Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria

Redox states of the mitochondrial coenzyme Q pool, which reacts with the electron transfer system, reflect the balance between (1) reducing capacities of electron flow from fuel substrates converging at the Q-junction, (2) oxidative capacities downstream of Q to O2, and (3) the load on the OXPHOS system utilizing or dissipating the protonmotive force. A three-electrode sensor (Rich 1988; Moore et al 1988) was implemented into the NextGen-O2k to monitor continuously the redox state of CoQ2 added as a Q-mimetic simultaneously with O2 consumption. The Q-Module was optimized for high signal-to-noise ratio, minimum drift, and minimum oxygen diffusion. CoQ2 equilibrates in the same manner as Q at Complexes CI, CII and CIII. The CoQ2 redox state is monitored amperometrically with the working electrode, which is poised at CoQ2 redox peak potentials determined by cyclic voltammetry. The voltammogram also provides quality control of the Q-sensor and reveals chemical interferences. The CoQ2 redox state and O2 consumption were measured simultaneously in isolated mouse cardiac and brain mitochondria. CoQ2 ― and by implication mitochondrial Q ― was more oxidized when O2 flux was stimulated by coupling control: when energy demand increased from LEAK to OXPHOS and electron transfer capacities in the succinate pathway. In contrast, CoQ2 was more reduced when O2 flux was stimulated by pathway-control of electron input capacities, increasing from the NADH (N)- to succinate (S)-linked pathway which converge at the Q-junction, with CI-Q-CIII and CII-Q-CIII segments, respectively. N- and S- respiratory pathway capacities were not completely additive, compatible with partitioning of Q intermediary between the solid-state and liquid-state models of supercomplex organization. The direct proportionality of CoQ2 reduction and electron input capacities through the CI-Q-CIII and CII-Q-CIII segments suggests that CoQ2 is accurately mimicking mitochondrial Q-redox changes

Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle

AIMS/HYPOTHESIS: Insulin resistance and type 2 diabetes are associated with mitochondrial dysfunction. The aim of the present study was to test the hypothesis that oxidative phosphorylation and electron transport capacity are diminished in the skeletal muscle of type 2 diabetic subjects, as a result of a reduction in the mitochondrial content. MATERIALS AND METHODS: The O(2) flux capacity of permeabilised muscle fibres from biopsies of the quadriceps in healthy subjects (n = 8; age 58 ± 2 years [mean±SEM]; BMI 28 ± 1 kg/m(2); fasting plasma glucose 5.4 ± 0.2 mmol/l) and patients with type 2 diabetes (n = 11; age 62 ± 2 years; BMI 32 ± 2 kg/m(2); fasting plasma glucose 9.0 ± 0.8 mmol/l) was measured by high-resolution respirometry. RESULTS: O(2) flux expressed per mg of muscle (fresh weight) during ADP-stimulated state 3 respiration was lower (p < 0.05) in patients with type 2 diabetes in the presence of complex I substrate (glutamate) (31 ± 2 vs 43 ± 3 pmol O(2) s(−1) mg(−1)) and in response to glutamate + succinate (parallel electron input from complexes I and II) (63 ± 3 vs 85 ± 6 pmol s(−1) mg(−1)). Further increases in O(2) flux capacity were observed in response to uncoupling by FCCP, but were again lower (p < 0.05) in type 2 diabetic patients than in healthy control subjects (86 ± 4 vs 109 ± 8 pmol s(−1) mg(−1)). However, when O(2) flux was normalised for mitochondrial DNA content or citrate synthase activity, there were no differences in oxidative phosphorylation or electron transport capacity between patients with type 2 diabetes and healthy control subjects. CONCLUSIONS/INTERPRETATION: Mitochondrial function is normal in type 2 diabetes. Blunting of coupled and uncoupled respiration in type 2 diabetic patients can be attributed to lower mitochondrial content

Observations on the distribution, daytime migrations and daily food intake rates of Chaoborus flavicans (Meigen) in the Goggausee [Translation from: Carinthia II 165(85), 184-190, 1975]

