Properties of the human long and short isoforms of the uncoupling protein‐3 expressed in yeast cells

Hinz, Willy; Grüninger, S.; Pover, Alain De; Chiesi, Michele

doi:10.1016/s0014-5793(99)01568-9

Cited by 47 publications

(46 citation statements)

References 26 publications

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“…Mitochondria were suspended at 0.15 mg of protein/ml in 2 ml of electrode buffer (10 mM Tris/maleate, 0.6 M mannitol, 0.5 mM EGTA, 2 mM MgCl 2 , 10 mM K 2 HPO 4 , 0.1% bovine serum albumin, pH 6.8) containing 3 mM NADH in a Rank oxygen electrode. Respiratory control ratios (rate with FCCP/ rate without) for control mitochondria were around 7 (Table II), comparable to published studies (1,14,16). Additions were made as solutions in water (NADH, GDP), or methanol (FCCP, fatty acids).…”

Section: Methodssupporting

confidence: 85%

Physiological Levels of Mammalian Uncoupling Protein 2 Do Not Uncouple Yeast Mitochondria

Stuart

Harper

Brindle

et al. 2001

Journal of Biological Chemistry

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We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 g/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 g/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity. Uncoupling protein 1 (UCP1)1 uncouples brown adipose tissue mitochondria, causing physiologically important, hormonally regulated, thermogenic proton cycling across the inner membrane. The functions of the UCP1 homologues, UCP2 and UCP3 (1-4), are currently uncertain (5-12). They have been demonstrated to uncouple mitochondrial oxidative phosphorylation in a number of experimental models, including proteoliposomes (13), yeast heterologous expression systems (1, 2, 14 -16), and transgenic mice (17). It is clear that, under some experimental conditions, heterologous or transgenic expression of these proteins can cause an increase in the proton conductance of the inner membrane (16, 18). However, it is less obvious whether these experimental observations of uncoupling are due to a native protein activity of the UCP1 homologues, or represent a more general disruption of mitochondrial function. None of the effects observed in genetically manipulated model systems has been repeated in natural systems where changes in the levels of UCP2 and/or UCP3 occur as a response to some environmental or physiological condition (19 -21).We have demonstrated that expression of UCP1 in yeast mitochondria can cause a nonspecific uncoupling that is not due to protein activity per se (22,23). This uncoupling artifact is present only at higher levels of UCP1 expression. At these levels, UCP1 expression in yeast also interferes with mitochondrial substrate oxidation. Similarly, Heidkaemper et al. (24) have concluded that both UCP1 and UCP3 can be expressed in an incompetent form that interferes with ATP production. They suggest that most of the UCP3 expressed in yeast mitochondria is nonfunctional. There is considerable evidence that, under some expression regimes, a substantial proportion o...

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Section: Methodssupporting

confidence: 85%

Physiological Levels of Mammalian Uncoupling Protein 2 Do Not Uncouple Yeast Mitochondria

Stuart

Harper

Brindle

et al. 2001

Journal of Biological Chemistry

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“…In this study, we have considered UCP1 in BAT to be the main system effector of thermogenesis; nevertheless, it should be taken into account that mitochondrial proton leak in other tissues, such as skeletal muscle, also contributes, to a large extent, to resting energy expenditure (55,66). In this way, other uncoupling proteins, particularly UCP3 in skeletal muscle, may function like UCP1, dissipating the mitochondrial membrane potential and contributing to energy expenditure (3,18,20,28,65). However, the role of these UCPs is a subject of significant controversy, and their physiological functions are yet to be determined (2,5,22,27).…”

