Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice

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© 2013. Published by The Company of Biologists Ltd

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Mitochondrial uncoupling prevents cold-induced oxidative stress: 1  

a case study using UCP1 knock-out mice 2  

3  

Short title: Increased metabolism & oxidative stress 4  

5  

Antoine Stier1,2*, Pierre Bize3†, Caroline Habold1,2, Frederic Bouillaud4, Sylvie Massemin1,2 6  

and François Criscuolo1,2 7  

8  

1University of Strasbourg, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France 9  

10  

2Département d’Ecologie, Physiologie et Ethologie (DEPE), CNRS UMR7178, 23 rue 11  

Becquerel, 67087 Strasbourg Cedex 2, France 12  

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3Department of Ecology and Evolution, University of Lausanne, Biophore 1015 Lausanne-14  

Dorigny, Switzerland. 15  

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4 Institut Cochin, Inserm UMRS 1016, CNRS UMR 8104, Université Paris Descartes, 75014 17  

Paris, France 18  

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*: corresponding author: [email protected] 20  

†: Present address: Institute of Biological and Environmental Sciences, University of 21  

Aberdeen, Aberdeen AB24 2TZ, UK22  

http://jeb.biologists.org/lookup/doi/10.1242/jeb.092700Access the most recent version at J Exp Biol Advance Online Articles. First posted online on 21 November 2013 as doi:10.1242/jeb.092700

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Abstract 23  

The relationship between metabolism and reactive oxygen species (ROS) production 24  

by the mitochondria has been often (wrongly) viewed as straightforward, with increased 25  

metabolism leading to higher pro-oxidants generation. Insights on mitochondrial functioning 26  

show that oxygen consumption is either principally coupled with energy conversion as ATP 27  

or as heat, depending on whether the ATP-synthase or the mitochondrial uncoupling protein 1 28  

(UCP1) is driving respiration. However, those two processes might greatly differ in terms of 29  

oxidative costs. We used a cold challenge to investigate the oxidative stress consequences of 30  

an increased metabolism achieved either by the activation  of  an  uncoupled  mechanism  (i.e.  31  

UCP1  activity)  in  the  brown  adipose  tissue  (BAT)  of  wild-­‐type  mice,  or  by ATP-dependent 32  

muscular shivering thermogenesis in mice deficient for UCP1. Although  both  mouse  strains  33  

increased   by   more   than   twofold   their   metabolism   when   acclimatised   for   4   weeks   to  34  

moderate   cold   (12°C),   only   mice   deficient   for   UCP1   suffered   from   elevated   levels   of  35  

oxidative  stress.  When  exposed  to  cold,  mice  deficient  for  UCP1  showed  an  increase  of  36  

20.2%  in  plasmatic  reactive  oxygen  metabolites,  81.8%  in  muscular  oxidized  glutathione  37  

and  47.1%  in  muscular  protein  carbonyls.  In  contrast,  there  was  no  evidence  of  elevated  38  

levels   of   oxidative   stress   in   the  plasma,  muscles   or  BAT  of  wild-­‐type  mice   exposed   to  39  

cold   despite   a   drastic   increase   in   BAT   activity.   Our   study   demonstrates   differing  40  

oxidative  costs  linked  to  the  functioning  of  two  highly  metabolically  active  organs  during  41  

thermogenesis.   It   urges   for   careful   considerations   of   mitochondrial   functioning   when  42  

investigating  the  links  between  metabolism  and  oxidative  stress.  43  

44  

Keywords: Uncoupling protein, oxidative stress, reactive oxygen species, cold, nonshivering 45  

thermogenesis, mitochondria46  

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Introduction 47  

The idea of a negative impact of high metabolic rate on longevity was first formulated 48  

almost a century ago by Raymond Pearl (Pearl, 1928) in his rate of living theory. Almost 30 49  

years later Denham Harman proposed as an underlying mechanism the fact that aerobic 50  

respiration leads to the inevitable by-production of damaging reactive oxygen species (ROS) / 51  

free radicals in his free radical theory of ageing (Harman, 1956). Ageing could then result 52  

from the accumulation of oxidative damage, caused by the imbalance between ROS 53  

production and antioxidant defences (i.e. oxidative stress), with the rate of ROS production 54  

being potentially coupled to whole organism oxygen consumption and, in turn, metabolic rate 55  