The Goggausee, in spite of its modest depth (Zmax = 12 metres), shows meromictic properties: autumn and spring circulation extend only to a depth of 8 metres. The water layers below about 10 metres are constantly oxygen-free, the critical zone with at least intermittent oxygen loss lies at a depth of between 6 and 10 metres. A limnological excursion in May 1974 offered an opportunity to investigate the daily vertical migration of the species Chaoborus flavicans with reference to its food supply of zooplankton as well as the chance to carry out some preliminary experiments on its rate of food intake. Among the studied features were the planktonic depth distribution of Chaoborus flavicans and the food intake of Chaoborus larvae under experimental conditions

Misconceptions about Aerobic and Anaerobic Energy Expenditure

The measurement of gas exchange has played an invaluable role in metabolic interpretation. The uptake of 1 liter of oxygen is often converted into an energy expenditure estimate of 21.1 kilojoules (e.g., 1 L O2 = 21.1 kJ or ~5 kcal). This article demonstrates both the importance of such a conversion and the potential for misinterpretation. Oxygen uptake during heavy and severe exercise will also be discussed

A Model of Brain Circulation and Metabolism: NIRS Signal Changes during Physiological Challenges

We construct a model of brain circulation and energy metabolism. The model is designed to explain experimental data and predict the response of the circulation and metabolism to a variety of stimuli, in particular, changes in arterial blood pressure, CO2 levels, O2 levels, and functional activation. Significant model outputs are predictions about blood flow, metabolic rate, and quantities measurable noninvasively using near-infrared spectroscopy (NIRS), including cerebral blood volume and oxygenation and the redox state of the CuA centre in cytochrome c oxidase. These quantities are now frequently measured in clinical settings; however the relationship between the measurements and the underlying physiological events is in general complex. We anticipate that the model will play an important role in helping to understand the NIRS signals, in particular, the cytochrome signal, which has been hard to interpret. A range of model simulations are presented, and model outputs are compared to published data obtained from both in vivo and in vitro settings. The comparisons are encouraging, showing that the model is able to reproduce observed behaviour in response to various stimuli

Mitochondrial Bioenergetic Alterations in Mouse Neuroblastoma Cells Infected with Sindbis Virus: Implications to Viral Replication and Neuronal Death

The metabolic resources crucial for viral replication are provided by the host. Details of the mechanisms by which viruses interact with host metabolism, altering and recruiting high free-energy molecules for their own replication, remain unknown. Sindbis virus, the prototype of and most widespread alphavirus, causes outbreaks of arthritis in humans and serves as a model for the study of the pathogenesis of neurological diseases induced by alphaviruses in mice. In this work, respirometric analysis was used to evaluate the effects of Sindbis virus infection on mitochondrial bioenergetics of a mouse neuroblastoma cell lineage, Neuro 2a. The modulation of mitochondrial functions affected cellular ATP content and this was synchronous with Sindbis virus replication cycle and cell death. At 15 h, irrespective of effects on cell viability, viral replication induced a decrease in oxygen consumption uncoupled to ATP synthesis and a 36% decrease in maximum uncoupled respiration, which led to an increase of 30% in the fraction of oxygen consumption used for ATP synthesis. Decreased proton leak associated to complex I respiration contributed to the apparent improvement of mitochondrial function. Cellular ATP content was not affected by infection. After 24 h, mitochondria dysfunction was clearly observed as maximum uncoupled respiration reduced 65%, along with a decrease in the fraction of oxygen consumption used for ATP synthesis. Suppressed respiration driven by complexes I- and II-related substrates seemed to play a role in mitochondrial dysfunction. Despite the increase in glucose uptake and glycolytic flux, these changes were followed by a 30% decrease in ATP content and neuronal death. Taken together, mitochondrial bioenergetics is modulated during Sindbis virus infection in such a way as to favor ATP synthesis required to support active viral replication. These early changes in metabolism of Neuro 2a cells may form the molecular basis of neuronal dysfunction and Sindbis virus-induced encephalitis

Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production

Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches

Modulation of Astrocytic Mitochondrial Function by Dichloroacetate Improves Survival and Motor Performance in Inherited Amyotrophic Lateral Sclerosis

Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1G93A mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1G93A mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1G93A astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1G93A mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1G93A mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1G93A mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS