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1 From the Department of Medicine, University of Toronto.
2 Supported by MRC grant MT-10885. 3 Address reprint requests to KN Jeejeebhoy, Medical Sciences Building, Room 6352, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: khush.jeejeebhoy{at}utoronto.ca.
ABSTRACT
Background: Previous studies suggested that cell energetics are altered by malnutrition.
Objective: We hypothesized that nutritional manipulations influence mitochondrial enzyme activities of the electron transport chain in both skeletal muscle and blood mononuclear cells.
Design: After a gastrostomy tube was inserted, 44 rats were randomly assigned to 1 of 4 experimental groups: control fed (CF; 364 kJ/d for 7 d), hypoenergetic fed (HF; 92 kJ/d for 7 d), hypoenergetic protein refed (HPR; 92 kJ/d for 7 d and then 129 kJ/d for 1 d), and hypoenergetic glucose refed (HGR; 92 kJ/d for 7 d and then 129 kJ/d for 1 d). The protein and glucose contents of the liquid formulas were different for the HPR and HGR groups. After mitochondria were isolated from the soleus muscle, the activities of complexes I–IV were measured spectrophotometrically. Because of the lack of available tissue, only the activity of complex I was measured in the mononuclear cell extract.
Results: The recovery of complex activities in the CF and HF groups was not significantly different in the mitochondrial fraction of the soleus muscle. Compared with that in the CF group, the activities of complexes I–III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cells were significantly lower in the HF group. The activities of complexes I–III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cells were significantly higher in the HPR than in the HF group. The activity of complex IV was generally not affected by nutritional manipulations.
Conclusion: Malnutrition decreases activities of mitochondrial complexes, which are restored by protein but not glucose refeeding.
Key Words: Protein-energy restriction • malnutrition • refeeding • mitochondrial complexes I-IV • electron transport chain • rats
INTRODUCTION
Hypoenergetic feeding is associated with significant changes in the fatigability of skeletal muscle and with a reduction in some key enzyme activities of the glycolytic pathway and the Krebs cycle (1, 2). Together, these findings suggest that muscle cell energetics are altered by protein-energy restriction. Consistent with this hypothesis are the findings of 31P nuclear magnetic resonance studies, which show a slower rephosphorylation of ADP in the muscles of malnourished rats than in control rats (3, 4). These abnormalities were corrected by increasing protein and energy intakes for 7 d. Furthermore, Ardawi et al (5) showed reduced oxygen uptake by the mitochondria of hypoenergetically fed rats. These findings support the hypothesis that mitochondrial enzyme activities in the electron transport chain are reduced in the muscle of malnourished rats.
Malnutrition results in derangements of both humoral and cellular immunity, contributing to increased susceptibility to infection (6). The nutritional impairment of immunoglobulin synthesis, due to protein starvation, appears to be rapidly reversed by refeeding (7). In malnourished children, leukocyte potassium is reduced and leukocyte sodium transport is altered (8, 9). These findings are comparable with those observed in skeletal muscle during malnutrition (10). Therefore, it is likely that malnutrition may also alter the mitochondrial electron transport chain of mononuclear cells.
Mitochondria occupy a pivotal position in aerobic ATP production. All of the energy-producing reactions generate reducing equivalents in the form of NADH and reduced flavins (FADH2), which are ultimately oxidized by oxygen through a chain of oxidoreduction reactions (OXPHOS system) occurring in complexes I–IV in the inner mitochondrial membrane (electron transport chain). Complex I [NADH dehydrogenase (ubiquinone)] and complex II [succinate dehydrogenase (ubiquinone)] oxidize NADH and succinate, respectively, and the electron acceptor is coenzyme Q10. Reduced coenzyme Q10 is subsequently oxidized by complex III (ubiquinol cytochrome-c reductase) and the electron acceptor is cytochrome c. Reduced cytochrome c is oxidized by oxygen with the aid of complex IV (cytochrome-c oxidase) and the products are water and oxidized cytochrome c. These processes create a proton gradient across the inner mitochondrial membrane, which is used to drive ATP synthesis by complex V (F0F1 ATPase) (11).
Protein synthesis in skeletal muscle is subject to control by hormonal and nutritional factors, including insulin and amino acids (12, 13). In both mice and rats, starvation reduces protein synthesis in skeletal muscle by 50–60% (12, 14). Protein synthesis responds rapidly to refeeding, rising to 180% of that in freely fed animals within 3 h (14). Accordingly, we hypothesized that hypoenergetic feeding would reduce, and protein refeeding restore, the activities of mitochondrial complexes I–IV in both muscle and mononuclear cells.
The purpose of the present investigation was to establish whether nutritional manipulation alters the activities of mitochondrial complexes in both the soleus muscle (complexes I–IV) and mononuclear cells (complex I) of rats. Specifically, we compared the changes in mitochondrial complex activities after hypoenergetic feeding and after protein or glucose refeeding with the mitochondrial complex activities in normally fed rats.
MATERIALS AND METHODS
Animals and surgery protocol
Male Wistar rats (Charles River Canada, Inc, St Constant, Canada) were housed in individual cages in a temperature-controlled room (22°C) with a 12-h light-dark cycle. On arrival at the animal facility, rats weighed 200–220 g and were acclimatized to the facility for 6 d, during which time they had free access to a cereal-based diet (Purina Rodent Chow 5001; Ralston Purina Company, Strathroy, Canada).
After this initial period, the rats were put under general anesthesia (50 mg sodium pentobarbital/kg intraperitoneally) and a gastrostomy was performed by inserting a silicone elastomer catheter (PE-20) into the stomach. The catheter was secured by a flange made of larger diameter tubing slipped over the catheter. The flange was placed into the stomach and the organ closed over it. The catheter was then tunneled subcutaneously to exit at the back, where it was encircled by a protective wire spring secured to the rat with a stainless steel button (Instech Labs, Horsham, PA) and led through a spring guard. The entire procedure was performed in a laminar flow hood and lasted 40–50 min. Postoperatively, the rats were housed in plastic metabolic cages (Nalgen, Sevenoaks, United Kingdom) and maintained on the control liquid formula, which was given orally ad libitum during the recovery phase (7 d). The protocol was approved by the University of Toronto Animal Care Committee.
Diet
The composition of the different liquid formulas is given in Table 1. The nutrient content of the control liquid formula met the recommended requirements for rats and was previously shown to result in weight gain comparable with that in rats fed a stock diet (15). In addition, we recently confirmed that this formula sustains weight gain and normal body composition when provided for >2 mo (data not shown). To control precisely for nutrient intake, all nutrients were administered through a gastrostomy tube. The fluid volume and the quantity of electrolytes, vitamins, and trace elements given were similar in all feeding groups; the only variable was the intake of protein, energy, or both.
View this table:
TABLE 1.. Composition of the liquid formulas (per 60 mL) consumed by the 4 groups of rats1
Experimental design
On day 0 of the experiment, the catheter and spring were connected to a swivel device (Instech Labs) that allowed movement of the animal about the cage. The swivel was suspended above the metabolic cage by a metal rod and the catheter was connected to the pump. The liquid formulas were administered continuously for 7 or 8 d at a constant rate (2.5 mL/h) with a Harvard infusion pump (pump 22; Harvard Apparatus, Wellesley, MA). Rats were randomly assigned to 1 of 4 experimental groups.
The refeeding formulas resulted in a 40% greater energy intake in the HPR and HGR groups than in the HF group. All of the animals were carefully observed, weighed daily, and allowed free access to water throughout the experimental period.
At the end of the feeding period, the rats were anesthetized with pentobarbital (50 mg/kg intraperitoneally). Six milliliters of blood was drawn by cardiac puncture and layered over cell preparation tubes (Vacutainer CPT for mononuclear cell isolation; Becton Dickinson and Co, Franklin Lakes, Canada). The soleus muscle, which is composed predominantly of type I muscle fibers rich in mitochondria, was removed from each leg and placed on ice in preparation for mitochondria isolation.
Blood and muscle analysis
Soleus muscle
Soleus muscles were trimmed of fat and connective tissue, chopped finely with a pair of scissors, and used for mitochondrial isolation according to the method of Birch-Machin et al (16). Each aliquot was rinsed in ice-cold medium A [120 mmol KCl/L, 20 mmol HEPES/L, 2 mmol MgCl2/L, 1 mmol EGTA/L, and 5 g bovine serum albumin (BSA)/L; pH 7.4] to remove any residual blood. The disrupted muscle was made up to 20 volumes with respect to the original wet weight of tissue with medium A and homogenized with a hand-held borosilicate glass homogenizer. The homogenate was centrifuged at 600 x g for 10 min at 5°C. The pellet obtained after centrifugation was resuspended in 8 volumes of medium A and centrifuged (600 x g, 5°C, 10 min). The 2 supernatant fluids were combined and were subsequently recentrifuged at 17000 x g for 10 min at 5°C. The pellet containing the mitochondria was resuspended in 10 volumes of medium A and then centrifuged at 7000 x g for 10 min at 5°C. The pellet obtained after the last centrifugation was resuspended in 10 volumes of medium B (300 mmol sucrose/L, 2 mmol HEPES/L, 0.1 mmol EGTA/L; pH 7.4) and recentrifuged (3500 x g, 10 min, 5°C ). The resulting pellet, which contained soleus muscle mitochondria, was suspended in a small volume of medium B (25 g/L protein) and was stored at -70°C until analyzed.
Mononuclear cells
Mononuclear cells were isolated from peripheral blood by Ficoll-Hypaque density centrifugation (Vacutainer CPT for mononuclear cell isolation; Becton Dickinson and Co) (17). The cell preparation tubes containing the whole blood were placed upright at room temperature for 90 min and then centrifuged at 1600 x g for 20 min at room temperature. The plasma was immediately removed and discarded. The buffy coat containing the mononuclear cells was collected, suspended in 10 mL phosphate buffered saline (PBS; 20 mmol/L, pH 7.5), and centrifuged at 300 x g for 15 min at room temperature. The supernatant fluid was removed and the cells were resuspended in PBS (20 mmol/L, pH 7.5) by gentle agitation and centrifuged at 300 x g for 10 min at room temperature. This procedure was repeated as needed until the pellet was free of erythrocytes [3 times and controlled by microscopy with a hemocytometer after dilution (18)]. The supernatant fluid was discarded and the cell pellet was resuspended by gentle agitation (no vortex mixing) in a medium containing 0.3 mol sucrose/L, 1 mmol EDTA/L, 5 mmol MOPS/L, and 5 mmol KH2PO4 buffer/L (pH 7.4) and placed on ice (19).
Because of the limitation in the amount of mononuclear cells obtained after isolation, the mitochondrial fraction was not isolated. The fresh cell suspensions were sonicated for 15 s (3 bursts of 5 s each) at 30 W on ice (19). After sonication, the buffer solution (described above) was added to make a final volume of 2 mL. The sonicated cell samples were centrifuged at 10000 x g for 10 min at 5°C. The supernatant fluid was discarded and the pellets were suspended in 200 µL PBS (20 mmol/L, pH 7.2) and were stored at -70°C until analyzed.
Enzyme assays
Protein concentrations
Protein concentrations of the mononuclear cell extract and the mitochondrial fraction of the soleus muscle were determined by using the biuret method. The activities of complexes I–IV were measured in the mitochondrial fraction of the soleus muscle and the activity of complex I in the mononuclear cell extract. The mononuclear cell samples were diluted with PBS (20 mmol/L, pH 7.2) to a protein concentration of 5 g/L. For the assay of activities of complexes I and II, aliquots of the mitochondrial fraction from the soleus muscle were diluted with PBS (20 mmol/L, pH 7.2) to a protein concentration of 1.2 g/L. The samples were frozen and thawed 3 times to disrupt the mitochondrial membranes. Similarly, aliquots of the mitochondrial fraction to be assayed for activities of complexes III and IV were diluted with medium B (described above) to a protein concentration of 1.2 g/L.
Enzyme activities were measured spectrophotometrically under conditions of maximal reaction velocity at an optimal pH and at room temperature as described below. All assays were performed in duplicate to a final volume of 1 mL by using a double-beam spectrophotometer (Spectrophotometer DU Series 600; Beckman Instruments, Fullerton, CA).
