The effect of short-term methionine restriction on hydrogen peroxide metabolism in Fischer-344 rat skeletal muscle mitochondria
Introduction
Skeletal muscle is a major contributor to the whole-body level of metabolic demand in mammals, contributing approximately 20–25% of resting energy expenditure (Rolfe and Brown, 1997). However, skeletal muscle metabolism is also known to alter with aging, a topic that has received substantial study and continues to be an active area of research (reviewed in (Gensous et al., 2019, Hepple, 2014, Johnson et al., 2013, Peterson et al., 2012)). Many studies have found that mitochondrial respiratory capacity and the efficiency of energy transformation during oxidative phosphorylation declines with aging in skeletal muscle, although this association between muscle mitochondrial function and aging is not universally reported (Gensous et al., 2019, Hepple, 2014, Johnson et al., 2013, Peterson et al., 2012, Rasmussen et al., 2003). Moreover, although contentious, mitochondrial reactive oxygen species (ROS) production has long been implicated in longevity and lifespan (Barja, 2013, Herrero and Barja, 1998, Ku et al., 1993, Lambert et al., 2007); however, the direct association between excess mitochondrial ROS and aging continues to be debated (Liochev, 2013, Pérez et al., 2009, Sanz et al., 2006, Stuart et al., 2014, Vyssokikh et al., 2020).
Diet is well established to influence lifespan in rodents, as well as other animals, with methionine restriction (MR) being one of the more unusual lifespan extending dietary interventions (Simpson et al., 2017, Speakman et al., 2016). Most rodent studies on MR use a diet that is severely restricted in sulfur containing amino acids (supplied only as methionine in most cases) which leads to a marked reduction in growth rate (Hasek et al., 2010, Malloy et al., 2006, Orentreich et al., 1993, Richie et al., 1994, Zimmerman et al., 2003). Counterintuitive to this lower growth rate, rodents can become hyperphagic during MR and consume substantially more food per unit animal mass than ad libitum fed control animals (Hasek et al., 2010, Orentreich et al., 1993). This increase in mass-specific food consumption has also be shown to be concomitant with a marked elevation in metabolic rate (Hasek et al., 2010, Wanders et al., 2015), indicating that the excess energy intake is largely dissipated as heat rather than incorporated as tissue accretion. The elevation in metabolic rate in rodents under MR has been attributed to proliferation of brown adipose tissue (Hasek et al., 2013, Wanders et al., 2015), browning of white adipose tissue (Hasek et al., 2013, Patil et al., 2015) with some indication of mitochondrial proliferation in several tissues including skeletal muscle (Perrone et al., 2010). However, despite skeletal muscle making up such a substantial component of metabolic demand, the effect of MR on skeletal muscle mitochondrial energetics has not received much attention.
Important to the association between lifespan and mitochondrial ROS, MR leads to lower rates of ROS formation, measured as hydrogen peroxide (H2O2) emission, by isolated mitochondria from several tissues (Caro et al., 2008, Sanz et al., 2006). With respect to ROS metabolism, H2O2 is particularly important because it can both contribute to oxidative stress at high levels while likely acting as a signalling molecule at lower concentrations (Sies, 2017). Lower rates of mitochondrial H2O2 emission have been implicated with lower oxidant burden and possible lower oxidative stress in tissues during MR (Caro et al., 2008, Sanz et al., 2006). Yet here again skeletal muscle mitochondria have been largely overlooked in studies of MR. Recently we have shown that short-term MR increased mitochondrial proton permeability (proton leak) in the liver of rats which could contribute to the metabolic inefficiency induced by MR (Tamanna et al., 2019) as well as the observed decrease in mitochondrial ROS emission in liver (Caro et al., 2008, Sanz et al., 2006). If similar increases in basal proton leak occur in skeletal muscle this could contribute to both increased metabolic rates, helping dissipate dietary energy intake as heat, as well as poise skeletal muscle mitochondria towards lower ROS formation.
In the current study, we test if short-term MR influences skeletal muscle mitochondrial energetics and H2O2 metabolism in Fischer-344 rats fed a diet known to increase lifespan (Orentreich et al., 1993, Richie et al., 1994). We assess mitochondrial electron transport efficiency by determining relative proton leak rates and measure H2O2 metabolism by isolated mitochondria. Mitochondria have both the capacity to produce and consume H2O2, leading to marked underestimation of actual rates of production when measured as H2O2 emission from isolated mitochondria (Munro et al., 2016). Indeed, we have recently shown that underappreciation of the antioxidant pathways in mitochondria may have skewed much of the debate over mitochondrial ROS metabolism and longevity in comparative studies that included the exceptionally long-lived naked mole-rat (Munro et al., 2019). For this reason, when examining H2O2 metabolism, it is important to measure both the capacity for isolated mitochondria to form ROS (as H2O2 emission in this study) and their capacity to consume exogenously added H2O2.
Section snippets
Animals and diet
Animal husbandry was approved by the University of Manitoba animal care committee (protocol- F2013-036) and procedures are as described elsewhere (Tamanna et al., 2018) because the same groups of animals were used in the current study. Briefly, five-week-old male Fischer-344 (F-344) rats were purchased from Charles River laboratories (Quebec, Canada) and kept in shoebox cages at the Biological Sciences Animal holding Facility. One week later, they were housed individually in wire bottom cages
Growth and food intake of F-344 rats
This study confirms that MR has an effect on energy intake and growth of rats. The treatment significantly lowered body weight after only two weeks (Fig. 1A) and increased both gross and digestible food intake after three weeks (Fig. 1. B-C).
Mitochondrial oxygen consumption and proton leak
Skeletal muscle mitochondrial oxygen consumption rates were analysed in the presence of complex I-linked (glutamate-malate) or complex II respiratory substrates. Under all conditions of substrate and respiratory states (state 2, 3 and 4o) tested, the
Discussion
Despite the importance of skeletal muscle to overall energy demand in animals (reviewed in (Rolfe and Brown (1997)) and the established influence of MR on rodent energy balance and mitochondrial function in several tissues (Hasek et al., 2010, Malloy et al., 2006, Orentreich et al., 1993, Patil et al., 2015, Wanders et al., 2015), the influence of MR on skeletal muscle mitochondria has been relatively overlooked. In the current study we found short-term MR (up to eight weeks) had negligible
Conclusion
Short-term MR alters energy intake and growth in ways consistent with a marked shift in energy metabolism that wastes more energy intake as heat and partitions less nutrition toward growth than rats on a sulfur amino acid sufficient diet. Based on food intake and growth, our MR rats appear to have higher mass-specific metabolic rates and lower growth rates compared to control rats. However, we found MR had no effect on respiration rates, proton permeability of the inner mitochondrial membrane,
Acknowledgements
The authors thank the staff of the Biological Sciences animal holding facility for their assistance with husbandry and Kristen Braun for providing the protein carbonyl data. This study is supported by an NSERC Discovery Grant (418503-2012) and the Canada Research Chairs program (223744) as well as a postdoctoral fellowship to DM from the Fond de recherche du Québec – Nature et Technologies (FRQ-NT (Grant # 183703)).
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Cited by (2)
Review: Using isolated mitochondria to investigate mitochondrial hydrogen peroxide metabolism
2021, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular BiologyCitation Excerpt :While the experiments to establish steady-state conditions of H2O2 may be beyond what many comparative studies are able to conduct we have argued, and used, the ratio of production and consumption rates, termed the oxidant index or ratio, as a means of comparing across species and in particular different assay temperatures (Treberg et al., 2018b; Munro et al., 2019). Similarly, we recently concluded that methionine restriction, which increases life span in rodents, does not appear to alter mitochondrial H2O2 balance in skeletal muscle because neither production nor consumption capacity is altered (Tamanna et al., 2020). If mitochondria can act as localized regulators of cellular H2O2 concentrations, then can the values from our isolated mitochondria experiments be extrapolated to mitochondria within living cells?