Section: Discussionmentioning

confidence: 99%

Sex-related differences in energy balance in response to caloric restriction

Valle

Català-Niell

Colom

et al. 2005

American Journal of Physiology-Endocrinology and Metabolism

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related differences in energy balance were studied in young Wistar rats fed standard chow pellets either ad libitum or in restricted amounts (60% of ad libitum intake) for 100 days. Caloric intake, indirect calorimetry, organ and adipose tissue weights, energy efficiency, liver mitochondrial respiration rate, and brown adipose tissue (BAT) uncoupling protein-1 (UCP1) content were measured. Ad libitum-fed females showed greater oxygen consumption (V O2) and carbon dioxide production (V CO2) and lower energy efficiency than males. Caloric restriction induced a chronic drop of V O2 and V CO2 in females but not in males over the period studied. Restricted females showed a better conservation of metabolic active organ mass and a greater decrease in adipose depots than restricted males. Moreover, changes of BAT size and UCP1 content suggest that BAT may be the main cause responsible for sex differences in the response of energy balance to caloric restriction. In conclusion, our results indicate that females under caloric restriction conditions deactivate facultative thermogenesis to a greater degree than males. This ability may have obvious advantages for female survival and therefore the survival of the species when food is limiting. brown adipose tissue; sexual dimorphism; uncoupling protein-1; oxygen consumption ENERGY BALANCE DEPENDS ON THE MECHANISMS that regulate and coordinate food intake and the different components of energy expenditure, including basal metabolism, physical activity, and heat production. When food is in short supply, caloric intake is lower than energy expenditure, resulting in a negative energy balance (25). Caloric restriction (CR) is a frequent condition in nature; hence, throughout evolution, organisms have evolved mechanisms to respond and adapt to energy restriction. Several studies have reported that CR and starvation have a consistently greater effect on physical growth in males than in females (10,23,25,26,29,63,64). Furthermore, important sex-associated differences have been found in the response of white adipose mass to CR. These studies suggest that females use energy stored as fat to a greater extent than males (26, 44). However, the loss of lean body mass cannot wholly explain the energy conservation seen when female rats are subjected to CR (26).Restriction of physical activity could be another strategy to improve energy efficiency. Nevertheless, CR rats are as active as ad libitum-fed rats, indicating that they are not economizing their energy by restricting their activity (16,24,38,43).Another possibility to explain changes in energy efficiency related to sex implies differences in heat production. In rodents, nonshivering thermogenesis takes place mainly in brown adipose tissue (BAT), where thermogenesis activity depends on uncoupling protein-1 (UCP1), an inner-membrane mitochondrial protein expressed characteristically in this tissue, the function of which is to dissipate, as heat, the proton gradient energy generated by the respiratory chain (7, 48). These particular energ...

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“…The situation, however, is complicated by the existence of two forms of the UCP3 message, a short and a long form. 25 Whilst the short form is equally, 26 or even more, 27 able to uncouple yeast mitochondria, it is incorporated into the mitochondria of cultured cells more slowly than the longer form. 28 Proof of this hypothesis then requires knowledge of direct effects of the c-55t polymorphism on long and short UCP3 message transcription ef®ciency and relations between UCP3 activity and message concentration in vivo.…”

Section: Discussionmentioning

confidence: 99%

Uncoupling protein 3 genetic variants in human obesity: the c-55t promoter polymorphism is negatively correlated with body mass index in a UK Caucasian population

et al. 2001

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OBJECTIVE: To investigate whether genetic variation at the UCP3 locus contributes to human obesity. SUBJECTS: Ninety-one obese children (BMI b 4 standard deviations from age related mean) and 419 Caucasian adults from the Isle of Ely Study. DESIGN: Single strand conformation polymorphism (SSCP) analysis was used to scan the coding region of the UCP3 gene in 91 severely obese children. A common polymorphism identi®ed in this gene (c-55t) has been shown to associate with lower UCP3 mRNA expression. Polymerase chain reaction-based forced restriction digestion was used to detect this allele in Caucasian adults. Multiple regression analysis was used to determine associations between the c-55t genotype and anthropometric, energetic and biochemical indices relevant to obesity. MEASUREMENTS: For the obese children, SSCP analysis and sequencing of variants were carried out. For the Isle of Ely Study, c-55t genotype and anthropometric (body mass index, waist ± hip ratio, percentage body fat), energetic (dietary fat intake, physical activity index, adjusted metabolic rate, maximum oxygen consumption) and biochemical indices (pre-and postglucose challenge plasma triglycerides, non-esteri®ed fatty acids, insulin and glucose) were determined. RESULTS: A previously reported missense mutation (V102I) was detected in a single obese Afro-Carribean child. Twenty-one percent of the genes examined in the Isle of Ely study carried the c-55t promoter variant. Age-adjusted body mass index (BMI) was signi®cantly (P 0.0037) lower in carriers of this variant. CONCLUSION: Mutations in the coding sequence of UCP3 are unlikely to be a common monogenic cause of severe human obesity. In a Caucasian population the UCP3 c-55t polymorphism is negatively associated with BMI.

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Properties of the human long and short isoforms of the uncoupling protein‐3 expressed in yeast cells

Cited by 47 publications

References 26 publications

Physiological Levels of Mammalian Uncoupling Protein 2 Do Not Uncouple Yeast Mitochondria

Physiological Levels of Mammalian Uncoupling Protein 2 Do Not Uncouple Yeast Mitochondria

Sex-related differences in energy balance in response to caloric restriction

Uncoupling protein 3 genetic variants in human obesity: the c-55t promoter polymorphism is negatively correlated with body mass index in a UK Caucasian population

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