Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions

1994 ◽  
Vol 72 (8) ◽  
pp. 899-908 ◽  
Author(s):  
Mary-Ellen Harper ◽  
Martin D. Brand
Keyword(s):  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Thyroid Hormones ◽  
Rat Hepatocytes ◽  
Substrate Oxidation ◽  
Proton Leak ◽  
Control Analysis ◽  
Top Down ◽  
Sites Of Action ◽  
Kinetics Of

Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for −2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (Δψm) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.Key words: thyroid hormones, oxidative phosphorylation, mitochondria, proton leak, thermogenesis.

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

2004 ◽  
Vol 287 (4) ◽  
pp. R794-R800 ◽  
Author(s):  
M. E. Chamberlin
Keyword(s):  
Oxygen Consumption ◽  
Membrane Potential ◽  
Oxidative Phosphorylation ◽  
Substrate Oxidation ◽  
The State ◽  
Proton Leak ◽  
Control Analysis ◽  
Top Down ◽  
Temperature Ranges ◽  
Oxidation System

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm ( Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35°C. State 4 respiration displayed a Q10 of 2.4–2.7 when measured over two temperature ranges (15–25°C and 25–35°C). In state 3, the Q10s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.

Age-related increase in mitochondrial proton leak and decrease in ATP turnover reactions in mouse hepatocytes

1998 ◽  
Vol 275 (2) ◽  
pp. E197-E206 ◽  
Author(s):  
Mary-Ellen Harper ◽  
Shadi Monemdjou ◽  
Jon J. Ramsey ◽  
Richard Weindruch
Keyword(s):  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Metabolic Control ◽  
Respiratory Control ◽  
Substrate Oxidation ◽  
Membrane Lipid ◽  
Proton Leak ◽  
Control Analysis ◽  
Atp Turnover ◽  
Age Related

Age-related changes in mitochondria, including decreased respiratory control ratios and altered mitochondrial inner membrane lipid composition, led us to study oxidative phosphorylation in hepatocytes from old (30 mo) and young (3 mo) male C57BL/J mice. Top-down metabolic control analysis and its extension, elasticity analysis, were used to identify changes in the control and regulation of the three blocks of reactions constituting the oxidative phosphorylation system: substrate oxidation, mitochondrial proton leak, and the ATP turnover reactions. Resting oxygen consumption of cells from old mice was 15% lower ( P < 0.05) than in young cells. This is explained entirely by a decrease in oxygen consumption supporting ATP turnover reactions. At all values of mitochondrial membrane potential assessed, the proportion of total oxygen consumption used to balance the leak was greater in the old cells than in the young cells. Metabolic control coefficients indicate a shift in control over respiration and phosphorylation away from substrate oxidation toward increased control by leak and by ATP turnover reactions. Control of the actual number of ATP molecules synthesized by mitochondria for each oxygen atom consumed by the ATP turnover and leak reactions was greater in old than in young cells, showing that efficiency in older cells is more sensitive to changes in these two blocks of reactions than in young cells.

scholarly journals Control of oxidative phosphorylation during insect metamorphosis

2004 ◽  
Vol 287 (2) ◽  
pp. R314-R321 ◽  
Author(s):  
M. E. Chamberlin
Keyword(s):  
Membrane Potential ◽  
Oxidative Phosphorylation ◽  
Adenine Nucleotide ◽  
Substrate Oxidation ◽  
Proton Leak ◽  
Protonmotive Force ◽  
Control Analysis ◽  
Membrane Area ◽  
Early Stages ◽  
Oxidation System

The midgut of the tobacco hornworm ( Manduca sexta) is a highly aerobic tissue that is destroyed and replaced by a pupal epithelium at metamorphosis. To determine how oxidative phosphorylation is altered during the programmed death of the larval cells, top-down control analysis was performed on mitochondria isolated from the midguts of larvae before and after the commitment to pupation. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored to determine the kinetic responses of the substrate oxidation system, proton leak, and phosphorylation system to changes in the membrane potential. Mitochondria from precommitment larvae have higher respiration rates than those from postcommitment larvae. State 4 respiration is controlled by the proton leak and the substrate oxidation system. In state 3, the substrate oxidation system exerted 90% of the control over respiration, and this high level of control did not change with development. Elasticity analysis, however, revealed that, after commitment, the activity of the substrate oxidation system falls. This decline may be due, in part, to a loss of cytochrome c from the mitochondria. There are no differences in the kinetics of the phosphorylation system, indicating that neither the F1F0 ATP synthase nor the adenine nucleotide translocase is affected in the early stages of metamorphosis. An increase in proton conductance was observed in mitochondria isolated from postcommitment larvae, indicating that membrane area, lipid composition, or proton-conducting proteins may be altered during the early stages of the programmed cell death of the larval epithelium.

scholarly journals Top-down control analysis of temperature effect on oxidative phosphorylation

