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© 2006 American Society for Nutrition J. Nutr. 136:2194-2200, August 2006

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Diets High in Sugar, Fat, and Energy Induce Muscle Type–Specific Adaptations in Mitochondrial Functions in Rats1,2

Emilie Chanseaume3, Corinne Malpuech-Brugère4, Véronique Patrac3, Guy Bielicki5, Paulette Rousset3, Karine Couturier6, Jérôme Salles3, Jean-Pierre Renou5, Yves Boirie3,4,7 and Béatrice Morio3,*

3 INRA, UMR1019, Clermont-Ferrand, F-63000, France; 4 University Clermont 1, UFR Médecine, UMR1019, Clermont-Ferrand, F-63000, France; 5 INRA, STIM Unit, Clermont-Ferrand, F-63000, France; 6 Inserm, E0221, Grenoble, F-38000, France; Joseph Fourier University, Grenoble, F-38000, France; and 7 CHU Clermont-Ferrand, Clermont-Ferrand, F-63000, France

* To whom correspondence should be addressed. Email: morio{at}clermont.inra.fr.


   ABSTRACT
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 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Obesity is often associated with insulin resistance and mitochondrial dysfunction within skeletal muscles, but the causative factors are not clearly identified. The present study examined the role of nutrition, both qualitatively and quantitatively, in the induction of muscle mitochondrial defects. Two experimental diets [high sucrose (SU) and high fat (F)] were provided for 6 wk to male Wistar rats at 2 levels of energy [standard (N) and high (H)] and compared with a standard energy cornstarch-based diet (C). Insulin sensitivity (intraperitoneal glucose tolerance test, IPGTT) and intramyocellular triglyceride (IMTG) content (1H MRS) were determined at wk 5. Mitochondrial oxidative phosphorylation and superoxide anion radical (MSR) production were assessed on soleus (oxidative) and tibialis (glycolytic) muscles. Experimental diets induced hyperinsulinemia during IPGTT (P < 0.01 vs. C). Rats in the HSU and HF groups were hyperglycemic relative to the C group, P < 0.05 vs. C. The severity of insulin resistance paralleled IMTG accumulation (P < 0.05). In soleus, mitochondrial respiration and ATP production rates were lower in HSU and HF than in C (P < 0.05). By contrast, respiration was unaffected by the diets in tibialis, whereas ATP production tended to be lower in rats fed the experimental diets compared with C (P = 0.09). Mitochondrial adaptations were associated with more than a 50% reduction in MSR production in HSU and HF compared with C in both soleus (P < 0.05) and tibialis (P < 0.01). Changes in mitochondrial functions in the NSU and NF groups were intermediate and not significantly different from C. Therefore, excess fat or sucrose and more importantly, excess energy intake by rats is associated with muscle type–specific mitochondrial adaptations, which contribute to decrease mitochondrial production of ATP and reactive oxygen species.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Mitochondria are the most efficient source of energy required for cell survival and metabolism (1). Recent experiments showed that mitochondrial oxidative phosphorylation (OXPHOS)8 activity and expression of genes involved in OXPHOS are reduced in skeletal muscle of insulin-resistant patients suffering from obesity or type 2 diabetes (2–7). However, the mechanisms responsible for the decline in muscle mitochondrial OXPHOS activity remain to be elucidated. Several environmental, genetic, or physiological factors may be involved; for example, physical inactivity is associated with decreased muscle mitochondrial oxidative capacity (8). Therefore, sedentary behaviors may predispose subjects to develop insulin resistance in relation to decreased mitochondrial fat oxidation (8–10). Furthermore, the fact that young insulin-resistant offspring of individuals with type 2 diabetes had reduced mitochondrial oxidative capacity suggests the possible existence of an inherited defect (4,5). But insulin resistance may also be responsible for the decline in muscle mitochondrial functions because the anabolic effect of insulin on mitochondrial protein synthesis was shown to be reduced in the muscle of insulin-resistant elderly individuals compared with insulin-sensitive young adults (11). Finally, nutrition strongly modulates risk factors in the development of metabolic disorders and chronic diseases including obesity, type 2 diabetes, and cardiovascular diseases (12,13). Over the years, changes in daily dietary habits have led to the consumption of larger proportions of fatty and energy-dense food (14). Consumption of high-sucrose or high-fat diets was shown to induce insulin resistance in the skeletal muscle of rodents (15–17). We therefore hypothesized that quality and quantity of macronutrient intakes might be responsible for alterations in muscle mitochondrial OXPHOS activity in association with the induction of insulin resistance.

