Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo

Abstract

There is increasing interest in the effect of energy metabolism on oxidative stress, but much ambiguity over the relationship between the rate of oxygen consumption and the generation of reactive oxygen species (ROS). Production of ROS (such as hydrogen peroxide, H₂O₂) in the mitochondria is primarily inferred indirectly from measurements in vitro, which may not reflect actual ROS production in living animals. Here, we measured in vivo H₂O₂ content using the recently developed MitoB probe that becomes concentrated in the mitochondria of living organisms, where it is converted by H₂O₂ into an alternative form termed MitoP; the ratio of MitoP/MitoB indicates the level of mitochondrial H₂O₂ in vivo. Using the brown trout Salmo trutta, we tested whether this measurement of in vivo H₂O₂ content over a 24 h-period was related to interindividual variation in standard metabolic rate (SMR). We showed that the H₂O₂ content varied up to 26-fold among fish of the same age and under identical environmental conditions and nutritional states. Interindividual variation in H₂O₂ content was unrelated to mitochondrial density but was significantly associated with SMR: fish with a higher mass-independent SMR had a lower level of H₂O₂. The mechanism underlying this observed relationship between SMR and in vivo H₂O₂ content requires further investigation, but may implicate mitochondrial uncoupling which can simultaneously increase SMR but reduce ROS production. To our knowledge, this is the first study in living organisms to show that individuals with higher oxygen consumption rates can actually have lower levels of H₂O₂.

1. Introduction

Oxidative stress occurs when the generation of reactive oxygen species (ROS) exceeds the capacity of antioxidant defence and repair mechanisms, thereby generating oxidative damage to lipids, DNA and proteins [1]. Given their potential role in cellular senescence, ROS are proposed as being one of the main mediators of life-history trade-offs [2,3]. Most of the ROS present in cells are produced within the mitochondria as natural by-products of aerobic respiration [1]. This has led to the pervasive idea that increased energy expenditure towards one life-history trait will result in greater ROS production, leading to accelerated senescence [4–6]. However, it is still unclear whether higher aerobic respiration actually alters in vivo ROS levels [7,8].

Some of the oxygen consumed by the mitochondria (mtVO₂) is subsequently reduced to superoxide and other ROS such as hydrogen peroxide (H₂O₂) and hydroxyl radicals [1]. Despite the long-held belief that individual organisms consuming more oxygen have higher ROS production, the relationship between mtVO₂ and in vitro ROS production is unclear, with studies reporting positive, negative or no correlation between mtVO₂ and ROS among individuals [8]. However, several recent studies have raised reservations over the measurement of ROS production in vitro [8–10], since artificially high levels of metabolic substrates [11] and very high partial pressures of oxygen (20% in contrast to approx. 5% in vivo [10]) make extrapolations of in vitro results to the in vivo situation potentially problematic [7,8]. This is especially true when examining among-individual variation in mitochondrial traits, given that the in vitro conditions standardize the mitochondrial environment among individuals, which may hide the actual sources of variability in the relationship between oxygen consumption in vivo and mitochondrial ROS production [11].

To avoid some of these potential confounding effects in vitro, a ratiometric probe called MitoB has recently been developed to infer the level of mitochondrial H₂O₂ in vivo [12]. When this artificial probe compound is administered to the organism, it becomes concentrated within the mitochondria, where it is converted to its alternative form MitoP by H₂O₂. The level of mitochondrial H₂O₂ can then be expressed as the rate at which MitoB is converted to MitoP [12]. Here, we use the MitoB probe to directly quantify variation in H₂O₂ content in living animals and relate this to variation in whole animal oxygen consumption among individuals of similar age and under identical environmental and nutritional states, using brown trout (Salmo trutta), a species known to exhibit consistent individual differences in oxygen consumption [13].

2. Material and methods

Juvenile brown trout S. trutta were collected from the wild and then kept in individual compartments for 22 weeks under standard conditions of temperature (mean ± actual range: 11.5 ± 1°C) and photoperiod (12 L : 12 D), as described for these same individuals in [14]. Forty fish were randomly assigned to eight batches of five and fed daily with a specific ration calculated for each fish based on its weight [15].

At week 21, the standard metabolic rate (SMR), defined as the oxygen consumption of a resting and post-absorptive ectotherm at a given temperature, was measured over a 20 h period using flow-through respirometry (further details in [14] and electronic supplementary material). The SMR was calculated as the mean of the lowest 10th percentile of oxygen consumption measurements after controlling for body mass and is described hereafter as the residual SMR (rSMR), in mg O₂ h⁻¹.

Each fish was allowed a week of recovery after its SMR measurement before being injected with 50 nmol of MitoB (initial concentration of MitoB: 5.44 ± 0.21 nmol g⁻¹ of fish). The fish were then culled after 24 h, and aliquots of their liver were immediately flash frozen for subsequent extraction and quantification of the amounts of MitoB and MitoP [12]. The content in MitoP and MitoB was determined by high performance liquid chromatography-tandem mass spectrometry and used to estimate H₂O₂ levels as the ratio of MitoP/MitoB.

Citrate synthase (CS) and cytochrome c oxidase (COX) activities were measured to determine liver mitochondrial density [16]. We analysed the link between the rSMR and MitoP/MitoB ratio using a general linear mixed model approach. The model included the MitoP/MitoB ratio as the dependent variable and rSMR, CS, COX and initial concentration of MitoB in the fish as continuous predictors, with batch as a random effect (see the electronic supplementary material for details of all assay protocols and statistical analyses).

