Differences in mitochondrial efficiency explain individual variation in growth performance.

The physiological causes of intraspecific differences in fitness components such as growth rate are currently a source of debate. It has been suggested that differences in energy metabolism may drive variation in growth, but it remains unclear whether covariation between growth rates and energy metabolism is: (i) a result of certain individuals acquiring and consequently allocating more resources to growth, and/or is (ii) determined by variation in the efficiency with which those resources are transformed into growth. Studies of individually housed animals under standardized nutritional conditions can help shed light on this debate. Here we quantify individual variation in metabolic efficiency in terms of the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by liver and muscle mitochondria and examine its effects, both on the rate of protein synthesis within these tissues and on the rate of whole-body growth of individually fed juvenile brown trout (Salmo trutta) receiving either a high or low food ration. As expected, fish on the high ration on average gained more in body mass and protein content than those maintained on the low ration. Yet, growth performance varied more than 10-fold among individuals on the same ration, resulting in some fish on low rations growing faster than others on the high ration. This variation in growth for a given ration was related to individual differences in mitochondrial properties: a high whole-body growth performance was associated with high mitochondrial efficiency of ATP production in the liver. Our results show for the first time, to our knowledge, that among-individual variation in the efficiency with which substrates are converted into ATP can help explain marked variation in growth performance, independent of food intake. This study highlights the existence of inter-individual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in whole-animal performance.

Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of Tripeptidyl peptidase II.

In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.

The RCR and ATP/O Indices Can Give Contradictory Messages about Mitochondrial Efficiency.

Mitochondrial efficiency is typically taken to represent an animal's capacity to convert its resources into ATP. However, the term mitochondrial efficiency, as currently used in the literature, can be calculated as either the respiratory control ratio, RCR (ratio of mitochondrial respiration supporting ATP synthesis to that required to offset the proton leak) or as the amount of ATP generated per unit of oxygen consumed, ATP/O ratio. The question of how flexibility in mitochondrial energy properties (i.e., in rates of respiration to support ATP synthesis and offset proton leak, and in the rate of ATP synthesis) affects these indices of mitochondrial efficiency has tended to be overlooked. Furthermore, little is known of whether the RCR and ATP/O ratio vary in parallel, either among individuals or in response to environmental conditions. Using data from brown trout Salmo trutta we show that experimental conditions affect mitochondrial efficiency, but the apparent direction of change depends on the index chosen: a reduction in food availability was associated with an increased RCR (i.e., increased efficiency) but a decreased ATP/O ratio (decreased efficiency) in liver mitochondria. Moreover, there was a negative correlation across individuals held in identical conditions between their RCR and their ATP/O ratio. These results show that the choice of index of mitochondrial efficiency can produce different, even opposing, conclusions about the capacity of the mitochondria to produce ATP. Neither ratio is necessarily a complete measure of efficiency of ATP production in the living animal (RCR because it contains no assessment of ATP production, and ATP/O because it contains no assessment of respiration to offset the proton leak). Consequently, we suggest that a measure of mitochondrial efficiency obtained nearer to conditions where respiration simultaneously offsets the proton leak and produce ATP would be sensitive to changes in both proton leakage and ATP production, and is thus likely to be more representative of the state of the mitochondria in vivo.

Decreased mitochondrial metabolic requirements in fasting animals carry an oxidative cost.

Many animals experience periods of food shortage in their natural environment. It has been hypothesised that the metabolic responses of animals to naturally-occurring periods of food deprivation may have long-term negative impacts on their subsequent life-history.In particular, reductions in energy requirements in response to fasting may help preserve limited resources but potentially come at a cost of increased oxidative stress. However, little is known about this trade-off since studies of energy metabolism are generally conducted separately from those of oxidative stress.Using a novel approach that combines measurements of mitochondrial function with in vivo levels of hydrogen peroxide (H2O2) in brown trout (Salmo trutta), we show here that fasting induces energy savings in a highly metabolically active organ (the liver) but at the cost of a significant increase in H2O2, an important form of reactive oxygen species (ROS).After a 2-week period of fasting, brown trout reduced their whole-liver mitochondrial respiratory capacities (state 3, state 4 and cytochrome c oxidase activity), mainly due to reductions in liver size (and hence the total mitochondrial content). This was compensated for at the level of the mitochondrion, with an increase in state 3 respiration combined with a decrease in state 4 respiration, suggesting a selective increase in the capacity to produce ATP without a concomitant increase in energy dissipated through proton leakage. However, the reduction in total hepatic metabolic capacity in fasted fish was associated with an almost two-fold increase in in vivo mitochondrial H2O2 levels (as measured by the MitoB probe).The resulting increase in mitochondrial ROS, and hence potential risk of oxidative damage, provides mechanistic insight into the trade-off between the short-term energetic benefits of reducing metabolism in response to fasting and the potential long-term costs to subsequent life-history traits.

