The Use of Reactive Oxygen Species Production by Succinate-Driven Reverse Electron Flow as an Index of Complex 1 Activity in Isolated Brown Adipose Tissue Mitochondria.

We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.

The Importance of Calcium Ions for Determining Mitochondrial Glycerol-3-Phosphate Dehydrogenase Activity When Measuring Uncoupling Protein 1 (UCP1) Function in Mitochondria Isolated from Brown Adipose Tissue.

Glycerol-3-phosphate is an excellent substrate for FAD-linked mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) in brown adipose tissue mitochondria and is regularly used as the primary substrate to measure oxygen consumption and reactive oxygen consumption by these mitochondria. mGPDH converts cytosolic glycerol-3-phosphate to dihydroxyacetone phosphate, feeding electrons directly from the cytosolic side of the mitochondrial inner membrane to the CoQ-pool within the inner membrane. mGPDH activity is allosterically activated by calcium, and when calcium chelators are present in the mitochondrial preparation medium and/or experimental incubation medium, calcium must be added to insure maximal mGPDH activity. It was demonstrated that in isolated brown adipose tissue mitochondria (1) mGPDH enzyme activity is maximal at free calcium ion concentrations in the 350 nM-1 µM range, (2) that ROS production also peaks in the 10-100 nM range in the presence of a UCP1 inhibitory ligand (GDP) but wanes with further increasing calcium concentration, and (3) that oxygen consumption rates peak in the 10-100 nM range with rates being maintained at higher calcium concentrations. This article provides easy-to-follow protocols to facilitate the measurement of mGPDH-dependent UCP1 activity in the presence of calcium for isolated brown adipose tissue mitochondria.

Detection of UCP1 protein and measurements of dependent GDP-sensitive proton leak in non-phosphorylating thymus mitochondria.

Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.

Lack of activation of UCP1 in isolated brown adipose tissue mitochondria by glucose-O-ω-modified saturated fatty acids of various chain lengths.

We previously demonstrated that uncoupling protein 1 activity, as measured in isolated brown adipose tissue mitochondria (and as a native protein reconstituted into liposome membranes), was not activated by the non-flippable modified saturated fatty acid, glucose-O-ω-palmitate, whereas activity was stimulated by palmitate alone (40 nM free final concentration). In this study, we investigated whether fatty acid chain length had any bearing on the ability of glucose-O-ω-fatty acids to activate uncoupling protein 1. Glucose-O-ω-saturated fatty acids of various chain lengths were synthesized and tested for their potential to activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, and the results were compared with equivalent non-modified fatty acid controls. Here we demonstrate that laurate (12C), palmitate (16C) and stearate (18C) could activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, whereas there was no activation with glucose-O-ω-laurate (12C), glucose-O-ω-palmitate (16C), glucose-O-ω-stearate (18C), glucose-O-ω-arachidate (20C) or arachidate alone. We conclude that non-flippable fatty acids cannot activate uncoupling protein 1 irrespective of chain length. Our data further undermine the cofactor activation model of uncoupling protein 1 function but are compatible with the model that uncoupling protein 1 functions by flipping long-chain fatty acid anions.

Uncoupling protein 1 dependent reactive oxygen species production by thymus mitochondria.

We have previously shown that uncoupling protein 1 is present in thymus and has a role in T-cell development. As reactive oxygen species have been implicated in T-cell development, we set out to determine whether uncoupling protein 1 had the potential to regulate reactive oxygen species production in mitochondria isolated from thymus. This was achieved by inhibiting proton leak through uncoupling protein 1 using the purine nucleotide GDP and through ablation of uncoupling protein 1, measuring the amplex red sensitive reactive oxygen species production by mitochondria. In this work we demonstrate, for the first time, that uncoupling protein 1 has the potential to regulate reactive oxygen species production in thymus mitochondria. We also show that reverse electron transport is possible in thymus mitochondria respiring on succinate and glycerol-3-phosphate. The implications of this regulatory role for uncoupling protein 1 are discussed in the context of thymus function. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

A role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial uncoupling protein 1 (UCP1).

In this study we show that mitochondrial uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and thymus mitochondria can be ubiquitinylated and degraded by the cytosolic proteasome. Using a ubiquitin conjugating system, we show that UCP1 can be ubiquitinylated in vitro. We demonstrate that UCP1 is ubiquitinylated in vivo using isolated mitochondria from brown adipose tissue, thymus and whole brown adipocytes. Using an in vitro ubiquitin conjugating-proteasome degradation system, we show that the cytosolic proteasome can degrade UCP1 at a rate commensurate with the half-life of UCP1 (i.e. 30-72h in brown adipocytes and ~3h, in thymocytes). In addition, we demonstrate that the cytoplasmic proteasome is required for UCP1 degradation from mitochondria that the process is inhibited by the proteasome inhibitor MG132 and that dissipation of the mitochondrial membrane potential inhibits degradation of UCP1. There also appears to be a greater amount of ubiquitinylated UCP1 associated with BAT mitochondria from cold-acclimated animals. We have also identified (using immunoprecipitation coupled with mass spectrometry) ubiquitinylated proteins with molecular masses greater than 32kDa, as being UCP1. We conclude that there is a role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial UCP1. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue.

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.

Control of choline oxidation in rat kidney mitochondria.

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173+/-64 microM and a Vmax of 0.4+/-0.1 nmol/min/mg mitochondrial protein (at 10 degrees C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 degrees C (125+/-6 pmol O2/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96+/-4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.