Indirect measurement of mitochondrial proton leak and its application

Porter, Richard K.; Joyce, O. J. P.; Farmer, M. K.; Heneghan, R.; Tipton, Keith F.; Andrews, Jonathan; McBennett, S.; Lund, Martin; Jensen, Catrin H.; Melia, H.P.

doi:10.1038/sj.ijo.0800937

Cited by 37 publications

(16 citation statements)

References 42 publications

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“…Uncoupling effects of high doses of thyroid hormone have previously been reported in vitro (6, 7), and more recently T 3 -induced proton leaks in the inner mitochondrial membrane have been demonstrated in vitro in rat liver (27) and rat skeletal muscle (28,29). However, no evidence of a similar uncoupling effect in humans has yet been demonstrated, due to the difficulty in extrapolating in vitro measurements of isolated mitochondrial membrane permeability to the in vivo situation.…”

Section: Discussionmentioning

confidence: 90%

Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle

Lebon

Dufour²,

Petersen³

et al. 2001

J. Clin. Invest.

107

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IntroductionThe regulation of basal metabolic rate by thyroid hormone has been known since the 18th century (1), but the mechanism by which it occurs remains controversial. Thyroid hormone has been shown to increase energy expenditure by activating Na + /K + -ATPase, stimulating futile cycling as well as promoting mitochondrial biogenesis (2-6). However, in vitro observations have raised the possibility that thyroid hormone also promotes mitochondrial uncoupling of substrate oxidation from ATP synthesis (7,8). The absence of an identified mechanism that accounts for uncoupling and the inability to assess the degree of mitochondrial coupling in vivo have long hampered investigation into the regulation of this process. Recently, a skeletal muscle-specific uncoupling protein (UCP-3) has been cloned in humans (9). This protein is thought to create a pathway that allows exothermic movement of protons through the inner mitochondrial membrane. A positive correlation between the concentration of thyroid hormone in plasma and UCP-3 mRNA expression in skeletal muscle has been demonstrated in mice, rats, and humans (10-18), raising the possibility that UCP-3 might be involved in regulating energy expenditure and body weight in humans by promoting mitochondrial energy uncoupling.In order to examine whether thyroid hormone is capable of inducing mitochondrial uncoupling in human skeletal muscle, we developed a combined 13 C/ 31 P nuclear magnetic resonance (NMR) approach to assess mitochondrial energy coupling in vivo. The rate of tricarboxylic acid (TCA) cycle flux was assessed by monitoring the rate of appearance of 13 C label into the C4 and C2 carbon atoms of muscle glutamate during an intravenous infusion of [2-13 C] acetate (12). The rate of ATP synthesis was measured using 31 P NMR spectroscopy by direct observation of 31 P magnetization transfer between inorganic phosphate (P i ) and the γ phosphate resonance of ATP (γATP); mitochondrial energy coupling was determined by the ratio of ATP synthesis to TCA cycle oxidation. To assess the effect of thyroid hormone on mitochondrial energy coupling, these measurements were performed in healthy volunteers before and after three days of treatment with triiodothyronine (T 3 ) to induce a state of mild hyperthyroidism. MethodsProtocol. NMR studies were performed on seven healthy volunteers (three men and four women, aged 25 ± 2) before (basal) and after three days of T 3 treatment (100 µg orally every 12 hours). Whole-body oxygen consumption was measured using a DeltaTrac II indirect The mechanism underlying the regulation of basal metabolic rate by thyroid hormone remains unclear.Although it has been suggested that thyroid hormone might uncouple substrate oxidation from ATP synthesis, there are no data from studies on humans to support this hypothesis. To examine this possibility, we used a novel combined 13 C/ 31 P nuclear magnetic resonance (NMR) approach to assess mitochondrial energy coupling in skeletal muscle of seven healthy adults before and after three days of triiod...

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Section: Discussionmentioning

confidence: 90%

Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle

Lebon

Dufour²,

Petersen³

et al. 2001

J. Clin. Invest.

107

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“…The observation that glucose failed to increase ATP concentration in the ob/ob islets is consistent with the uncoupling of respiration. In ob/ob hepatocytes, which express ucp2 (Rashid et al 1999), proton leak is elevated compared with lean hepatocytes (Chavin et al 1999, Porter et al 1999, which do not express ucp2 (Rashid et al 1999). In islets, reduced ATP production leads to impaired closure of K ATP channels, thereby suppressing GSIS (Chan et al 2001).…”

Section: Discussionmentioning

confidence: 99%

Endogenous islet uncoupling protein-2 expression and loss of glucose homeostasis in ob/ob mice

Saleh¹,

Wheeler²,

Chan³

2006

Journal of Endocrinology

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We hypothesized that the loss of glucose homeostasis in ob/ob mice is associated with upregulation of islet uncoupling protein-2 (UCP2) expression, leading to impaired glucosestimulated insulin secretion (GSIS). Changes in glucose homeostasis in lean and ob/ob mice from 5 to 16 weeks were assessed by fasting blood glucose, plasma insulin, oral glucose tolerance, and tissue insulin sensitivity. In vitro GSIS and ATP content were assayed in isolated islets, while UCP2 expression was determined by quantitative real-time PCR and immunoblotting. Short-term reduction of UCP2 expression was achieved through transfection of islets with specific small interfering RNA. Insulin resistance was detected in 5-week-old ob/ob mice, but GSIS and blood glucose levels remained normal. By 8 weeks of age, ob/ob mice displayed fasting hyperglycemia, hyperinsulinemia and glucose intolerance, and also had elevated non-esterified fatty acid concentration in plasma. In vitro, GSIS and ATP generation were impaired in ob/ob islets. Islet UCP2 expression was elevated at 5 and 8 weeks of age. Short-term knockdown of islet UCP2 increased GSIS in islets of lean mice, but had no effect in islets from ob/ob mice. Loss of glucose homeostasis and impairment of insulin secretion from isolated islets at 8 weeks in ob/ob mice is preceded by an increase in UCP2 expression in islets. Moreover, the glucolipotoxic conditions observed are predicted to increase UCP2 activity, contributing to lower islet ATP and GSIS.

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“…Mitochondria were incubated in respiratory buffer [220 mM mannitol, 70 mM sucrose, 2.5 mM KH2PO4, 2 mM MgCl2, 1 mM EDTA, 2 mM HEPES, pH 7.4, with 0.1% fatty acid-free BSA, 2 M oligomycin to inhibit ATP synthase, 5 M rotenone to inhibit electron entry at complex I, and 0.1 M nigericin to abolish the ⌬pH (20) across the mitochondrial membrane]. H ϩ flux, under these conditions, is proton leak dependent and, with succinate as fuel, follows a 6:1 stoichiometry (H ϩ -O) with oxygen consumed (6,7,22,26). Succinate (5 mM) was added, followed by malonate in incremental amounts to final concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, and 2.0 mM to inhibit succinate dehydrogenase, thereby decreasing electrons available for the transport system and creating a range of membrane potentials (4).…”

Section: Methodsmentioning

confidence: 99%

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Fink

Reszka

Herlein

et al. 2005

American Journal of Physiology-Endocrinology and Metabolism

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Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.

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Indirect measurement of mitochondrial proton leak and its application

Cited by 37 publications

References 42 publications

Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle

Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle

Endogenous islet uncoupling protein-2 expression and loss of glucose homeostasis in ob/ob mice

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

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