The present study examined the consequences of 3 nutritional strategies, i.e., diets enriched in sucrose, fat, and excess energy, on mitochondrial OXPHOS activity and superoxide anion radical (MSR) production in skeletal muscles of 400-g male Wistar rats. Skeletal muscle is a heterogeneous tissue composed of 4 contractile fiber types, i.e., types I, IIa, IIx, and IIb, in which the relative importance of glycolysis and mitochondrial oxidative phosphorylation for energy production varies. The fast type IIx and IIb fibers are adapted to brief and intense contractions fuelled by the glycolytic pathway and immediate availability of phosphocreatine. The slow type I fibers can sustain prolonged low power work in association with oxidative metabolism. Finally, type IIa fibers are oxidoglycolytic and exhibit intermediate contractile function. Because of the differences in energy demands and reliance on mitochondrial OXPHOS activity between fiber types, fiber-type specific mechanisms of adaptations to nutritional factors can be expected. For these reasons, we chose to study diet-induced adaptations in mitochondrial OXPHOS activity in 2 muscles characterized by high dependence on either oxidative metabolism, i.e., the soleus, which contains ~100% type I fibers (18,19), or glycolytic metabolism, i.e., the tibialis which contains 77% type IIb fibers, 19% type IIa and 4% type I (18,19). The degree of insulin sensitivity also was evaluated.


   Materials and Methods
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 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Chemicals. All reagents were obtained from Sigma Chemical. The bioluminescence ATP assay kit (ENLITEN rLuciferase/Luciferine) was purchased from Promega and the insulin RIA kit from Schering (CIS BIO International).

    Animals and experimental diets. Male Wistar rats (n = 80; 3 mo old; CERJ) were maintained under standard conditions (12-h light:dark cycle, controlled-temperature room 20–22°C) and housed individually with free access to water. After 1 wk acclimatization, rats were randomly distributed into 5 groups as follows: standard energy control group (cornstarch-based diet), standard-energy high-sucrose group (NSU), high-energy high-sucrose group (HSU), standard-energy high-fat group (NF), and high-energy high-fat group (HF). High-energy groups received 33% more energy than standard energy groups. Diets were supplied for 6 wk and were similarly enriched in protein (casein), fibers (cellulose), vitamins, and minerals. Ingredients were purchased from UPAE (Table 1). Food consumption was controlled daily using individual ramekins. Rats were weighed 2 times/wk to record their body mass gain. The experiment was performed in accordance with the French guidelines on the care and use of animals and approved by the ethical committee for animal experimentation (CREFA Auvergne, CE 1–05).


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  		TABLE 1 Composition of the experimental diets

 
    Intraperitoneal glucose tolerance test (IPGTT). An IPGTT was performed 1 wk before slaughter. To minimize stress, rats were handled by the same operator during weighing and IPGTT. Food was removed from rats 18 h before they received an i.p. injection of glucose (1 g glucose/kg body weight, between 0900 and 1000). Blood samples were collected from the retroorbital plexus in heparinized tubes immediately before glucose administration and 15, 30, 60, and 120 min after. Glucose values were obtained after sampling using a glucose reagent strip and a glucometer (Glucotouch, Lifescan). After centrifugation at 3000 x g, plasma samples were stored at –20°C until insulin measurement by RIA. Insulin sensitivity was estimated by the product of the areas under the curve (AUCs) for glucose and insulin as previously used by Cortez et al. (20). To validate the test, a saline injection was performed by the same method (data not shown). During this experiment, glucose concentration did not differ from baseline at each time point.

    In vivo localized 1H magnetic resonance spectroscopy (1H MRS) technique. 1H MRS was performed using an horizontal 4.7-T Brucker system with 2 concentric surface coils 31P(diameter, 15 mm)/1H(diameter, 15 mm) positioned over the rat hindlimb; 6 rats/group were anaesthetized with 2 mL/h isoflurane throughout the duration of the measurement. Body temperature was maintained using an electric blanket, and breathing rate was continuously controlled. Rat hindlimb was fixed to prevent any movement. Shimming was performed by optimizing the proton signal from water.

During the initial scan, the positioning of voxel (volume of interest) was selected to be as reproducible as possible between rats. A voxel of ~8 mm3 was chosen within the gastrocnemius of the rat hindlimb. Peaks of water, extramyocellular lipid, and intramyocellular triglycerides (IMTG) were identified at 4.70, 1.45, and 1.35 ppm, respectively, and integrated using Peakfit software (Systat Software,). IMTG levels were analyzed relative to water. Total acquisition time for 1H MRS was ~30 min.

    Tissue collection. At the end of the feeding period, rats were killed by decapitation after anesthesia with an i.p. injection of ketamine and valium. Soleus (slow-oxidative fibers) and tibialis anterior (fast-glycolytic fibers) were quickly removed, and separated from fat and connective tissues; ~50 mg of each skeletal muscle was kept at 4°C until skinned fiber preparation. The remaining pieces of muscle were cooled in liquid nitrogen and kept at –80°C until further analyses.

    Skinned fiber preparation. Mitochondrial respiration and ATP production were studied in saponin-skinned fibers as described by Saks et al. (21). Fiber bundles were mechanically separated with tongs and permeabilized with saponin (60 mg/L, 20 min). Bundles were then washed 3 times for 10 min to remove ADP, creatine phosphate (PCr), soluble enzymes and metabolites.

    Measurement of mitochondrial respiration and ATP synthesis. Fiber respiration rates were measured at 25°C using an oxygraph system (Oxytherm, Hansatech Instruments). The respiratory buffer contained 20 µmol/L EDTA (MgATPase inhibitor) and 0.2% BSA. Respiratory substrates and ADP solutions were prepared in the respiratory buffer without BSA. Saponin and EDTA solutions were freshly prepared each experimental day.

Different substrates were used as follows: glutamate 5 mmol/L + malate 2 mmol/L (G/M); NADH-linked substrates, which enter the respiratory chain through complex I (NADH-ubiquinone oxidoreductase); succinate 5 mmol/L, FAD-linked substrate which enters through complex II (succinate-ubiquinone reductase) with inhibition of complex I by rotenone 0.1 mmol/L; glutamate 5 mmol/L + malate 2 mmol/L + succinate 5 mmol/L (G/M/S) to approach physiological situation.

State 2 was measured in the presence of respiratory substrates without ADP. State 3 was measured after the addition of 1 mmol/L ADP (22). The quality of fiber preparation (notably complete removal of free ADP during washing) was validated by the return to state 2 after the addition of 60 µmol/L atractyloside, a potent inhibitor of the ATP/ADP carrier, adenine nucleotide translocator. When substrates G/M/S were used, ATP synthesis was measured as described by Ouhabi et al. (22) using a luminometer (Luminoskan Ascent, ThermoLabsystems) and ATP reagent kit. Finally, fibers were dried for 24 h at 110°C and weighed. Measurements were performed in duplicate with 0.5–1.5 mg dried fibers.

State 2 and state 3 respiration rates were expressed as nanoatoms (nat) O/(min·mg dried fiber). Respiratory control ratio (RCR) was calculated by dividing state 3 by state 2 respiration rate. ATP production rate was expressed as nmol/(min·mg dried fiber). ATP/O evaluated coupling of ATP production [nmol/(min·mg] to maximal oxygen consumption (state 3 in nmol O2/(min·mg).

    Muscle enzyme activities. Frozen muscle (30 mg) was homogenized in ice-cold buffer consisting of 0.25 mol/L sucrose, 2 mmol/L EDTA, and 10 mmol/L Tris HCl (pH 7.4) using a Polytron PT 1200C homogenizer (Bioblock Scientific) for a few seconds at maximum power. Citrate synthase (CS) and cytochrome c oxidase (COX) activities were assayed spectrophotometrically as described by Morio et al. (23). Maximal activity of CS and COX was expressed in µmol coenzyme/(min·mg wet tissue).

    Mitochondrial superoxide anion radical (MSR) production. Chemiluminescence elicited by superoxide radical production in the presence of lucigenin (Bis-N-methylacridinium) was measured according to a procedure adapted from Li and al. (24) on mitochondria isolated from soleus and tibialis as previously described (25). Mitochondria were placed in microwell plates containing lucigenin (25 µmol/L), and substrates G/M/S (5 mmol/L, 2 mmol/L, 5 mmol/L) with or without rotenone 0.1 mmol/L. Experiments were performed using 0.1 mg protein. All manipulations were conducted in the dark with minimal light and at 37°C. Microwells were counted for 40 min using a luminometer (Luminoskan Ascent, ThermoLabsystems). Results were expressed as the area under the curves of lucigenin luminescence in (40 min·arbitrary unit)/µg protein.

    Statistical analysis. The AUCs for glucose, insulin and MSR were calculated using the trapezoidal rule. Data are provided as means ± SD. Statistical analyses were performed using Statview, version 5.0 (SAS Institute). Data were evaluated by ANOVA and Fisher's Protected Least Significant Difference post hoc test. Differences were considered significant at the 5% level.


   Results
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 Introduction
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Discussion
 LITERATURE CITED
 
    Food consumption and body weight gain. Energy intake (kJ/d) was calculated for each group as the product of the diet's energy density (kJ/g) and daily food consumption (g/d). Energy intake was similar within the isocaloric groups (Supplemental Table 1). Body weights did not differ among the groups at the beginning of the study (data not shown). Body weight gain was greater in rats fed the high-energy diets compared with the control group (HSU +8% and HF +17%, P < 0.05). Furthermore, body weight gain was greater in the HF than in the NSU and NF groups (P < 0.05).

    Intraperitoneal glucose tolerance test results (IPGTT). The basal plasma glucose concentration was greater in the NSU, HSU, and HF groups compared with control (P < 0.01), whereas the plasma insulin concentration was greater in rats fed all experimental diets compared with control (P < 0.05). Experimental diets induced hyperinsulinemia during IPGTT (P < 0.01 vs. C). Rats in the HSU and HF groups were hyperglycemic relative to the C group, P < 0.05 vs. C. Insulin resistance was greatest in rats fed the experimental diets and decreased in the order of HSU > HF > NSU > NF > control (P < 0.001, Fig. 1A).


Figure 1
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  		Figure 1  Insulin resistance (A) and IMTG content in gastrocnemius muscle (B) in rats fed diets varying in sugar, fat, and energy levels for 6 wk. Values are means ± SD, n = 6. Means without a common letter differ, P < 0.05.

 
    Intramyocellular triglyceride (IMTG) content. The NF, HF, and HSU groups had 100–140% greater gastrocnemius IMTG levels than controls (P < 0.05), whereas that in the NSU group tended to increase by 87% (P = 0.16, Fig. 1B).

    Mitochondrial OXPHOS activity on permeabilized fibers. Preservation of outer membrane structure and complete removal of ADP and PCr were validated by the return to state 2 after the addition of ATR at the end of each measurement (Table 2).


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  		TABLE 2 Mitochondrial respiration rates and RCR in permeabilized fibers from soleus and tibialis muscles of rats fed diets varying in sugar, fat, and energy levels for 6 wk1

 
In soleus, the mitochondrial ATP production rate with substrates G/M/S tended to be slower in the NSU (–28%) and NF (–20%) groups compared with control, P = 0.10 and was 46 and 40% slower in HSU and HF, respectively (P < 0.05, Table 3). There were diet-induced changes in mitochondrial oxidative capacity with all substrates (G/M, succinate and G/M/S) in HSU and HF compared with control (P < 0.05, Tables 2, 3). More specifically, the state 2 respiration rate was lower in HSU and HF compared with standard energy groups with (G/M; P < 0.05). State 2 respiration using succinate was lower in the NSU and HF groups than in the others using succinate (P < 0.05), but when G/M/S was used, the groups did not differ. State 3 respiration rate was lower in the HSU and HF groups with all substrates compared with control (P < 0.05), except for HSU when succinate was used. Reductions in state 3 respiration rate were 24 and 31% in HSU and HF compared with control using G/M and G/M/S (P < 0.05). Finally, RCR (Table 2) and ATP/O (Table 3) did not different among the groups.


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  		TABLE 3 OXPHOS coupling in permeabilized fibers from soleus and tibialis muscles of rats fed diets varying in sugar, fat, and energy levels for 6 wk

 
In tibialis, the mitochondrial ATP production rate in the experimental groups tended to be lower than in control (P = 0.09, Table 3). Furthermore, the groups did not differ in state 2 and state 3 respiration rates, RCR, or ATP/O regardless of the substrates used (Tables 2, 3).

    COX and citrate synthase CS maximal activities in muscle homogenates. In soleus, the maximal activity of COX was reduced in all experimental groups compared with control (P < 0.001), whereas the maximal activity of CS did not differ among the groups (Table 4). By contrast, in tibialis, COX and CS did not differ from the control in any of the experimental groups.


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  		TABLE 4 Maximal activity of CS and COX from soleus and tibialis muscles of rats fed diets varying in sugar, fat, and energy levels for 6 wk

 
    MSR production on isolated mitochondria. In soleus, MSR production was not affected in NF, whereas it was reduced in NS compared with control without rotenone (P < 0.05; Table 5). MSR production was reduced by 64–86% without rotenone (P < 0.0001) and 73–78% with rotenone (P < 0.0001) in HSU and HF, respectively, compared with control. In tibialis, MSR production was not affected in NSU and NF compared with control, whereas it was reduced by 52–64% without rotenone (P < 0.01) and 64–90% with rotenone (P < 0.01) in HSU and HF, respectively, compared with control.


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  		TABLE 5 Mitochondrial superoxide anion radical production by isolated mitochondria from soleus and tibialis muscles of rats diets varying in sugar, fat, and energy levels for 6 wk

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Defects in mitochondrial functions were identified in skeletal muscles of insulin-resistant patients suffering from obesity or type 2 diabetes (2–7). The present study examined whether nutrition, both qualitatively and quantitatively, may be responsible for part of the induction of these defects. Data demonstrated that high-energy intake promoted muscle type–specific alterations in mitochondrial OXPHOS activity. By contrast, diets enriched in sucrose or fat, but equal in energy to the control diet, did not affect mitochondrial OXPHOS activity. Because duration of the diet may be critical, prolonged dietary interventions would be required to determine whether standard energy experimental diets may induce disturbances similar to those found in the high-energy groups.

Muscle mitochondrial OXPHOS activity was evaluated on permeabilized skinned fiber instead of isolated mitochondria for the following reasons: 1) very small tissue samples are required, 2) all cellular populations of mitochondria can be investigated, and 3) mitochondria are studied in their natural surrounding (21,22). RCR values were similar to those obtained by Saks and collaborators (21,22) under the same conditions, i.e., when measurements are performed at 25°C. Furthermore, ATP/O values, the amount of ATP produced per atom of oxygen consumed, were similar to those obtained with isolated mitochondria (26), demonstrating that measured ATP production originated mainly from mitochondria.

Supporting our hypothesis, mechanisms of diet-induced alterations in mitochondrial OXPHOS activity were muscle type specific. In soleus, alterations in state 3 respiration rates with substrates G/M, succinate and G/M/S demonstrated that the high-energy diets (i.e., HSU and HF) significantly reduced overall respiratory chain activity compared with control. These alterations were closely associated with a reduction in mitochondrial ATP production rate in the high-energy groups compared with control. In agreement with the significant decline in state 3 respiration in HSU and HF but not with the lack of changes in NSU and NF, maximal activity of COX, a key oxidative enzyme of the respiratory chain, was significantly reduced from all experimental groups compared with the control. However, the maximal activity of CS, a key oxidative enzyme of the tricarboxylic acid cycle, remained unchanged in all groups. If one considers the maximal activity of this latter enzyme as an index of mitochondrial density, this result indicates that mitochondrial biogenesis might not be altered after 6 wk of consuming experimental diets in the oxidative muscle but that synthesis and/or functionality of complexes of the respiratory chain may be more specifically modified.

By contrast with soleus, no significant adaptation in response to experimental diets was evidenced in the respiratory chain activity of mitochondria from tibialis muscle fibers. This was demonstrated through unchanged state 3 respiration rates with the substrates G/M, succinate, and G/M/S and through unchanged COX maximal activity. It should be noted that ATP synthesis tended to be lower in the experimental groups, suggesting that mild OXPHOS uncoupling might exist with the experimental diets. The difficulty in presenting evidence of OXPHOS uncoupling may be due to the method used. Assessment of the P/O ratio on isolated mitochondria would be more relevant for demonstrating such a mechanism (27). Given the increased IMTG content in rats fed the experimental diets, decreased ATP synthesis rate may be due to fatty acid–mediated proton leak uncoupling and/or increased uncoupling proteins (UCPs) content (28). In agreement with this assumption, 1.6- to 2-fold increases in UCP-2 and -3 gene expression were described in gastrocnemius (88% type IIb and 12% type IIa, (18)) of rats fed a high-fat diet (29,30).