(Beckman and Ames, 1998). The production of mitochondrial ROS has sometimes been 56  

assumed to be a fixed percentage of total oxygen consumption, falling somewhere between 57  

0.1 and 4% according to in vitro experiments (Golden and Melov, 2001; Nicholls et al., 58  

2002), but whether the same values are found under in vivo circumstances remain to be 59  

demonstrated. Nevertheless, one common prediction of the free radical theory of ageing has 60  

been that an increase in metabolic rate (i.e. oxygen consumption) should lead to an increase in 61  

mitochondrial ROS production and concomitant rate of ageing (Beckman and Ames, 1998). 62  

However, recent evidences, mostly coming from our more accurate understanding of 63  

mitochondrial functioning, are arguing against a trivial, monotonic, relationship between 64  

oxygen consumption and ROS production (reviewed in Murphy, 2009; Lambert and Brand 65  

2009; Speakman and Selman, 2011). During mitochondrial respiration, some electrons can 66  

escape the electron transport chain and react directly with molecular oxygen and form ROS. 67  

The energy associated with the electron flow through the respiratory chain is used to pump 68  

protons against their electrochemical gradient across the mitochondrial innermembrane, and 69  

the backflow of protons through the Fo/F1 ATP synthase is responsible for the conversion of 70  

cellular energy as ATP. Hence, mitochondria couple respiration to ATP synthesis through an 71  

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electrochemical proton gradient (Divakaruni and Brand, 2011). A growing number of studies 72  

show that electron loss from the respiratory chain and concomitant ROS production is highly 73  

sensitive to changes in mitochondrial innermembrane potential, ROS production being 74  

sharply declined at low membrane potential (Barja, 2007; Murphy, 2009; Mookerjee et al., 75  

2010). Several pathways including the inducible uncoupling proteins (UCP1 to 3, reviewed in 76  

Ricquier and Bouillaud, 2000) might lower the mitochondrial membrane potential by 77  

increasing its proton permeability, thereby uncoupling respiration from ATP production and 78  

releasing energy as heat. The occurrence of such a mitochondrial uncoupling can then 79  

increase oxygen consumption while lowering ROS production, which in turn might lead to a 80  

negative association between metabolism, ROS production and rate of ageing, as stated by the 81  

uncoupling to survive hypothesis (Brand, 2000; see also Speakman et al., 2004). 82  

In such context, experimental increases of the uncoupling state of mitochondria, either 83  

obtained through a pharmacological uncoupling treatment (2,4-dinitrophenol; Caldeira Da 84  

Silva et al., 2008) or through the ectopic (i.e. muscular) expression of the uncoupling protein 85  

UCP1 (Gates et al., 2007) in mice have been shown to extend lifespan. Nevertheless, the 86  

relevance of mitochondrial uncoupling in the control of ROS production under normal 87  

physiological conditions remains unclear and controversial (Shabalina et al., 2011). Some of 88  

these controversies could be attributable to the fact that the impact of UCP1 on oxidative 89  

stress levels in situations where UCP1 expression / activity is naturally triggered (e.g. chronic 90  

cold exposure) has been under-evaluated so far, despite the fact that UCP1 is the only 91  

acknowledged UCP with a physiologically relevant uncoupling activity (Shabalina et al., 92  

2011). Early evidence suggested that UCP1 ablation had no impact on ROS production and 93  

various markers of oxidative stress in brown adipose tissue (BAT) mitochondria (Shabalina et 94  

al., 2006). However, recent studies demonstrated that UCP1 activity can reduce ROS 95  

production of BAT mitochondria at least under in vitro circumstances (Dlasková et al., 2010; 96  

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Oelkrug et al., 2010). Because these studies only focused on BAT mitochondria, we are still 97  

lacking vital information about the impact of UCP1 activity on oxidative homeostasis at the 98  

scale of the organism, knowing that UCP1 seems crucial to survive during long-term cold 99  

exposure (Golozoubova et al., 2001). 100  

The main function of UCP1 is to uncouple ATP synthesis from respiration, leading to 101  

heat production (i.e. UCP1 was first referred to as thermogenin: Cannon and Nedergaard, 102  

1982). UCP1 is the most abundant protein in the BAT, and this tissue has a central role in the 103  

progressive substitution of muscular shivering in response to cold exposure by an endogenous 104  

production of heat (Fig. 1A), a process referred as to the adaptive nonshivering thermogenesis 105  

(NST, Klingenberg, 1990; Cannon and Nedergaard 2004). In the present study, we used an 106  

experimental design where metabolism was increased with or without the triggering of an 107  

uncoupled mitochondrial state (UCP1 activity). To do so, we compared the metabolic rate and 108  

oxidative stress markers of wild type (WT) mice and of mice deficient for the uncoupling 109  

protein 1 (UCP1 KO) housed at 26°C or after a four-week exposure to moderate cold (12°C). 110  

Cold exposure is known to trigger NST in small mammals, but this response cannot be set-up 111  

by UCP1 KO mice, which instead have to rely mostly on muscular shivering thermogenesis 112  

and on an efficient production of ATP (i.e. coupled respiration) to fuel muscle activity 113  

(Golozoubova et al., 2001). Based on the existing literature, WT mice were expected to rely 114  

only on UCP1-dependent thermogenesis after approximately 3 weeks of cold acclimation 115  

since NST replace progressively shivering (Fig. 1A, Cannon and Nedergaard, 2004), whilst 116  

UCP1 KO were expected to have to maintain high-intensity shivering on the long-term (Fig. 117  

1B, Golozoubova et al., 2001). This two-by-two experimental design (genotype: WT vs. 118  

UCP1 KO and temperature: 12°C vs. 26°C) allowed us to determine oxidative damage, 119  

oxidative challenge (GSSG/GSH ratio, see methods for details) and antioxidant capacities 120  

across four groups of mice, both at the plasmatic level and in the two main thermogenic 121  

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tissues (i.e. skeletal muscles and BAT). We predicted that a cold-induce rise in metabolism 122  

should be associated with higher oxidative damage in UCP1 KO mice, whereas no such 123  

relationship should be observed in WT mice relying on UCP1-dependent nonshivering 124  

thermogenesis. These two alternative predictions predict strong genotype by temperature 125  

interactions on markers of oxidative stress. 126  

127  

Experimental procedures 128  

(a) Animal treatment 129  

The study complied with the ‘Principles of Animal Care’ publication no.86-23, revised 130  

1985 of the National Institute of Health, and with current legislation (L87-848) on animal 131  

experimentation in France. The experiment started with 40 non-reproductive male and female 132  

mice C57 black 6 from our animal husbandry unit (temperature = 26 ± 1 °C). Half of the 133  

animals were wild type (WT) mice and the other half were UCP1 knockout mice (UCP1 KO). 134  

The founder mice (C57BL/6 J) UCP1 knock out for establishing our colony were originally 135  

provided by the CNRS (UPR-9078) and were backcrossed and genotyped according to The 136  

Jackson Laboratory protocol. During five weeks, 10 mice per genotype were maintained at 137  

26°C (groups WT26 and KO26), and 10 mice per genotype were exposed at 12°C (groups 138  

WT12 and KO12) during four weeks, after one week of progressive cooling (2°C per day). 139  

The cold exposure was chosen to be moderate (12°C - 4/5 weeks) for two main reasons: 1) to 140  

avoid premature death of UCP1 KO mice since their longevity is markedly reduced when 141  

exposed to 4°C (Golozoubova et al., 2001); 2) to avoid differences between WT and KO mice 142  

in terms of metabolic rate since it has been shown that below 12°C UCP1 KO mice might 143  

exhibit higher metabolic rates (Ukropec et al., 2006). We used an equal number of male and 144  

female mice in each experimental group (5 males / 5 females) and the animals did not differ 145  

between groups in terms of mass (GLM, p = 0.97) and age (GLM, p = 0.49) at the beginning 146  

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of the experiment. Animals were maintained on a 12 L : 12 D light cycle, and food (SAFE 147  

A03) and water were provided ad libitum. 148  

At the end of the experimental period, animals were culled (between 1:00pm and 4:00pm 149  

to restrict circadian bias in oxidative stress parameters) by cervical dislocation followed by 150  

decapitation in order to collect blood in heparinised micro tubes, as well as to collect the BAT 151  

and skeletal muscles (i.e. a mix of thigh and abdominal muscles). Immediately after 152  

collection, blood samples were centrifuged to separate plasma from cells, and tissues samples 153  

were snap frozen in liquid nitrogen. Samples were subsequently stored at -80°C until 154  

laboratory analyses. 155  

(b) Oxygen consumption measurements 156  

Oxygen consumption (VO2 expressed in mL O2 consumed per minute) was determined 157  

twice for eight animals of the WT12 and KO12 groups. The first measurement was conducted 158  

before the experimental period close to thermoneutrality (26°C), and the second one-week 159  

before the end of the experiment during the moderate (12°C) cold exposure. We recorded O2 160  

consumption (open-circuit indirect calorimetry system, Sable System, USA) during 5 hours 161  

after one-night of acclimation (without food but with water ad libitum). We used the average 162  

of these 5 hours to obtain the mean VO2. 163  

(c) Plasmatic oxidative stress measurements 164  

The antioxidant capacity and the concentration of Reactive Oxygen Metabolites (ROMs) 165  

were measured using the OXY-Adsorbent (5 µL of 1:100 diluted plasma) and d-ROMs tests 166  

(5 µL of plasma, Diacron International, Italy) following the manufacturer protocol. OXY 167  

adsorbent test allows quantifying the ability of the plasma antioxidant components to buffer 168  

massive oxidation through hypochlorous acid, while the d-ROMs test measures mostly 169  

hydroperoxydes, as a marker of global early oxidative damages (see Stier et al., 2012 for a 170  

review of the literature of previous experiments using those two markers of oxidative stress). 171  

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Antioxidant barrier is expressed as mM of HClO neutralised and d-ROMs as mg of H2O2 172  

equivalent/dL. Mean ± SE intra-individual coefficient of variation based on duplicates was 173  

2.23 ± 0.30% for the OXY test and 1.90 ± 0.26% for the d-ROMs test. Inter-plate coefficient 174  

of variation based on a standard sample repeated over plates was 4.16% for the OXY test and 175  

2.75% for the d-ROMs test. 176  

(d) Tissue oxidative stress measurements 177  

Glutathione content and proportion of oxidized glutathione in BAT and muscle was 178  

determined using DetectX® Glutathione fluorescent detection kit (Arbor Assays, USA), 179  

following manufacturer instructions. Glutathione (GSH) plays a key role in many biological 180  

processes including the protection of cells against oxidation. GSH is used as a reductant by 181  

the enzyme glutathione peroxidase to scavenge deleterious hydrogen peroxide. The oxidized 182  

form of glutathione (GSSG) can be restored into GSH by the action of the enzyme glutathione 183  

reductase. We evaluated the total glutathione content as an indicator of antioxidant protection 184  

and the ratio GSSG/total glutathione (which represent the proportion of oxidized glutathione) 185  

as an indicator of the oxidative challenge (i.e. the pro-oxidant power buffered by the 186  

glutathione system). Values are respectively expressed as nmol total glutathione/mg protein, 187  

and as a ratio of oxidized glutathione / total glutathione (0 meaning that all glutathione is free 188  

GSH, and 1 meaning that all glutathione is oxidized (GSSG)). Mean ± SE intra-individual 189  

coefficient of variation based on duplicates was 4.13 ± 0.42%. 190  

To assess oxidative damage on protein in BAT and skeletal muscle, we determined 191  

protein carbonylation using OxiselectTM protein carbonyl spectrophotometric assay kit (Cell 192  

Biolabs Inc., USA) following manufacturer instructions. This method allows quantifying 193  

carbonyl content, which is a common form of ROS-induced protein oxidation. All samples 194  

were measured on the same plate. Values are expressed as nmol protein carbonyl/mg protein. 195  

Mean ± SE intra-individual coefficient variation based on duplicates was 5.13 ± 0.78%. Total 196  

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protein content of tissues homogenates was determined in duplicates using a PierceTM BCA 197  

protein assay (Thermo Scientific, USA). 198  

(e) Statistical analysis 199  

We investigated genotype and temperature effect on metabolic rate (VO2) by running a 200  

repeated ANOVA. We used individual as subject, temperature as within-subject factor, 201  

genotype and the interaction between genotype and temperature as fixed factors, and mass as 202  

a covariate. 203  

We investigated the effects of genotype (WT vs. KO), temperature (26°C vs. 12°C), and 204  

the interaction between genotype and temperature on oxidative stress parameters with GLMs 205  

(General Linear Models), after testing residuals of each model for normality and 206  

homoscedasticity. When a significant interaction between genotype and temperature was 207  

revealed, we ran a post-hoc analysis to determine statistical differences between our four 208  

experimental groups. 209  

Age and sex were initially included in statistical models but were not significant; they 210  

were removed in order to clarify statistical models. Repeated ANOVA and GLMs were fitted 211  

with a normal error distribution (SPSS 18.0). Analyses were two-tailed tests and p values ≤ 212  

0.05. Means are quoted ± S.E. 213  

214  

Results 215  

(a) Metabolism 216  

Exposure to moderate cold ambient temperature increased mean oxygen consumption 217  

(VO2) by more than twofold (Fig. 2, Temperature: F = 1509.8, p < 0.001), but independently 218  

of mouse genotype (Genotype: F = 0.23, p = 0.64; Interaction: F = 1.43, p = 0.24). Body 219  

mass was entered as a covariate in the model to control for the increase in VO2 with body 220  

mass (Body mass: F = 15.03, p = 0.001). 221  

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(b) Oxidative stress 222  

Our experimental exposure to moderate cold of WT mice and mice deficient for UCP1 223  

revealed strongly significant genotype by temperature interactions (p-values 0.007) on 224  

markers of oxidative stress measured in the plasma (i.e. d-ROMs) and the skeletal muscles 225  

(i.e. proportion of glutathione oxidized and protein carbonyl content) but no in the BAT 226  

(Table 1, Figs 3-5). These interactions were explained by the increase in the aforementioned 227  

markers of oxidative stress in response to moderate cold exposure for UCP1 KO mice (Figs 228  

3A, 5B, 5C). In contrast, WT mice showed no or only moderate cold-induced increase in 229  

oxidative stress restricted to the proportion of glutathione oxidized in skeletal muscles (Fig. 230  

5B). Here, note that the activity of the antioxidant enzyme Glutathione Reductase (GR) in 231  

skeletal muscle was significantly affected both by the genotype (UCP1 KO > WT) and by the 232  

temperature (12°C > 26°C, see Electronic Supplementary Material [ESM] for details on 233  

methods and results). Plasma antioxidant capacity and tissue total GSH content did not 234  

significantly differ according to mouse genotype or temperature (Table 1, Figs 3B, 4A, 5A). 235  

Discussion 236  

Cold exposure has been previously used to assess how an increased metabolism may 237  

impact ageing in rodents (Holloszy and Smith, 1986; Topp et al., 2000; Selman et al., 2002; 238  

Kaushik and Kaur, 2003; Venditti et al., 2004; Selman et al., 2008; Vaanholt et al., 2009). 239  

Several studies found significant short- to mid-term effects (from 10 hours to 3 weeks) of cold 240  

challenge on oxidative stress markers, with for example increased oxidative damage ( Topp et 241  

al., 2000; Selman et al., 2002; Kaushik and Kaur, 2003; Venditti et al., 2004) or tissue-242  

specific modifications of antioxidant defences, which globally reflect a situation of oxidative 243  

stress (Kaushik and Kaur, 2003; Venditti et al., 2004). However, while always inducing a rise 244  

in metabolism, mid to long-term cold challenge experiments were producing contrasting 245  

results. Despite higher metabolic rate in the cold, oxidative stress markers and ultimately 246  

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individual survival were not markedly affected by long-term (i.e. throughout adult life) cold 247  

exposure in small rodents (Holloszy and Smith, 1986; Selman et al., 2008; Vaanholt et al., 248  

2009). Thermoregulatory mechanisms implicated in the cold response may be, at least 249  

partially, responsible for those discrepancies. An underestimated phenomenon is that 250  

thermogenesis is primarily achieved through muscular shivering in the hours to days of 251  

exposure to cold, but progressively replaced by the adaptive nonshivering thermogenesis (Fig. 252  

1A; Janský, 1973; Klingenspor, 2003; Cannon and Nedergaard, 2004; Ouellet et al., 2012). 253  

This latter process is achieved through mitochondrial uncoupling via UCP1 in the brown 254  

adipose tissue (extensively reviewed by Cannon and Nedergaard, 2004). Interestingly, 255  

longevity is shortened in UCP1 KO mice during prolonged cold exposure, with a median 256  

survival of ca. 13 weeks compared to more than 24 weeks for WT mice (Golozoubova et al., 257  

2001). Following a period of acclimation at 18°C, mice lacking UCP1 could maintain body 258  

temperature and resist cold temperatures through continuous shivering but apparently at a cost 259  

for longevity. We confirmed here that a moderate cold exposure (26°C to 12°C) increases 260  

approximately twice the metabolic rate, but independently of the mice genotype as previously 261  

demonstrated (Golozoubova et al., 2001; Meyer et al., 2010). The longevity effect observed in 262  

(Golozoubova et al., 2001) was probably not mediated by differences in terms of metabolic 263  

rate per se, but our results shed in light that cold-induced oxidative stress occurs for mice 264  

lacking UCP1, which may contribute to explain the reduced longevity of these mice in the 265  

cold. 266  

Because cold-induced increase in metabolism at the whole organism level is related to 267  

the higher activity of few, specific, tissues (i.e. muscular shivering thermogenesis and/or BAT 268  

nonshivering thermogenesis), the impact of cold-induced high metabolism on the oxidative 269  

balance is likely to be tissue-dependent (Kaushik and Kaur, 2003). Accordingly, following 270  

cold exposure UCP1 KO mice showed greater levels of oxidative stress in the blood and in 271  

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skeletal muscles, but not in BAT, compared to WT mice. In the absence of a cold challenge, 272  

WT and UCP1 KO mice had similar levels of BAT/muscles total glutathione content, 273  

oxidative challenge (proportion of glutathione oxidized) or oxidative damage on proteins. 274  

Hence, our results suggest first that, in the absence of a cold challenge and concomitant over-275  

expression of UCP1, BAT has no major influence on oxidative stress (as previously suggested 276  

by (Shabalina et al., 2006)). Furthermore, once UCP1 expression is triggered, we found that 277  

BAT metabolism activation during NST has no local (i.e. on BAT) but also no systemic pro-278  

oxidant deleterious effect (i.e. on the muscles and plasma). This is remarkable given that BAT 279  

metabolism is dramatically increased during cold exposure (Cannon and Nedergaard, 2004) 280  

and that brown adipocytes contain numerous mitochondria and then have a high oxidative 281  

capacity (Ricquier and Bouillaud, 2000). Therefore, even if UCP1 over-expression does not 282  

directly reduce oxidative stress following cold exposure, it might likely reduce the proportion 283  

of ROS generated per unit of oxygen consumed. The induced uncoupling mitochondrial state 284  

due to UCP1 activity could be one of these processes, and contributes to maintain redox 285  

homeostasis in the BAT during thermogenesis. Such UCP1 secondary effect (i.e. in addition 286  

to its thermogenic effect) in brown adipocytes is supported by in vitro experiments (Dlasková 287  

et al., 2010; Oelkrug et al., 2010) showing that UCP1 expression reduces ROS production in 288  

isolated mitochondria. Our results are also in line with a recent report of beneficial health 289  

effects of over expression of the tumour suppressor Pten in transgenic mice, those health 290  

effects being associated with striking hyperactivity of BAT and increased levels of UCP1, 291  

which in turn were leading to high metabolic rate but low levels of oxidative damage and 292  

lifespan extension (Ortega-Molina et al., 2012). 293  

Although we did not measure shivering per se in the present study, previous studies on 294  

cold acclimation between WT and UCP1 KO mice point out that after more than 4 weeks in 295  

the cold WT mice are expected to rely only on NST whilst UCP1 KO mice are expected to 296  

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still rely on shivering (Fig. 1, Golozoubova et al., 2001; Cannon and Nedergaard, 2004). 297  

Therefore, our results suggest that NST and muscular shivering thermogenesis led to similar 298  

cold-induced increase in metabolic rate after 4 weeks of mild cold exposure. The protective 299  

effect of NST in terms of oxidative stress could be indirect, by limiting the thermal 300  

dependency of animals upon the shivering process during prolonged cold exposure. Indeed, 301  

muscular shivering thermogenesis relies on muscle contractile activity, which itself relies on 302  

strong ATP production to fuel this activity. Contractile activity was previously reported to be 303  

positively related to ROS production and to a transient decrease in thiols content, followed by 304  

increased levels of various antioxidant enzymes (McArdle et al., 2001). Our results show that 305  

both WT and UCP1 deficient mice exhibited an increased proportion of oxidized glutathione 306  

after cold exposure, in a significantly higher extent in UCP1-KO mice. Given that muscle 307  

glutathione reductase activity reached the same level in both groups in cold conditions (see 308  

ESM) and that total glutathione did not significantly differ between groups, it implies that 309  

ROS production of muscle mitochondria might have been increased in the cold. Nevertheless, 310  

a direct measurement of muscle ROS production is required to ascertain this hypothesis. This 311  

potential increase in ROS production seems to have a different final impact (i.e. oxidative 312  

damage) depending on mice genotype. The slight rise of ROS production in WT mice, which 313  

could be attributed to a low or transient shivering activity or to a switch in pro-oxidant 314  

metabolic substrate (i.e. lipid mobilization; St-Pierre et al., 2002), had no impact on protein 315  

carbonyl levels. On the contrary, UCP1 KO mice exposed to 12°C showed a larger oxidative 316  

imbalance and higher protein carbonyl content in skeletal muscle. Interestingly, recent work 317  

has demonstrated a rise in muscular mitochondrial ROS production for UCP1 KO mice 318  

acclimated to 5°C but not for WT mice, which is in support to our results (Oelkrug, 2013). 319  

Furthermore, the idea that NST can indirectly protect the muscle from an overloading ROS 320  

production is in agreement with previous studies reporting that muscle antioxidant enzymes 321  

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activities decreased over time in WT mice exposed to cold (Petrovic et al., 2008) and that life 322  

long exposure to cold caused no significant muscle oxidative damage in wild derived rodents 323  

(Selman et al., 2008). Note also that it has been recently demonstrated that physical activity 324  

can induce the production by the muscle of irisin, and that this hormone stimulates UCP1 325  

expression and a brown-fat-like development of white adipose cells (Boström et al., 2012). 326  

Hence, such a system may act as a negative feedback to mitigate the deleterious impact of 327  

prolonged muscle shivering, such as oxidative stress (present study) or defective calcium 328  

handling (Aydin et al., 2008). 329  

330  

Conclusion 331  

Insights on mitochondrial functioning have shown that oxygen consumption is either 332  

principally coupled with energy conversion as ATP or as heat, depending on whether the ATP 333  

synthase or the mitochondrial UCP1 is driving respiration. There is however growing 334  

evidence that those two processes might lead to differing oxidative costs (Brand, 2000). 335  

According to one common expectation of the free radical theory of ageing, our results show 336  

that the high metabolism of UCP1 KO mice acclimated to cold, which was coupled to high 337  

ATP-dependent muscular shivering thermogenesis, was associated with increased levels of 338  

oxidative stress/damage in the muscles and in the blood. Alternatively and in agreement with 339  

expectations of the uncoupling to survive hypothesis, we found that the cold-induced 340  

activation of UCP1 in the BAT (i.e. NST) allowed WT mice to increase their metabolism to 341  

generate heat while preventing them from oxidative damage. Therefore, we suggest that 342  

determining the accurate nature of the mitochondrial mechanisms implicated in the control of 343  

metabolism in a given environmental condition (present study), but also in the determination 344  

of life history trajectories (Salin et al., 2012a, 2012b), are important milestones in our 345  

understanding of the determinants of longevity. 346  

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347  

Funding 348  

We are grateful to 2 anonymous reviewers for providing interesting and constructive 349  

comments on a previous draft of the paper, and to the CNRS (PICS, grant n° 5296), the 350  

French Ministry of Research and the University of Strasbourg for funding. P.B. is funded by 351  

the Swiss National Research Foundation (grant n° 31003A_124988). 352  

353  

Competing  interests:  354  

The  authors  declare  that  they  have  no  competing  interests.  355  

 356  

Authors’  contributions  357  

AS  designed   the  study.  AS  &  CH  collected   the  data.  AS,  FC,  PB,  SM  and  FB  took  part   in  358  

data  analyses  and  interpretations.  AS,  PB  and  FC  wrote  the  paper.  All  authors  have  read  359  

and  approved  the  final  version  of  the  manuscript. 360  

361  

References 362  

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Tables & Figures 486  

487  

Table 1: Results of GLMs (General Linear Models) testing the effects of genotype (WT 488  

vs. UCP1 KO), temperature (26 vs. 12°C) and the interaction between genotype and 489  

temperature on oxidative stress parameters. Results are presented for a general marker 490  

(plasma) and for the two main thermogenic organs (brown adipose tissue and skeletal 491  

muscle). Significant effects are reported in bold characters (N = 40, 10 mice per genotype and 492  

temperature). 493  

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Oxidative stress markers Genotype Temperature Genotype*Temperature

ROMs (damage) F = 7.44 p = 0.010 F = 3.26 p = 0.079 F = 8.27 p = 0.007 Plasma

OXY (antioxidants) F = 3.39 p = 0.074 F = 0.64 p = 0.430 F = 0.08 p = 0.784

Total glutathione F = 3.82 p = 0.058 F = 0.01 p = 0.906 F < 0.01 p = 0.974

Proportion of glutathione oxidized F < 0.01 p = 0.990 F < 0.01 p = 0.933 F = 0.04 p = 0.842 BAT

Protein carbonyl content F = 2.39 p = 0.130 F < 0.01 p = 0.934 F = 1.39 p = 0.246

Total glutathione F = 2.52 p = 0.121 F = 0.24 p = 0.623 F = 0.37 p = 0.546

Proportion of glutathione oxidized F = 14.63 p = 0.001 F = 41.22 p < 0.001 F = 8.41 p = 0.006 Muscle

Protein carbonyl content F = 9.23 p = 0.004 F = 9.86 p = 0.003 F = 17.39 p < 0.001

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Fig. 1: Theoretical time course and relative importance of shivering and nonshivering 502  

thermogenesis for (A) WT and (B) UCP1 KO mouse during a prolonged cold exposure 503  

(adapted from Golozoubova et al. 2001 and Cannon & Nedergaard 2004). 504  

505  

Fig. 2: Mean oxygen consumption expressed as VO2 (ml O2 consumed per minute) for 506  

WT mouse and UCP1 deficient mouse (UCP1-KO) exposed to 26°C or 12°C. White bars 507  

and black bars respectively represent normal and cold temperature. Different letters indicate 508  

significant differences between groups according to a repeated ANOVA model (see text for 509  

statistics, N = 8 per genotype and condition). Means are plotted ± SE. 510  

511  

Fig. 3: Plasmatic oxidative stress markers for WT and UCP1 deficient mouse exposed to 512  

26°C or 12°C for 4 weeks. (A) Plasmatic oxidative damage (Reactive Oxygen 513  

Metabolites) (B) Plasmatic antioxidant barrier (total capacity). White bars and black bars 514  

respectively represent normal and cold temperature. Different letters indicate significant 515  

differences (p ≤ 0.05) between groups according to GLMs models and associated post-hoc 516  

tests (N = 10 per genotype and temperature). Means are plotted ± SE. 517  

518  

Fig. 4: Brown Adipose Tissue (BAT) oxidative stress markers for WT and UCP1 519  

deficient mouse exposed to 26°C or 12°C for 4 weeks. (A) Total glutathione content; (B) 520  

Proportion of glutathione oxidized; (C) Protein carbonylation level. White bars and black 521  

bars respectively represent normal and cold temperature. Different letters indicate significant 522  

differences (p ≤ 0.05) between groups according to GLMs models and associated post-hoc 523  

tests (N = 10 per genotype and temperature). Means are plotted ± SE. 524  

525  

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Fig. 5: Muscle oxidative stress markers for WT and UCP1 deficient mouse exposed to 526  

26°C or 12°C for 4 weeks. (A) Total glutathione content; (B) Proportion of glutathione 527  

oxidized; (C) Protein carbonylation level. White bars and black bars respectively represent 528  

normal and cold temperature. Different letters indicate significant differences (p ≤ 0.05) 529  

between groups according to GLMs models and associated post-hoc tests (N = 10 per 530  

genotype and temperature). Means are plotted ± SE. 531  

532