The effect of short-term methionine restriction on hydrogen peroxide metabolism in Fischer-344 rat skeletal muscle mitochondria

Highlights

• Skeletal muscle mitochondria bioenergetics during methionine restriction are comparable to control Fischer-344 rats.

• The rates of H₂O₂ formation and consumption by mitochondria were unaltered by methionine restriction.

• Unlike other organs, mitochondria from skeletal muscle may show limited response to short-term methionine restriction.

Abstract

Skeletal muscle, a significant contributor to resting energy expenditure and reactive oxygen species, may play a critical role in body-weight regulation and aging processes. Methionine restriction (MR) is a dietary intervention which extends lifespan, lowers body-weight and enhances energy expenditure in rodents, all of which have been linked to mitochondrial function in various tissues including liver, kidney, heart and brown adipose tissue; however, mitochondrial responses to MR in skeletal muscle is largely unknown. Given the importance of skeletal muscle on energy metabolism and aging-related processes, we investigated if there are changes in skeletal muscle mitochondrial energetics in response to MR. Although MR lowers body-weight in rats, neither respiration, proton leak nor hydrogen peroxide metabolism were altered in isolated skeletal muscle mitochondria. This suggests that mitochondrial function in skeletal muscle remains conserved while MR alters metabolism in other tissues.

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.