Insulin resistance was observed in all groups fed the experimental diets compared with the controls, even in the NSU group for which 28% of starch was replaced by sucrose, with other nutrients similar to those of the control diet. Indeed, the addition of sucrose or fat to standard energy diets induced insulin resistance, characterized by a normoglycemic profile associated with a hyperinsulinemic response to IPGTT. Furthermore, high-energy diets induced glucose intolerance, characterized by relative insulinopenia and hyperglycemic profile in response to IPGTT. In that context, the reduction in ATP production in both muscles (although not significant in tibialis) of rats fed the high-energy diets is in agreement with the decreased ATP synthesis rate observed on isolated mitochondria from muscle of insulin resistant offspring of diabetic patients (4,5). The decline in mitochondrial respiratory chain activity in soleus of rats fed the high-energy diets corroborates the reduced mitochondrial oxidative capacity described in muscle of insulin-resistant patients (2,4,6,31,32). In these patients, the decrease in muscle mitochondrial oxidative capacity was related to the downregulation of genes involved in oxidative phosphorylation and of PGC-1{alpha}, which is a major transcriptional coactivator controlling mitochondrial biogenesis and oxidative capacity (3,33). Muscle PGC-1{alpha} mRNA level was shown to be decreased when plasma free fatty acid levels increased (34,35). In addition, an inverse relation was described between PGC-1{alpha} protein expression and IMTG accumulation in red and white gastrocnemius tissues of rodents (35). Therefore, because the experimental diets promoted IMTG storage in the present study compared with control, we hypothesize that the high-energy diet-induced alterations in soleus mitochondrial oxidative capacity may be related in part to decreased PGC-1{alpha} gene expression and/or activity.

Interestingly, adaptations in mitochondrial OXPHOS activity in rats receiving high-energy diets were associated with a reduction in mitochondrial superoxide anion production in both muscle types. Given their crucial role in energy homeostasis, does it make sense for mitochondria to be decreased in situations of excess macronutrient supply. Mitochondria are an important source of reactive oxygen species (ROS) and unavoidably, the direct target. Decreasing respiratory chain activity in oxidative muscle may not only be considered detrimental to ATP synthesis but may also be responsible for reduced ROS production (36). Similarly, mild uncoupling of substrate oxidation from ATP production may also be protective against excess ROS release (37). With (MSR without rotenone) or without (MSR with rotenone) consideration for reversed electron transfer (38), combined data from the present study suggest that mitochondrial adaptations to excess macronutrient intake might be protective against excess superoxide production. This could be related to the decreased levels of mitochondrial oxidative protein damage observed in the muscle of diabetic Sprague-Dawley rats (39).

In conclusion, nutrition quality and, most importantly, quantity are important factors modulating mitochondrial OXPHOS activity within skeletal muscle. The mechanisms of adaptation of mitochondrial OXPHOS activity are muscle type specific and affect primarily the oxidative muscle fibers. In these muscle fibers, mitochondrial adaptations were characterized by a reduction in the respiratory chain activity and consequently, in ATP production rate. By contrast, in glycolytic muscle fibers, 6 wk of consuming the experimental diets did not affect the respiratory chain activity although ATP production rate tended to be lower. Because changes in mitochondrial OXPHOS activity are associated with a concomitant decrease in mitochondrial superoxide production in the 2 muscle fiber types, the present data indicate that muscle mitochondrial adaptations induced by recurrent excess energy intake might be protective against excess production of mitochondrial ROS.


   ACKNOWLEDGMENTS
 
The authors thank Christophe Giraudet and Alexandre Teynie for their skilful technical assistance. They are grateful to Laurence Kay, Monique Malgat, Valdur Saks, and Jean-Pierre Mazat for their valuable assistance regarding the mastering of the permeabilized fiber technique. Jean-Pierre Donnat is also acknowledged for his contribution to MRS probe construction.


   FOOTNOTES
 
1 Supported by research founds from Institut National de la Recherche Agronomique (INRA). Back

2 Supplemental Tables 1 and 2 are available with the online posting of this paper at jn.nutrition.org. Back

8 Abbreviations used: AUC, area under the curve; COX, cytochrome c oxidase; CS, citrate synthase; 1H MRS, 1H magnetic resonance spectroscopy; HF high-energy-high fat group; HSU, high-energy high-sucrose group; IMTG, intramyocellular triglyceride; IPGTT, intraperitoneal glucose tolerance test; MSR, mitochondrial superoxide anion radical; NF, standard energy high-fat group; NSU, standard energy high-sucrose group; OXPHOS, oxidative phosphorylation; PGC-1{alpha}, peroxisome proliferator-activated receptor-{gamma} co-activator-1{alpha}; RCR, respiratory control ratio; ROS, reactive oxygen species. Back

Manuscript received 24 March 2006. Initial review completed 24 April 2006. Revision accepted 31 May 2006.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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