1996 ◽  
Vol 314 (3) ◽  
pp. 743-751 ◽  
Author(s):  
Sylvie DUFOUR ◽  
Nicole ROUSSE ◽  
Paul CANIONI ◽  
Philippe DIOLEZ
Keyword(s):  
Membrane Potential ◽  
Oxidative Phosphorylation ◽  
Substrate Oxidation ◽  
Leakage Rate ◽  
Effect Of Temperature ◽  
Rat Liver Mitochondria ◽  
Protonmotive Force ◽  
Control Analysis ◽  
Top Down ◽  
Proton Leakage

The effects of temperature on the control of respiration rate, phosphorylation rate, proton leakage rate, the protonmotive force and the effective ATP/O ratio were determined in isolated rat liver mitochondria over a range of respiratory conditions by applying top-down elasticity and control analyses. Simultaneous measurements of membrane potential, oxidation and phosphorylation rates were performed under various ATP turnover rates, ranging from state 4 to state 3. Although the activities of the three subsystems decreased with temperature (over 30-fold between 37 and 4 °C), the effective ATP/O ratio exhibited a maximum at 25 °C, far below the physiological value. Top-down elasticity analysis revealed that maximal membrane potential was maintained over the range of temperature studied, and that the proton leakage rate was considerably reduced at 4 °C. These results definitely rule out a possible uncoupling of mitochondria at low temperature. At 4 °C, the decrease in ATP/O ratio is explained by the relative decrease in phosphorylation processes revealed by the decrease in depolarization after ADP addition [Diolez and Moreau (1985) Biochim. Biophys. Acta 806, 56–63]. The change in depolarization between 37 and 25 °C was too small to explain the decrease in ATP/O ratio. This result is best explained by the changes in the elasticity of proton leakage to membrane potential between 37 and 25 °C, leading to a higher leak rate at 37 °C for the same value of membrane potential. Top-down control analysis showed that despite the important changes in activities of the three subsystems between 37 and 25 °C, the patterns of the control distribution are very similar. However, a different pattern was obtained at 4 °C under all phosphorylating conditions. Surprisingly, control by the proton leakage subsystem was almost unchanged, although both control patterns by substrate oxidation and phosphorylation subsystems were affected at 4 °C. In comparison with results for 25 and 37 °C, at 4 °C there was evidence for increased control by the phosphorylation subsystem over both fluxes of oxidation and phosphorylation as well as on the ATP/O ratio when the system is close to state 3. However, the pattern of control coefficients as a function of mitochondrial activity also showed enhanced control exerted by the substrate oxidation subsystem under all intermediate conditions. These results suggest that passive membrane permeability to protons is not involved in the effect of temperature on the control of oxidative phosphorylation.

scholarly journals Control of the effective P/O ratio of oxidative phosphorylation in liver mitochondria and hepatocytes

1993 ◽  
Vol 291 (3) ◽  
pp. 739-748 ◽  
Author(s):  
M D Brand ◽  
M E Harper ◽  
H C Taylor
Keyword(s):  
Oxygen Consumption ◽  
Liver Mitochondria ◽  
Substrate Oxidation ◽  
Proton Leak ◽  
Rat Liver Mitochondria ◽  
Protonmotive Force ◽  
Control Analysis ◽  
Control Coefficient ◽  
Oxidation Reactions ◽  
In Cells

The control exerted by substrate oxidation reactions, by ATP turnover and by the proton leak over the oxygen consumption rate, the phosphorylation rate, the proton leak rate and the protonmotive force (delta p) in isolated rat liver mitochondria under a range of conditions between non-phosphorylating (State 4) and maximum phosphorylation (State 3) was investigated by using the top-down approach of metabolic control analysis. The experiments were carried out with saturating concentrations of the substrates succinate, glutamate with malate, or pyruvate with malate. The distribution of control was very similar with each of the three substrates. The effective P/O ratio (i.e. not corrected for leak reactions) was also measured; it varied from zero in State 4 to 80-90% of the maximum theoretical P/O ratio in State 3. Under most conditions control over the effective P/O ratio was shared between proton leak (which had negative control) and the phosphorylating subsystem (which had roughly equal positive control); near State 4, substrate oxidation reactions also acquired some control over this ratio. In resting hepatocytes the effective P/O ratio was only 50% of its maximum theoretical value, corresponding to an effective P/O ratio of only 1.3 for complete oxidation of glucose. The effective P/O ratio for intracellular mitochondrial oxygen consumption was 64% of the maximum value. The control coefficient of the mitochondrial proton leak over the effective P/O ratio in cells was -0.34; the control coefficient of phosphorylation reactions over this ratio was 0.31 and the control coefficient of substrate oxidation reactions over the ratio was 0.03, showing how the coupling efficiency in cells can respond sensitively to agents that change the proton leak or the ATP demand, but not to those that change substrate oxidation.

