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Changes Induced by Physical Activity and Weight Loss in the Morphology of Intermyofibrillar Mitochondria in Obese Men and Women

Division of Endocrinology and Metabolism (F.G.S.T., D.E.K.), Department of Medicine, and the Structural Biology Imaging Center (S.W.), Department of Cell Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213

Address all correspondence and requests for reprints to: Frederico G. S. Toledo, 807N Montefiore Hospital, 3459 5th Avenue, Pittsburgh, Pennsylvania 15213. E-mail: toledofs{at}upmc.edu.

	Abstract

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Context: In obesity, skeletal muscle insulin resistance may be associated with smaller mitochondria.

Objective: Our objective was to examine the effect of a lifestyle-modification intervention on the content and morphology of skeletal muscle mitochondria and its relationship to insulin sensitivity in obese, insulin-resistant subjects.

Design: In this prospective interventional study, intermyofibrillar mitochondrial content and size were quantified by transmission electron microscopy with quantitative morphometric analysis of biopsy samples from vastus lateralis muscle. Systemic insulin sensitivity was measured with euglycemic hyperinsulinemic clamps.

Setting: The study took place at a university-based clinical research center.

Participants: Eleven sedentary, overweight/obese volunteers without diabetes participated in the study.

Intervention: Intervention included 16 wk of aerobic training with dietary restriction of 500-1000 kcal/d.

Main Outcome Measures: We assessed changes in mitochondrial content and size and changes in insulin sensitivity.

Results: The percentage of myofiber volume occupied by mitochondria significantly increased from 3.70 ± 0.31 to 4.87 ± 0.33% after intervention (P = 0.01). The mean individual increase was 42.5 ± 18.1%. There was also a change in the mean cross-sectional mitochondrial area, increasing from a baseline of 0.078 ± 0.007 to 0.091 ± 0.007 µm² (P < 0.01), a mean increase of 19.2 ± 6.1% per subject. These changes in mitochondrial size and content highly correlated with improvements in insulin resistance (r = 0.68 and 0.72, respectively; P = 0.01).

Conclusions: A combined intervention of weight loss and physical activity in previously sedentary obese adults is associated with enlargement of mitochondria and an increase in the mitochondrial content in skeletal muscle. These findings indicate that in obesity with insulin resistance, ultrastructural mitochondrial plasticity is substantially retained and, importantly, that changes in the morphology of mitochondria are associated with improvements in insulin resistance.

	Introduction

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IN HUMAN OBESITY, there is increased lipid accumulation in skeletal muscle, a feature that correlates well with insulin resistance. The etiology of increased lipid accumulation in muscle is yet to be fully elucidated. Reduced mitochondrial oxidative capacity has been postulated as a contributing factor and has been shown to correlate with systemic insulin resistance (1, 2, 3). Under transmission electron microscopy (TEM), skeletal muscle intermyofibrillar mitochondria are significantly smaller in obesity and type 2 diabetes mellitus, being also associated with reduced activity of the electron transport chain (ETC) (3). These observations indicate that poor mitochondrial capacity is associated with insulin resistance. However, there are very few studies on the impact of interventions on the relationship between reduced oxidative capacity and insulin resistance.

Mitochondrial content and morphology can vary with changes in energy demands (4, 5). In skeletal muscle, malleability of oxidative capacity in response to exercise is regarded as a classic example of the plasticity of muscle in adaptation to changes in energy expenditure (4, 6, 7). For instance, using TEM and quantitative image analysis, higher mitochondrial density has been observed in physically active individuals (8, 9, 10).

Recently, our laboratory reported the results of a combined dietary weight loss and physical training intervention upon skeletal muscle oxidative capacity in obese individuals (11, 12, 13). Key findings noted were increases in systemic insulin sensitivity and skeletal muscle ETC activity (13), even though fiber-type distribution did not change (12). It was concluded that in obesity there is a substantial retained ability to increase skeletal muscle oxidative capacity. However, whether such functional plasticity is accompanied by modulation of myofibrillar mitochondrial content and morphology was not previously addressed. In a review of the literature, we found that the impact of lifestyle modifications upon mitochondrial content and morphology in obese, sedentary subjects has not been reported. The aim of this report was to study the hypothesis that mitochondrial content and morphology are affected by a lifestyle modification in obesity and to explore whether a relationship to insulin sensitivity exists. We report here the results of such intervention on mitochondria analyzed by TEM from muscle tissue.

	Subjects and Methods

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Research volunteers

Sedentary, weight-stable, nondiabetic volunteers with a body mass index (BMI) greater than 27 kg/m² were recruited after informed consent as previously reported (11). The data in this report are from 11 subjects (six men and five women) of that cohort who had sufficient muscle samples for TEM analysis.

After preintervention metabolic, aerobic fitness, and body composition studies and muscle biopsy, volunteers entered a 16-wk lifestyle-modification program as previously described (11). Briefly, volunteers participated in more than two exercise sessions per week (60–70% of maximal heart rate). Daily caloric intake was reduced by 500-1000 kcal with orientation by nutritionists. Preintervention measurements were repeated after the intervention was completed.

Subjects were admitted to an inpatient bed the night before metabolic studies and received a standardized dinner (7 kcal/kg, 50% carbohydrates, 20% protein, and 30% fat), followed by an overnight period of fasting of 12–14 h, during which a urine collection was obtained to estimate protein oxidation. Fasting respiratory quotient (RQ) was determined by indirect calorimetry using an open-circuit spirometry metabolic hood system (DeltaTrac, Anaheim, CA) during 30 min preceding the insulin infusion and during the last 30 min of the clamp. Insulin sensitivity was determined using euglycemic clamps and is expressed as the mean glucose infusion rate during the final 30 min of a 4-h insulin infusion (40 mU/m²·min). Fat and lean mass were assessed by dual-energy x-ray absorptiometry (Lunar-DPX-L, Madison, WI). Maximal oxygen consumption (VO₂ max) was measured using an incremental protocol on an electronically braked cycle ergometer.

TEM

A portion of vastus lateralis muscle obtained by percutaneous needle biopsy was cut into small pieces (1 x 1 x 2 mm), fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon for TEM. For each sample, 10 random, independent longitudinal images of intermyofibrillar regions were taken at x36,000 (JEM-1210; JEOL-Ltd., Tokyo, Japan). To measure mitochondrial volume density, i.e. the fraction of cell volume occupied by mitochondria, we employed digital morphometry and the point-sampling technique of classical stereology (14, 15). Briefly, a grid with 144 equally and symmetrically spaced intersection points was drawn on each micrograph. Volume density for each micrograph was calculated as the percentage of points overlaid by mitochondria from the total number of grid points contained within the myofiber cytosolic compartment. Mitochondrial cross-sectional area was determined by digital imaging software (Metamorph 6.3; Molecular Devices Corp., Sunnyvale, CA). We measured mitochondrial areas in all 10 micrographs of each sample (average, 73; range, 42–134). Numerical density, i.e. the frequency of individual mitochondrial profiles per area of cytoplasm, was calculated as the quotient of volume density and mean mitochondrial area (14, 15).

Statistics

Mean ± SEM were used, unless otherwise indicated, with P < 0.05 considered statistically significant. Paired t tests were used to examine the effects of intervention. Signed-rank test was used when data did not conform to a normal distribution. Spearman correlation analysis was used to examine associations between physiological variables and mitochondrial characteristics.

	Results

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Clinical characteristics (Table 1)

In response to the intervention, there were substantial reductions in weight, BMI, and fat mass. There were significant improvements in aerobic fitness (VO₂ max), insulin sensitivity, and fat oxidation after an overnight fast, as reflected in a decrease in fasting RQ.

Mean mitochondrial volume density (Vm), i.e. the fraction of muscle fiber occupied by mitochondria, was 3.70 ± 0.31% at baseline. This value increased to 4.87 ± 0.33% after intervention (P = 0.01), with a mean 42.5 ± 18.3% increase per subject. The average mitochondrial area also increased after intervention, from 0.078 ± 0.007 to 0.091 ± 0.007 µm² (P < 0.01). The mean within-subject increase was 19.2 ± 6.10%. Numerical density, i.e. the concentration of individual mitochondrial profiles per area of cytosol, changed from 489 ± 33.2 to 558 ± 36.8 mitochondria/1000 µm² (P = 0.056). There was no effect of gender, as changes in Vm resulting from intervention were similar between men (34%) and women (29%) (data not shown). Changes in VO₂ max and insulin sensitivity were also comparable. Representative micrographs are shown in Fig. 1A.

View larger version (106K): [in this window] [in a new window]   FIG. 1. A, Representative transmission electron micrographs of skeletal muscle obtained from a volunteer before and after intervention. Larger and more abundant intermyofibrillar mitochondria can be observed after intervention. B, Correlation plot between changes in insulin sensitivity, expressed as the change in glucose infusion rate corrected for lean body mass (LBM), vs. the corresponding within-subject change in Vm. C, Correlation plot between changes in insulin sensitivity and mitochondrial area.

We examined which physiological parameters best predicted changes in Vm (Vm). The change in insulin sensitivity was the only predictor (Fig. 1B). There were no statistically significant correlations between Vm and changes in VO₂ max, fat mass, percent fat mass, BMI, and fasting RQ. Among these variables, the change in mitochondrial area correlated with the change in insulin sensitivity (r = 0.68; P < 0.01; Fig. 1C) and less so with percent fat mass (r = –0.61; P = 0.04) and BMI (r = –0.61; P = 0.04). The average number of calories spent per exercise session did not correlate with changes in Vm or mitochondrial area.

	Discussion

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It has been known for decades that athletes have more numerous and larger mitochondria in skeletal muscle, adaptations that occur as a response to greater metabolic demands in skeletal muscle. Both mitochondrial content and size can be augmented by intense endurance training (10, 16, 17), but the intensity of the exercise stimulus is likely to be important for the extent of mitochondrial adaptations. In our previous study, we showed that mitochondrial ETC activity can be increased by exercise training incorporated in a typical lifestyle intervention recommended for obesity. However, whether the mitochondria became more abundant and larger was not previously addressed. In fact, there have not been TEM studies examining the effects of training on skeletal muscle mitochondrial ultrastructure in obesity. The present study was sought to address this knowledge gap. We observed that substantial capacity for changing mitochondrial content is retained in obesity. Mitochondria were also larger. Larger mitochondria may provide an environment for greater capacity for oxidative phosphorylation, and a larger size has been postulated to favor more rapid shuttling of high-energy phosphates to the sites of ATP use, such as the contractile apparatus and the sarcoplasmic reticulum (6).

An important aspect of our study is the confirmation of mitochondrial atrophy in obesity. Before intervention, the mitochondrial area (0.077 µm²) was nearly identical to that previously observed (0.076 µm²) in another study with obese volunteers (3). By comparison, the mean area appeared to be 0.114 µm² in lean subjects in that same study (3). In a review of the literature, this normal mitochondrial size is quite similar to that reported in normal young men (0.111 ± 0.060 µm²) (17) and healthy adults (0.102 ± 0.01 µm²) (18). Thus, our data indicate that in obesity there is atrophy of mitochondrial size, but lifestyle modification can significantly reverse it.

In skeletal muscle, mitochondria can be interconnected as a network (19, 20). Thus, a direct count of individual mitochondrial figures (numerical density) may not accurately express mitochondrial content. Therefore, Vm has been traditionally employed to more accurately express mitochondrial content (5, 8, 9). We found that in obesity, Vm increases after lifestyle modification as a result of mitochondria becoming both larger and more numerous. We found a modest 17.5 ± 9.86% increase in numerical density, a figure remarkably concordant with the modest increase of 16 ± 8% in mitochondrial DNA, previously reported in this intervention (13). These observations indicate that in obesity individual mitochondrial proliferation after training represents a minor component of mitochondrial biogenesis, underestimating the total increase in mitochondrial content.

What are the implications of the plasticity of muscle mitochondrial content for obesity? Exercise physiology studies tell us that changes in muscle mitochondrial content represent a cellular adaptation to the metabolic demands for oxidative ATP generation during exercise. It is plausible that skeletal muscle mitochondrial content may also have other metabolic implications, and one might be insulin sensitivity. In fact, there is emerging evidence linking reduced mitochondrial oxidative capacity to insulin resistance in muscle (1). However, to date, this association has been exclusively found in cross-sectional studies. In contrast, intervention studies are one of the most effective means to examine the importance of baseline correlations, because proportional changes should occur in parallel between variables that are physiologically linked. A key and novel finding of our study is that changes in mitochondrial content and size predicted changes in insulin sensitivity, accounting for approximately 50% of the variance in the change in insulin sensitivity. To our knowledge, these data constitute the first demonstration, in a prospective fashion, that modulation of mitochondrial content is associated with respective changes in insulin sensitivity. These findings strengthen the concept that the health of skeletal muscle mitochondria may be important for systemic insulin sensitivity in humans. However, a limitation of our study is the inability to discern whether improved mitochondria resulted in improved insulin sensitivity or the opposite. The former, though, is compatible with the concept that improved mitochondrial content and function could theoretically result in improved fat oxidation, reduced intramuscular lipotoxicity, and thus improved insulin sensitivity. We did observe a reduced fasting RQ, indicating improved systemic fat oxidation. Although we cannot determine whether this specifically occurred in skeletal muscle, this might have been the case because intramyocellular lipid droplets were smaller after intervention in this cohort (12).

In conclusion, we found that aerobic exercise training with weight loss reverses the ultrastructural atrophy of skeletal muscle mitochondria in obesity, a modulation that appears to be closely associated with systemic insulin sensitivity.

	Footnotes

 
This investigation was supported by funding from the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (DK49200), by the Obesity and Nutrition Research Center (P30DK462), and by the University of Pittsburgh General Clinical Research Center (5 M01RR00056).

First Published Online May 9, 2006

Abbreviations: BMI, Body mass index; ETC, electron transport chain; RQ, respiratory quotient; TEM, transmission electron microscopy; Vm, mitochondrial volume density; VO₂ max, maximal oxygen consumption.

Received January 3, 2006.

Accepted May 3, 2006.

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