A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast

2001 ◽  
Vol 356 (3) ◽  
pp. 779-789 ◽  
Author(s):  
Jeff A. STUART ◽  
James A. HARPER ◽  
Kevin M. BRINDLE ◽  
Mika B. JEKABSONS ◽  
Martin D. BRAND
Keyword(s):  
Uncoupling Protein ◽  
Mitochondrial Protein ◽  
Physiological Property ◽  
Growth Defect ◽  
Yeast Mitochondria ◽  
Proton Conductance ◽  
Uncoupling Protein 1 ◽  
Mitochondrial Uncoupling ◽  
Mitochondrial Carrier ◽  
Brown Adipose

Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the specific (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochondrial uncoupling were assessed. UCP1 provides a GDP-inhibitable model system to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1μg/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating proton conductance that was completely GDP-sensitive. The catalytic-centre activity of UCP1 in these yeast mitochondria was similar to that in mammalian brown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11μg/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates and an increased proton conductance that was partly GDP-insensitive. Thus, although UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is not a physiological property of the native protein and is therefore artifactual. This observation might be important in the interpretation of results from experiments in which the functions of UCP1homologues are verified by their ability to uncouple yeast mitochondria.

The thermogenic and metabolic responses to photoperiod manipulations in Apodemus chevrieri

2013 ◽  
Vol 63 (3) ◽  
pp. 241-255 ◽  
Author(s):  
Wan-long Zhu ◽  
Lin Zhang ◽  
Zheng-kun Wang
Keyword(s):  
Body Mass ◽  
Uncoupling Protein ◽  
Resting Metabolic Rate ◽  
Mitochondrial Protein ◽  
Short Photoperiod ◽  
Uncoupling Protein 1 ◽  
Mitochondrial Uncoupling ◽  
Adipose Tissues ◽  
Serum Leptin ◽  
Brown Adipose

Environmental cues play important roles in the regulation of an animal’s physiology and behavior. In the present study, we examined the effects of short photoperiod on body weight as well as on several physiological, hormonal, and biochemical measures indicative of thermogenic capacity to test our hypothesis that short photoperiod stimulates increases in thermogenesis without cold stress in Apodemus chevrieri. A. chevrieri were randomly assigned to either a long or short photoperiod for 4 weeks at constant temperature. The short photoperiod group of A. chevrieri showed increases in resting metabolic rate and nonshivering thermogenesis during the 4-week photoperiod acclimation. At the end, A. chevrieri at short photoperiod had lower body weights, higher levels of mitochondrial protein content and cytochrome C oxidase activity in liver and brown adipose tissues, and had higher levels of mitochondrial uncoupling protein-1 contents in brown adipose tissues. No difference in serum leptin levels were found between short and long photoperiod groups, but serum leptin levels were positively correlated with body mass and body fat mass, and negatively correlated with energy intake and uncoupling protein-1 content in brown adipose tissues, respectively. All results suggest that the short photoperiod may induce an increased thermogenesis capacity in A. chevrieri and that leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in A. chevrieri.

Effects of intravenous isoproterenol (β-agonist) administration on expression and synthesis of ovine uncoupling protein-1

1999 ◽  
Vol 1999 ◽  
pp. 164-164
Author(s):  
D.S. Finn ◽  
P. Trayhurn ◽  
J. Struthers ◽  
M.A. Lomax
Keyword(s):  
Adipose Tissue ◽  
Cold Stress ◽  
Uncoupling Protein ◽  
Uncoupling Protein 1 ◽  
Mitochondrial Uncoupling ◽  
Mitochondrial Uncoupling Protein ◽  
Neonatal Life ◽  
Adrenoceptor Agonist ◽  
Brown Adipose

A crucial factor in the prevention of hypothermia in the neonatal lamb is the functional activitation of a mitochondrial uncoupling protein (UCP1) in brown adipose tissue. UCP1 disappears from lamb brown fat over the first 14 days of life (Finn et al., 1998), but it is not known whether this process can be modulated in lambs by the release of catecholamines which have been established in rodents as a mediator of the response to cold stress. This study examines the effect of administering a β-adrenoceptor agonist on the disappearance of UCP1 and UCP1 mRNA during early neonatal life, using immunohistochemistry and in situ hybridization.

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria

2001 ◽  
Vol 361 (1) ◽  
pp. 49-56 ◽  
Author(s):  
James A. HARPER ◽  
Jeff A. STUART ◽  
Mika B. JEKABSONS ◽  
Damien ROUSSEL ◽  
Kevin M. BRINDLE ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Uncoupling Protein ◽  
Mitochondrial Protein ◽  
Yeast Mitochondria ◽  
Rat Skeletal Muscle ◽  
Muscle Mitochondria ◽  
Skeletal Muscle Mitochondria ◽  
Uncoupling Protein 3 ◽  
Brown Adipose

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14μg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200–700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24–84μg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

scholarly journals Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not physiologically relevant for their uncoupling activity