Activity of complex I
The activity of complex I was measured by monitoring the amount of NADH oxidized to NAD, which results in a change in optical density of 340 nm (16). Briefly, mitochondria were added to a buffer containing PBS (25 mmol/L, pH 7.2), 5 mmol MgCl2/L, 2 mmol KCN/L, 2.5 g BSA/L (fraction V), 2 µg antimycin A/L, 0.13 mmol NADH/L, and 65 µmol ubiquinone 1/L. The activity of NADH dehydrogenase (ubiquinone) was measured for 4 min. Then, 2 mg rotenone/L was added, after which the activity was measured for an additional 3 min. The specific activity of complex I was rotenone-sensitive NADH dehydrogenase (ubiquinone) activity.
Activity of complex II
The activity of complex II was measured by following the reduction of 2,6-dichlorophenolindophenol, indicated by the slope of the change in optical density at 600 nm (16). Mitochondria were preincubated in a buffer containing PBS (25 mmol/L, pH 7.2), 5 mmol MgCl2/L, and 20 mmol succinate/L for 10 min. Antimycin A (2 mg/L), rotenone (2 mg/L), KCN (2 mmol/L), and dichlorophenolindophenol (50 µmol/L) were added and the nonenzymatic activity was recorded for 3 min. The reaction was started by adding ubiquinone 1 (65 µmol/L) and the change in optical density was subsequently followed for 4 min. The specific activity of complex II was calculated by subtracting the nonenzymatic activity from that observed after the addition of ubiquinone 1.
Activity of complex III
The activity of complex III was determined by monitoring the reduction of cytochrome c at 550 nm with 580 nm as the reference wavelength (20). Briefly, mitochondria were equilibrated at room temperature in a buffer containing PBS (50 mmol/L, pH 8), 0.1 mmol EDTA/L, 2 g defatted BSA/L, 3 mmol sodium azide/L, and 60 µmol ferricytochrome c/L for 3 min. The reaction was initiated by adding 100 µmol decylubiquinol/L and the optical density was measured for 4 min. The nonenzymatic reduction in cytochrome c was measured under the same conditions after addition of 10 mg antimycin A/L and subtracted from the total activity of complex III to calculate the activity specifically due to complex III.
Activity of complex IV
The activity of complex IV was measured by monitoring the oxidation of cytochrome c at 550 nm with 580 nm as the reference wavelength (16, 21). Mitochondria (0.1–0.25 g protein/L) were preincubated for 5 min on ice in a buffer containing PBS (100 mmol/L, pH 7.2), 25 mmol NaCl/L, and 15 g dodecylmaltoside/L. Ten microliters of the preincubated mitochondrial sample was added to PBS (50 mmol/L, pH 7.2) and 1 mmol dodecylmaltoside/L. The reaction was initiated by adding 15 µmol ferrocytochrome c/L (oxidized form) and the specific activity of complex IV was estimated from the apparent first-order rate constant of the decrease in absorbance.
To test whether the recovery of mitochondrial complex activities and the integrity of the mitochondria were similar in CF and HF rats after differential centrifugation, the following experiment was performed. Mitochondria were isolated from the soleus muscles of 2 HF rats and 2 CF rats as described previously. During the isolation procedure, the recovery of activities of mitochondrial complexes I–IV was determined in aliquots taken at each step:
In addition, an aliquot at the end of the isolation from the soleus muscle of a CF rat was used for electron microscopy to evaluate the integrity of the mitochondrial membrane. The mitochondrial pellet was placed in universal fixative (gluteraldehyde:formalin, 4:1 by vol), dehydrated in graded ethanol, and embedded in Epon/araldite epoxy resin (Marivac Ltd, Halifax, Canada) according to standard techniques. Ultrathin sections were stained with saturated aqueous uranyl acetate and Reynold's lead citrate and were examined by using a model 1200EXII microscope (JEOL, Tokyo).
Data analysis
Soleus muscle
To determine whether the recovery of activities of complexes I–IV was the same between the CF and HF rats, the activities were calculated for the homogenate (step 1) and at steps 2–6 of the mitochondrial isolation procedure and were expressed as nmol•min-1•mg soleus muscle-1 for complexes I–III and as min-1•mg soleus muscle-1 for complex IV. The calculated recoveries at each step were the activities of complexes I–IV at that step divided by the complex activity in the homogenate (step 1) and were expressed as a percentage. For the nutritional study, the activities of complexes I–III were expressed as nmol•min-1•mg mitochondrial protein-1, whereas the activity of complex IV was expressed as the first-order rate constant-1 (min-1•mg mitochondrial protein-1).
Mononuclear cell extracts
In view of the limited amount of blood (6 mL) that could be obtained from each animal, the number of mononuclear cells obtained after purification was sufficient for the measurement of complex I only in the mononuclear cell extract. These results were expressed as nmol•min-1•mg mononuclear cell protein-1. To evaluate the linearity of our measurement in the CF and HF groups, we measured the activity of complex I when 2 different amounts of homogenate were added to the reaction mixture. In the CF group, the activity of complex I was 2.93 ± 1.20 and 3.21 ± 0.09 nmol•min-1•mg protein-1 (NS) when the mean amount of homogenate protein added to the assay was 0.173 ± 0.03 and 0.324 ± 0.07 mg, respectively. Similarly, the activity of complex I in the HF group was 0.96 ± 0.60 and 1.09 ± 0.54 nmol•min-1•mg protein-1 (NS) when the mean amount of homogenate protein added to the assay was 0.242 ± 0.09 and 0.464 ± 0.15 mg, respectively. Hence, the activity of mononuclear cell complex I was not at the lower limit of the assay.
Statistical analysis
All results are presented as means ± SDs. CIs were calculated for activities of complexes I–IV. The differences between the CF, HF, HPR, and HGR groups were tested by one-way analysis of variance (ANOVA). If the ANOVA showed significance (P < 0.05), the differences between group means were tested by using Tukey's honestly significant difference test. The nonparametric Wilcoxon test was used to compare the absolute weight for each group. The Spearman correlation coefficient was used to determine the relation between the activities of the mitochondrial complexes in the mitochondrial fraction of the soleus muscle and that in the mononuclear cell extract as well as between the body weight and the activities of mitochondrial complexes in both muscle and mononuclear cells. The STATISTICA program for WINDOWS was used for the statistical analyses (StatSoft, Tulsa, OK).
RESULTS
Recovery of activities of complexes I–IV at the different steps of the isolation of mitochondria from soleus muscle
The recovery of activities of complexes I–IV in the mitochondria isolated from the soleus muscle of CF and HF rats at the different steps of the isolation procedure are given in Table 2. Activities were not significantly different between the 2 groups. The percentages of activities of complexes I–IV lost in the pellet after the low-speed centrifugation (step 2) in the HF and CF rats were 24 ± 5% and 23 ± 7%, respectively. The activities of complexes I–IV remaining in the supernatant fluid containing the mitochondrial fraction (step 3) in the HF and CF rats were 62 ± 4% and 69 ± 9%, respectively. Similarly, losses in the supernatant fluid after high-speed centrifugation (step 4) were extremely low and similar in both the HF and CF rats. Furthermore, losses in the supernatant fluid after the 2 washes with mediums A and B (steps 5 + 6) were negligible. Electron microscopy of the final mitochondrial pellet from the soleus muscle of a CF rat showed well-defined organelles with intact membranes (data not shown).
View this table:
TABLE 2.. Activities of complexes I–IV at the different steps of the procedure for mitochondria isolation from the soleus muscle of rats in the control fed (CF) and hypoenergetic fed (HF) groups1
Body weight
At the initiation of enteral feeding (7 d after surgery), body weights were not significantly different between the 4 groups of rats. During the 7 d of enteral feeding, the weight of the CF group increased from 246.1 ± 16.0 to 293.4 ± 18.2 g (P < 0.01), corresponding to a gain of 2.7 ± 0.5%/d. The weights of the HF, HPR, and HGR groups decreased from 250.2 ± 14.3 to 204.2 ± 15.21 g (P < 0.01), from 256.5 ± 12.2 to 212.6 ± 8.1 g (P < 0.01), and from 264.2 ± 16.0 to 216.4 ± 18.5 g (P < 0.01), corresponding to daily losses of 2.6 ± 0.3%, 2.4 ± 0.3%, and 2.6 ± 0.6%, respectively. The weight of the HP group did not change significantly after 1 d of increased protein feeding. On the other hand, the body weight of the HGR group decreased after 1 d of increased glucose feeding (P < 0.02).