3. Results

The MitoP/MitoB ratio 24 h after injection with the MitoB probe varied up to 26-fold among individual fish (2.29 × 10⁻⁴–59.76 × 10⁻⁴). As the size of the fish at the time of injection ranged from 5.05 to 13.95 g (mean ± s.e. = 9.56 ± 0.29), the initial concentration of MitoB varied three-fold among individuals (3.59–9.90 nmol g⁻¹), but as in a previous study [12], variation in this initial MitoB concentration did not explain the subsequent variation in the MitoP/MitoB ratio (F_1,25.33 = 0.81, p = 0.38). However, a significant effect of the rSMR on the MitoP/MitoB ratio was observed (F_1,30.55 = 12.04, p = 0.002; figure 1); fish with a higher rSMR had a lower MitoP/MitoB ratio compared with individuals with a lower rSMR. MitoP/MitoB ratios were independent of mitochondrial density, regardless of whether this was quantified in terms of COX activity (F_1,30.14 = 1.78, p = 0.19) or CS activity (F_1,25.17 = 0.20, p = 0.66).

Figure 1.

The MitoP/MitoB ratio, a proxy of in vivo mitochondrial H₂O₂ levels, as a function of mass-independent SMR in brown trout (Salmo trutta) at 12°C. Values for the MitoP/MitoB ratio are adjusted to control for the random effects of processing batch; see the electronic supplementary material for calculation of the MitoP/MitoB ratio. Solid line indicates the regression line; see text for statistical analysis.

4. Discussion

Measurement of ROS levels within living organisms make it possible to avoid potential biases introduced when using in vitro assays [17]. Development of the MitoB probe has enabled in vivo estimations of H₂O₂ levels over a period of several hours in mice, Drosophila and Caenorhabditis elegans [12]. We now demonstrate that this method can also be used successfully in fish over a 24 h period. Our findings reveal for the first time that individuals with a high SMR have a lower level of H₂O₂. The negative relationship between SMR and the level of H₂O₂ was independent of mitochondrial density estimates.

The MitoP/MitoB ratio represents the H₂O₂ content within the mitochondria and reflects the balance between the H₂O₂ generated by mitochondria during aerobic respiration and that scavenged by antioxidants such as mitochondrial glutathione peroxidase (GPx) [1,3]. Consequently, lower H₂O₂ levels may be attributed to a lower rate of mitochondrial H₂O₂ generation and/or greater antioxidant scavenging capacity [1,3]. The synthesis of GPx, an endogenous antioxidant, may be costly in term of resources [3,6]. Food intake in this experiment was limited and similar between individuals, so it is feasible that individuals with a lower H₂O₂ level may have allocated more resources towards antioxidant defences. Alternatively, they may have had a lower rate of ROS production. H₂O₂ arises in the mitochondria from the enzymatic conversion of superoxide anions that are produced by the respiratory chain. In order for the mitochondria to reduce oxygen to the superoxide anion, the mitochondrial respiratory chain must be in a highly reduced state [7,8]. Previous studies have shown that natural variation in mitochondrial function can have a significant influence on mitochondrial H₂O₂ production, but also on the relationship between oxygen consumption and H₂O₂ generation [7,8,18]. One key parameter capable of influencing such a relationship is the degree of mitochondrial uncoupling: higher uncoupling can lead to lower membrane potentials and greater rates of electron and oxygen flow in the respiratory chain (so making it less reduced) [7]. Uncoupling is known to simultaneously increase SMR and decrease ROS generation in vitro [7,19,20]. Our study is the first to report a negative relationship between oxygen consumption and H₂O₂ levels in vivo, but measurements of H₂O₂ production and H₂O₂ scavenging are now required in order to understand the mechanisms underlying variability in H₂O₂ levels.

The SMR of brown trout varies considerably among individuals of the same age and size within a population [13]; this level of interindividual variation in oxygen consumption is common across a broad range of taxa [21]. We demonstrate that a lower level of mitochondrial H₂O₂ is associated with a higher respiration rate, which must increase the rate at which energy substrates are oxidized. A lower H₂O₂ may therefore carry a cost in terms of a decrease in resources available for other traits such as body reserves or growth [2,3,6]. However, alleviation of the accumulation of oxidative damage may be a selective advantage for individual trout that have a higher rate of aerobic respiration and less H₂O₂ [7,19,20]. If oxidative damage is an important contributor to ageing [1], then those individuals with a high SMR may benefit through slower ageing but at a cost of reduced resource allocation to other traits, although this requires further study [22].

Ethics

Experiments were approved by the university of Glasgow ethical panel (project licence 60/4292).

Data Accessibility

Data are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.t5c10.

Authors' contributions

K.S., S.K.A., C.S. and N.B.M. conceived the study. K.S., S.K.A., A.M.R., G.J.A., A.G.C. and W.M. undertook the experiments; A.G.C. and R.C.H. selected and synthesized the probe compounds. K.S. drafted the manuscript and all authors contributed to manuscript revisions. All authors approved the final version of the manuscript.

Competing interests

The authors declare they have no competing interests.

Funding

We acknowledge financial support from a European Research Council Advanced grant (no. 322784 to N.B.M.).