Top-down control analysis of the cadmium effects on molluscan mitochondria and the mechanisms of cadmium-induced mitochondrial dysfunction

2011 ◽  
Vol 300 (1) ◽  
pp. R21-R31 ◽  
Author(s):  
Ilya O. Kurochkin ◽  
Markus Etzkorn ◽  
David Buchwalter ◽  
Larry Leamy ◽  
Inna M. Sokolova
Keyword(s):  
Mitochondrial Function ◽  
Adenine Nucleotide ◽  
Eastern Oyster ◽  
Substrate Oxidation ◽  
Proton Leak ◽  
Control Analysis ◽  
Proton Conductance ◽  
Inner Mitochondrial Membrane ◽  
Top Down ◽  
Atp Production

Cadmium (Cd) is a toxic metal and an important environmental pollutant that can strongly affect mitochondrial function and bioenergetics in animals. We investigated the mechanisms of Cd action on mitochondrial function of a marine mollusk (the eastern oyster Crassostrea virginica ) by performing a top-down control analysis of the three major mitochondrial subsystems (substrate oxidation, proton leak, and phosphorylation). Our results showed that the substrate oxidation and proton leak subsystems are the main targets for Cd toxicity in oyster mitochondria. Exposure to 12.5 μM Cd strongly inhibited the substrate oxidation subsystem and stimulated the proton conductance across the inner mitochondrial membrane. Proton conductance was also elevated and substrate oxidation inhibited by Cd in the presence of a mitochondrially targeted antioxidant, MitoVitE, indicating that Cd effects on these subsystems were to a large extent ROS independent. Cd did not affect the kinetics of the phosphorylation system, indicating that it has negligible effects on F1, FO ATP synthase and/or the adenine nucleotide transporter in oyster mitochondria. Cd exposure altered the patterns of control over mitochondrial respiration, increasing the degree of control conferred by the substrate oxidation subsystem, especially in resting (state 4) mitochondria. Taken together, these data suggest that Cd-induced decrease of mitochondrial efficiency and ATP production are predominantly driven by the high sensitivity of substrate oxidation and proton leak subsystems to this metal.

scholarly journals Effect of 3,5-di-iodo-L-thyronine on the mitochondrial energy-transduction apparatus

1998 ◽  
Vol 330 (1) ◽  
pp. 521-526 ◽  
Author(s):  
Assonta LOMBARDI ◽  
Antonia LANNI ◽  
Maria MORENO ◽  
D. Martin BRAND ◽  
Fernando GOGLIA
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Respiration ◽  
Atp Synthesis ◽  
Substrate Oxidation ◽  
Energy Transduction ◽  
Energy Utilization ◽  
Proton Leak ◽  
Elasticity Analysis ◽  
Top Down ◽  
Respiratory State

We examined the effect of a single injection of 3,5-di-iodo-l-thyronine (3,5-T2) (150 μg/100 g body weight) on the rat liver mitochondrial energy-transduction apparatus. We applied ‘top-down’ elasticity analysis, which allows identification of the site of action of an effector within a metabolic pathway. This kinetic approach considers oxidative phosphorylation as two blocks of reactions: those generating the mitochondrial inner-membrane potential (Δψ; ‘substrate oxidation’) and those ‘consuming’ it (‘proton leak’ and ‘phosphorylating system’). The results show that 1 h after the injection of 3,5-T2, state 4 (respiratory state in which there is no ATP synthesis and the exogenous ADP added has been exhausted) and state 3 (respiratory state in which ATP synthesis is at maximal rate) of mitochondrial respiration were significantly increased (by approx. 30%). ‘Top-down’ elasticity analysis revealed that these increases were due to the stimulation of reactions involved in substrate oxidation; neither ‘proton leak’ nor the ‘phosphorylating system’ was influenced by 3,5-T2. Using the same approach we divided the respiratory chain into two blocks of reactions: cytochrome c reducers and cytochrome c oxidizers. We found that both cytochrome c reducers and cytochrome c oxidizers are targets for 3,5-T2. The rapidity with which 3,5-T2 acts in stimulating the mitochondrial respiration rate suggests to us that di-iodo-L-thyronine may play an important role in the physiological conditions in which rapid energy utilization is required, such as cold exposure or overfeeding.

scholarly journals Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica

2021 ◽  
Author(s):  
Jennifer B. M. Steffen ◽  
Fouzia Haider ◽  
Eugene P. Sokolov ◽  
Christian Bock ◽  
Inna M. Sokolova
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Substrate Oxidation ◽  
Hypoxia Tolerance ◽  
Oxygen Flux ◽  
Proton Leak ◽  
Control Analysis ◽  
Oxygen Species ◽  
Arctica Islandica ◽  
Normoxic Control

Oxygen fluctuations are common in marine waters, and hypoxia/reoxygenation (H/R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h &lt;0.01% O­2) and subsequent reoxygenation (1.5 h 21% O­2) compared to normoxic control animals (21% O­2). We demonstrated that flux capacities of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased oxygen flux capacities of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H/R stress, whereas the control of the proton leak subsystem over LEAK respiration increased during hypoxia and returned to normoxic level during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, while suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

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