Les restrictions alimentaires sont courantes dans le milieu naturel et peuvent impacter de nombreux animaux. Il a été émis l'hypothèse que les animaux, face à ces épisodes de restriction alimentaire, mettaient en place des réponses métaboliques pouvant affecter leurs histoires de vie future.En particulier, si une diminution des besoins énergétiques lors du jeûne peut contribuer à préserver les réserves de l'animal cela peut néanmoins entraîner une augmentation du stress oxydant. Ce type de compromis n'a toutefois pas encore été démontré car l'étude du métabolisme énergétique est généralement réalisée séparément de celle du stress oxydant.Par une nouvelle approche combinant des mesures du fonctionnement mitochondrial et des niveaux in vivo de peroxyde d'hydrogène (H2O2) chez la truite commune (Salmo trutta), nous montrons ici que le jeûne entraîne une économie d'énergie dans un tissu métaboliquement très actif tel que le foie, mais au coût d'une augmentation significative en H2O2, une forme majeure des espèces réactives de l'oxygène.Après deux semaines de jeûne, les truites communes ont réduit leurs capacités respiratoires mitochondriales (état 3, état 4 et l'activité de la cytochrome c oxydase) principalement du fait d'une réduction de la taille du foie (et donc du nombre total de mitochondries). Une compensation a été observée au niveau de la mitochondrie. Cela se traduit par une augmentation de la respiration en état 3 et une diminution concomitante de celle en état 4, suggérant une augmentation sélective des capacités de production de l'ATP sans augmentation parallèle de l'énergie dissipée par la fuite de protons. La diminution des capacités métaboliques du foie chez les poissons à jeun était associée in vivo à des niveaux quasiment doubles de H2O2 mitochondriaux (mesurés par la sonde MitoB).Cette augmentation en espèces réactives de l'oxygène dans les mitochondries, avec son risque inhérent de dommages oxydatifs, apporte une vision mécanistique du compromis entre les bénéfices énergétiques à court terme d'une réduction métabolique en réponse au jeûne et les possibles coûts à long terme sur leurs traits histoires de vie futurs. A http://onlinelibrary.wiley.com/doi/10.1111/1365-2435.13125/suppinfo is available for this article.

Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress.

In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H2O2) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H2O2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H2O2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings.

Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios.

The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO2) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of "uncorrected" ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO2 for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO2 in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food-deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.

Is there an alternative to the proteasome in cytosolic protein degradation?

While it is clear that the proteasome is the major player in degradative proteolysis in the nucleus and cytosol, there is a lack of complete agreement on whether there are alternative proteolytic pathways or activities responsible for a significant degradation of cytosolic/nuclear substrates. Particularly relevant is the case of the aminopeptidase TPPII (tripeptidyl peptidase II), which has been suggested to be able to perform some of the proteasome functions. However, the current evidence seems to support only a limited role for these cytosolic alternatives. On the other hand, there is evidence of an alternative, autophagy, a pathway involving the delivery of cytosolic substrates to the lysosome for degradation.

Tripeptidyl peptidase II is dispensable for the generation of both proteasome-dependent and proteasome-independent ligands of HLA-B27 and other class I molecules.

A significant fraction of the HLA-B27-bound peptide repertoire is resistant to proteasome inhibitors. The possible implication of tripeptidyl peptidase II (TPPII) in generating this subset was analyzed by quantifying the surface re-expression of HLA-B*2705 after acid stripping in the presence of two TPPII inhibitors, butabindide and Ala-Ala-Phe-chloromethylketone. Neither decreased HLA-B27 re-expression under conditions in which TPPII activity was largely inhibited. This was in contrast to a significant effect of the proteasome inhibitor epoxomicin. The failure of TPPII inhibition to decrease surface re-expression was not limited to HLA-B27, since it was also observed in several HLA-B27-negative cell lines, including Mel JuSo. Actually, HLA class I re-expression in Mel JuSo cells increased as a function of butabindide concentration, which is consistent with an involvement of TPPII in destroying HLA class I ligands. Inhibition of TPPII with small interfering RNA also failed to decrease the surface expression of HLA class I molecules on 143B cells. Our results indicate that TPPII is dispensable for the generation of proteasome-dependent HLA class I ligands and, without excluding its role in producing some individual epitopes, this enzyme is not involved to any quantitatively significant extent, in generating the proteasome-independent HLA-B27-bound peptide repertoire.

Need for tripeptidyl-peptidase II in major histocompatibility complex class I viral antigen processing when proteasomes are detrimental.

CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic tripeptidyl-peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminopeptidases, probably ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes.

TCR dynamics in human mature T lymphocytes lacking CD3 gamma.

The contribution of CD3gamma to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3gamma is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3gamma-deficient (gamma(-)) individuals. In contrast to gamma(-) mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural gamma(-) T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3gamma. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3gamma-dependent, as it was restored by retroviral transduction of CD3gamma. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3gamma contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.