2020 ◽  
Author(s):  
Mathilde S. Piel ◽  
Sandrine Masscheleyn ◽  
Frédéric Bouillaud ◽  
Karine Moncoq ◽  
Bruno Miroux
Keyword(s):  
Uncoupling Protein ◽  
Brown Adipocyte ◽  
Acid Oxidation ◽  
Long Chain Fatty Acids ◽  
Proton Conductance ◽  
Uncoupling Protein 1 ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Uncoupling ◽  
Mitochondrial Uncoupling Proteins

AbstractUncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocyte. In the presence of long-chain fatty acids (LCFA), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Several atomic models of UCP1 and UCP2 have been obtained by NMR in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiment on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of S. cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S and K56S/K269S mutants did not display any default in activation, which shows that the NMR experiments in DPC detergent are not relevant to understand UCP1 function.

scholarly journals Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria

2004 ◽  
Vol 379 (2) ◽  
pp. 309-315 ◽  
Author(s):  
Telma C. ESTEVES ◽  
Karim S. ECHTAY ◽  
Tanya JONASSEN ◽  
Catherine F. CLARKE ◽  
Martin D. BRAND
Keyword(s):  
Proton Transport ◽  
Uncoupling Protein ◽  
Coenzyme Q ◽  
Yeast Mitochondria ◽  
Proton Conductance ◽  
Wild Type ◽  
Uncoupling Protein 1 ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Strains ◽  
Wild Type Yeast

Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609–613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63–65]. In the present study, yeast (Saccharomyces cerevisiae) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENΔCOQ3 and CENΔCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria.

scholarly journals Cold acclimation enhances UCP1 content, lipolysis, and triacylglycerol resynthesis, but not mitochondrial uncoupling and fat oxidation, in rat white adipocytes

2019 ◽  
Vol 316 (3) ◽  
pp. C365-C376 ◽  
Author(s):  
Diane M. Sepa-Kishi ◽  
Shailee Jani ◽  
Daniel Da Eira ◽  
Rolando B. Ceddia
Keyword(s):  
Fatty Acids ◽  
Cold Acclimation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Uncoupling Protein ◽  
Fat Oxidation ◽  
Uncoupling Protein 1 ◽  
Mitochondrial Uncoupling ◽  
Palmitate Oxidation ◽  
White Adipocytes ◽  
Brown Adipose

The objective of this study was to investigate whether cold-induced browning of the subcutaneous (Sc) inguinal (Ing) white adipose tissue (WAT) increases the capacity of this tissue to oxidize fatty acids through uncoupling protein 1 (UCP1)-mediated thermogenesis. To accomplish that, rats were acclimated to cold (4°C for 7 days). Subsequently, interscapular and aortic brown adipose tissues (iBAT and aBAT, respectively), epididymal (Epid), and Sc Ing WAT were used for adipocyte isolation. In BAT adipocytes, cold acclimation increased UCP1 content and palmitate oxidation either in the absence or presence of oligomycin, whereas in Sc Ing adipocytes glucose and palmitate oxidation were not affected, although multilocular adipocytes were formed and UCP1 content increased upon cold acclimation in the WAT. Furthermore, isoproterenol-stimulated cold Sc Ing adipocytes exhibited significantly lower rates of palmitate oxidation than control cells when exposed to oligomycin. These findings provide evidence that, despite increasing UCP1 levels, cold acclimation essentially reduced mitochondrial uncoupling-mediated fat oxidation in Sc Ing adipocytes. Conversely, glycerol kinase and phosphoenolpyruvate carboxykinase levels, isoproterenol-induced lipolysis, as well as glycerol and palmitate incorporation into lipids significantly increased in these cells. Therefore, instead of UCP1-mediated mitochondrial uncoupling, cold acclimation increased the capacity of Sc Ing adipocytes to export fatty acids and enhanced key components of the triacylglycerol resynthesis pathway in the Sc Ing WAT.

scholarly journals Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

2006 ◽  
Vol 395 (3) ◽  
pp. 619-628 ◽  
Author(s):  
Telma C. Esteves ◽  
Nadeene Parker ◽  
Martin D. Brand
Keyword(s):  
Proton Transport ◽  
Uncoupling Protein ◽  
Significant Activity ◽  
Proton Conductance ◽  
Uncoupling Protein 1 ◽  
Physiological Models ◽  
Synergistic Activation ◽  
Brown Adipose ◽  
The Individual ◽  
Kinetics Of

The kinetics of proton transport through mammalian UCP1 (uncoupling protein 1) expressed in yeast mitochondria were measured. There was little or no UCP1 activity in the absence of added palmitate, but significant activity in its presence. The activator 4-HNE (4-hydroxy-2-nonenal) had little effect when added alone, but significantly enhanced proton conductance in the presence of added palmitate. Activation of the proton conductance of UCP1 was synergistic: proton conductance in the presence of both palmitate and 4-HNE was significantly greater than the sum of the individual effects. Mitochondria from control yeast transformed with empty vector showed no such synergy, showing that synergy is a property of UCP1. Activation by the 4-HNE analogue trans-cinnamate showed essentially the same characteristics as activation by 4-HNE. Mitochondria from brown adipose tissue also showed synergistic activation of GDP-sensitive proton conductance by palmitate and 4-HNE. These results show that reactive alkenals activate the proton conductance of UCP1 more strongly when fatty acids are also added, with implications for both mechanistic and physiological models of UCP1 activation.

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