosphorylating) respiration, was significantly lower during torpor compared with normothermia, suggesting that active regulated changes occur during daily torpor. Using top-down elasticity analysis, we determined that these active changes in torpor included a reduced substrate oxidation capacity and an increased proton conductance of the inner mitochondrial membrane. At 15 degrees C, mitochondrial respiration was at least 75% lower than at 37 degrees C, but there was no difference between normothermia and torpor. This implies that the active regulated changes are likely more important for reducing respiration at high temperatures (i.e., during entrance) and/or have effects other than reducing respiration at low temperatures. The decrease in respiration from 37 degrees C to 15 degrees C resulted predominantly from a considerable reduction of substrate oxidation capacity in both torpid and normothermic animals. Temperature-dependent changes in proton leak and phosphorylation kinetics depended on metabolic state; proton leakiness increased in torpid animals but decreased in normothermic animals, whereas phosphorylation activity decreased in torpid animals but increased in normothermic animals. Overall, we have shown that both active and passive changes to oxidative phosphorylation occur during daily torpor in this species, contributing to reduced metabolic rate.
AJP Regulatory Integrative and Comparative Physiology 12/2007; 293(5):R1833-45. · 3.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Abstract Daily torpor results in an ∼70% decrease in metabolic rate (MR) and a 20%-70% decrease in state 3 (phosphorylating) respiration rate of isolated liver mitochondria in both dwarf Siberian hamsters and mice even when measured at 37°C. This study investigated whether mitochondrial metabolic suppression also occurs in these species during euthermic fasting, when MR decreases significantly but torpor is not observed. State 3 respiration rate measured at 37°C was 20%-30% lower in euthermic fasted animals when glutamate but not succinate was used as a substrate. This suggests that electron transport chain complex I is inhibited during fasting. We also investigated whether mitochondrial metabolic suppression alters mitochondrial reactive oxygen species (ROS) production. In both torpor and euthermic fasting, ROS production (measured as H(2)O(2) release rate) was lower with glutamate in the presence (but not absence) of rotenone when measured at 37°C, likely reflecting inhibition at or upstream of the complex I ROS-producing site. ROS production with succinate (with rotenone) increased in torpor but not euthermic fasting, reflecting complex II inhibition during torpor only. Finally, mitochondrial ROS production was twofold more temperature sensitive than mitochondrial respiration (as reflected by Q(10) values). These data suggest that electron leak from the mitochondrial electron transport chain, which leads to ROS production, is avoided more efficiently at the lower body temperatures experienced during torpor.
Physiological and Biochemical Zoology 84(5):467-80. · 2.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: During fasting, mice (Mus musculus) undergo daily bouts of torpor, considerably reducing body temperature (Tb) and metabolic rate (MR). We examined females of different laboratory strains (Balb/c, C57/6N, and CD1) to determine whether liver mitochondrial metabolism is actively reduced during torpor. In all strains, we found that state 3 (phosphorylating) respiration rate measured at 37 °C was reduced up to 35% during torpor for at least one of the substrates (glutamate and succinate) used to fuel respiration. The extent of this suppression varied and was correlated with Tb at sampling. This suggests that, at the biochemical level, the transition to and from a hypometabolic torpid state is gradual. In fasted non-torpid animals, Tb and MR still fluctuated greatly: Tb dropped by as much as 4 °C and MR was reduced up to 25% compared to fed controls. Changes in Tb and MR in fasted, non-torpid animals were correlated with changes in mitochondrial state 3 respiration rate measured at 37 °C. This suggests that fasting mice may conserve energy even when not torpid by occasionally reducing Tb and mitochondrial oxidative capacity to reduce MR. Furthermore, proton conductance was higher in torpid compared to non-torpid animals when measured at 15 °C (the lower limit of torpid Tb). This pattern is similar to that reported previously for daily torpor in Phodopus sungorus.
Biochimica et Biophysica Acta (BBA) - Bioenergetics.
-
[show abstract]
[hide abstract]
ABSTRACT: The toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) appears to be due to a mismatch between ATP supply and demand in lamprey, depleting glycogen stores and starving the nervous system of ATP. The cause of this TFM-induced ATP deficit is unclear. One possibility is that TFM uncouples mitochondrial oxidative phosphorylation, thus impairing ATP production. To test this hypothesis, mitochondria were isolated from the livers of sea lamprey and rainbow trout, and O2 consumption rates were measured in the presence of TFM or 2,4-dinitrophenol (2,4-DNP), a known uncoupler of oxidative phosphorylation. TFM and 2,4-DNP markedly increased State IV respiration in a dose-dependent fashion, but had no effect on State III respiration, which is consistent with uncoupling of oxidative phosphorylation. To determine how TFM uncoupled oxidative phosphorylation, the mitochondrial transmembrane potential (TMP) was recorded using the mitochondria-specific dye rhodamine 123. Mitochondrial TMP decreased by 22% in sea lamprey, and by 28% in trout following treatment with 50 μmol L− 1 TFM. These findings suggest that TFM acted as a protonophore, dissipating the proton motive force needed to drive ATP synthesis. We conclude that the mode of TFM toxicity in sea lamprey and rainbow trout is via uncoupling of oxidative phosphorylation, leading to impaired ATP production.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology.
-
[show abstract]
[hide abstract]
ABSTRACT: Lower ROS release rate in long-lived species is likely caused by decreased reduction of electron transport chain (ETC) complexes, but how this is achieved remains largely unknown. We compared liver mitochondrial H2O2 release rates among endotherms of comparable size and metabolic rate: house sparrow and big brown bat (both long-lived) and house mouse (short-lived). We hypothesized that low ROS release rates in long-lived species result from (i) lower mitochondrial respiration rate, (ii) increased mitochondrial proton conductance (‘uncoupling to survive’), and/or (iii) increased ETC oxidative capacity (‘spare oxidative capacity’). H2O2 release rate was 70% lower in bats than mice despite similar respiration rates. Consistent with ‘uncoupling to survive’, proton leakiness was 3-fold higher in bats at membrane potentials above 130 mV. Basal H2O2 release rate and respiration rates were 2-fold higher in sparrows than mice. Consistent with ‘spare oxidative capacity’, subsaturating succinate decreased H2O2 release rate in sparrows but not mice. Moreover, succinate:Cytochrome c oxidoreductase activity was 3-fold higher in sparrows, and ETC inhibitors increased ROS release rate 20–27-fold in sparrows (with glutamate or subsaturating succinate) but only 4–5-fold in mice. Taken together these data suggest that complexes I and III are less reduced under physiological conditions in sparrows. We conclude that different long-lived species may use distinct mechanisms to lower mitochondrial ROS release rate.
Mechanisms of Ageing and Development.