Comparison of the activities of complexes I–IV between the 4 groups
Mitochondrial fraction of the soleus muscle
The activities of complex I (-73%), complex II (-68%), and complex III (-92%) were significantly lower in the HF group than in the CF group (Figures 1–3). In contrast, there was no significant difference in the activity of complex IV between the CF and HF groups (Figure 4). After 1 d of protein refeeding, the activities of complexes I–III were significantly higher in the HPR group than in the HF group (Figures 1–3). In contrast, 1 d of glucose refeeding did not significantly affect the activities of complexes I–III. The activity of complex IV was not significantly influenced by 1 d of protein refeeding in the HPR group but was significantly lower after 1 d of glucose refeeding in the HGR group than in the CF group (Figure 4).
FIGURE 1.. Mean (±SD) activity of complex I in the mitochondrial fraction of the soleus muscle of the control fed (CF), hypoenergetic fed (HF), hypoenergetic protein refed (HPR), and hypoenergetic glucose refed (HGR) groups. n = 11 rats/group. *Significantly different from CF and HPR, P < 0.001 (one-way ANOVA and Tukey's honestly significant difference test). 95% CIs in brackets.
FIGURE 2.. Mean (±SD) activity of complex II in the mitochondrial fraction of the soleus muscle of the control fed (CF), hypoenergetic fed (HF), hypoenergetic protein refed (HPR), and hypoenergetic glucose refed (HGR) groups. n = 11 rats/group. One-way ANOVA and Tukey's honestly significant difference test: *Significantly different from CF, P < 0.001; **Significantly different from HPR, P < 0.01; ***Significantly different from HPR, P < 0.05. 95% CIs in brackets.
FIGURE 3.. Mean (±SD) activity of complex III in the mitochondrial fraction of the soleus muscle of the control fed (CF), hypoenergetic fed (HF), hypoenergetic protein refed (HPR), and hypoenergetic glucose refed (HGR) groups. n = 11 rats/group. One-way ANOVA and Tukey's honestly significant difference test: *Significantly different from CF, P < 0.001; **Significantly different from CF, P < 0.005; ***Significantly different from HPR, P < 0.04. 95% CIs in brackets.
FIGURE 4.. Mean (±SD) activity of complex IV in the mitochondrial fraction of the soleus muscle of the control fed (CF), hypoenergetic fed (HF), hypoenergetic protein refed (HPR), and hypoenergetic glucose refed (HGR) groups. n = 11 rats/group. *Significantly different from CF, P < 0.04 (one-way ANOVA and Tukey's honestly significant difference test). 95% CIs in brackets.
Mononuclear cell extracts
The activity of complex I in mononuclear cells was significantly lower in the HF group than in the CF group (-74%; Figure 5). After protein refeeding, the activity of complex I was significantly higher in the HPR group than in the HF group but was not significantly different from that of the CF group. Glucose refeeding did not significantly change the activity of complex I in the HGR group.
FIGURE 5.. Mean (±SD) activity of complex I in the mononuclear cell extract of the control fed (CF), hypoenergetic fed (HF), hypoenergetic protein refed (HPR), and hypoenergetic glucose refed (HGR) groups. n = 11 rats/group. *Significantly different from CF and HPR, P < 0.005 (one-way ANOVA and Tukey's honestly significant difference test). 95% CIs in brackets.
Relation between the activities of complexes I–IV in soleus muscle and activity of complex I in mononuclear cells
The activity of complex I in the mononuclear cell extract correlated significantly with the activity of complex I in the mitochondrial fraction of the soleus muscle in the CF and HF groups combined (Figure 6). Similar relations were observed for activities of complexes II and III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cell extract (data not shown). There was no significant correlation between the activity of complex I in mononuclear cell extract and that of complex IV in the mitochondrial fraction of soleus muscle. In the HPR group, the activity of complex I in mononuclear cell extract correlated significantly with the activity of complex I in the mitochondrial fraction of the soleus muscle (Figure 7).
FIGURE 6.. Relation between the activity of complex I in the mononuclear cell extract and in the mitochondrial fraction of the soleus muscle in the control fed (CF, ) and hypoenergetic fed (HF, ) groups. n = 11 rats/group. The correlation line is for the CF and HF groups combined.
FIGURE 7.. Relation between the activity of complex I in the mononuclear cell extract and in the mitochondrial fraction of the soleus muscle in the hypoenergetic protein refed (HPR) group. n = 11 rats.
Relation between body weight and the activities of complexes I–IV in soleus muscle and complex I in mononuclear cells
Mitochondrial fraction of the soleus muscle
At the time the rats were killed, the activities of complexes I–III correlated with body weight (r = 0.69, 0.76, and 0.69, respectively; P < 0.02) in the CF and HF groups combined but not in the HPR and HGR groups. Moreover, the activity of complex IV did not correlate with body weight in any of the groups.
Mononuclear cell extract
The activity of complex I in the CF and HF groups combined correlated with body weight at the time of sacrifice (r = 0.81, P < 0.01). In contrast, there was no correlation between the activity of complex I and body weight at the time rats in the HPR or HGR group were killed.
DISCUSSION
The recovery of activities of complexes I–IV was similar in the soleus muscle of the HF and CF rats. In a single preliminary measurement in the CF group, the isolated mitochondria were structurally intact. The activity of complex I in mononuclear cells was lower than that in the soleus muscle because the former was expressed per mg protein of mononuclear cell extract, whereas the latter was expressed per mg protein of isolated mitochondria. Therefore, changes in complex activities due to malnutrition and refeeding shown herein were not due to differences in mitochondrial recovery or to structural damage.
Effect of nutritional deprivation on activities of mitochondrial complexes I–IV
Our study is the first to show that protein-energy restriction, which is not confounded by micronutrient or electrolyte deficiency, selectively reduces the activities of complexes I–III in the mitochondrial fraction of the soleus muscle and the activity of complex I (the only complex measured) in the mononuclear cell extract. One week of protein-energy restriction did not influence the activity of complex IV in the soleus muscle. Therefore, our results support a selective reduction in mitochondrial enzyme activities concerned with electron transport during protein-energy restriction. The observation that these complex activities are reduced in mitochondria from both muscle and mononuclear cells is consistent with a more generalized effect of nutritional restriction. This conclusion is reinforced by the correlation between the activity of complex I in the mononuclear cell extract and in the mitochondrial fraction of the soleus muscle (Figure 6).
Relation between deficiencies of activities of complexes I–III and ATP synthesis
The previous studies concerning deficiencies in rat mitochondria focused on oxidative phosphorylation by liver mitochondria (22) and soleus mitochondria (5) after 2–3 wk of protein-energy deprivation. These studies showed that with protein-energy deprivation, the oxidation of substrates, the phosphorylation of ADP, and the respiratory control index are significantly lower at the 3 coupling sites in mitochondria. In the soleus muscle, the ratio of ADP to oxygen did not change with malnutrition, suggesting that protein-energy restriction did not alter the ability of the mitochondrial membrane to maintain a normal proton gradient. However, none of these studies measured activities of complexes I–IV and it is not clear whether the profound reduction in the activities of the complexes observed herein would result in reduced respiration and ATP synthesis.
When inhibition of the activity of complex I was plotted against the respiration rates and the amount of ATP synthesized, there was an abrupt decrease in these indexes only when the activity of complex I was inhibited by 80% in skeletal muscle mitochondria (23) and in nonsynaptic brain mitochondria (24). Similar results were obtained for activities of muscle mitochondrial complexes III and IV (50–70% inhibition) (25–27). In a recent publication (28) it was shown that the oxygen-consumption flux in muscle was controlled at the level of the electron transport chain (complexes I–III). Because the data produced in this study showed a reduction in the activities of complexes I–III of 68% after 1 wk of protein-energy restriction, it is likely that respiration and ATP synthesis would be significantly reduced. However, our preliminary data do not rule out the possibility that other steps known to contribute to the control of mitochondrial respiration, such as the adenine translocator, phosphate carrier, dicarboxylate carrier, proton leak, the ADP-regenerating system, and calcium may also be influenced by protein-energy deprivation (24, 29).
Effect of refeeding (glucose and protein) on activities of complexes I–IV
Traditionally, the effects of refeeding have been defined in terms of the restoration of total lean body mass. However, muscle performance responds earlier than do traditional indicators of body composition to refeeding. Russell et al (30) showed that 4 wk of refeeding normalized muscle function in anorexia nervosa patients, whereas body weight remained at 71% of ideal body weight and body nitrogen was 78% of normal. The rapid improvement in muscle function before observed changes in body mass was confirmed by Rigaud et al (31). In addition, Castaneda et al (32) showed that protein restriction profoundly influenced muscle function despite adequate energy intake. Consistent with these clinical observations, we found that protein refeeding, but not glucose refeeding, restored the activities of mitochondrial complexes I–III in the soleus muscle and the activity of complex I in mononuclear cells without any associated gain in body weight.
Effect of nutritional manipulation on the activities of mitochondrial complexes I–IV: possible mechanisms
The possible mechanisms by which protein-energy restriction and protein refeeding influence activities of mitochondrial complexes I–IV remain speculative. Because nutritional manipulations alter protein metabolism, it is likely that the effects on activities of complexes I–III are due to changes in mitochondrial protein synthesis, breakdown, or both. It is not known whether the apparent stability of complex IV activity is based on differences in transcription, translation, or protein turnover between the different enzymes of the electron transport chain or on the key role of complex IV in the regulation of energy production (33).
The rapid and specific effect of protein, but not of glucose refeeding, suggests that increased amino acid availability rather than increased insulin concentrations (glucose effect) is important in restoring the activities of mitochondrial complexes. In mice deprived of food for 18 h, refeeding of a complete diet stimulated protein synthesis in skeletal muscle in contrast with a diet lacking in protein or amino acids (34). Similarly, subjects who had fasted overnight and were then fed isoenergetic meals consisting of either carbohydrate and lipid or carbohydrate, lipid, and amino acids, had identical increases in plasma insulin concentrations (35). Nevertheless, whole-body protein synthesis increased only in those individuals who consumed the amino acid–containing meal. The role of insulin in stimulating protein synthesis in muscle was questioned in a recent study in diabetic mice (type 1 and type 2). The results showed that the response of protein synthesis was the same in healthy mice as in the diabetic mice after refeeding (14). Moreover, anti-insulin attenuated significantly the muscle protein synthesis in response to oral feeding (14). These different studies suggest that the stimulation of protein synthesis in response to food intake is not mediated by insulin alone but perhaps by a combination of an increase in plasma amino acid and insulin concentrations.
Clinical significance of findings
The effect of malnutrition and refeeding on mitochondrial enzyme activities in mononuclear cells is of clinical significance. Because the activity of complex I in both tissues correlated with body weight (in the CF and HF groups combined) and because the activity of complex I in soleus muscle correlated with the activity of complex I in mononuclear cells (Figures 6 and 7), it may be possible to examine the effect of malnutrition and refeeding on the mitochondria of humans by using mononuclear cells without having to resort to repeated muscle biopsies. Because the metabolic activity of rats is from 6 to 11 times faster than that of humans, the 20% weight loss observed over 7 d of hypoenergetic feeding corresponds to a similar weight loss in humans over 80 d (36, 37). On the basis of the same logic, we expect that the metabolic change in activity of mitochondrial complex I in humans would be maximal 7–14 d after refeeding begins. This time interval should be less than that required for a response in muscle function after refeeding in anorexia nervosa patients (30).
Conclusion
Our study showed that complex activities of the mitochondrial electron transport chain in the soleus muscle and mononuclear cells were depressed selectively after 1 wk of protein-energy deprivation when compared with normally fed rats. Protein refeeding was particularly effective in restoring activities of mitochondrial complexes to control levels without a corresponding weight gain. These findings may explain the mechanism of reduced muscle energetics during reduced protein-energy intakes.
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