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Rat Myocellular and Perimysial Intramuscular Triacylglycerol: A Histological Approach

BELMONTE, MÔNICA A.; AOKI, MARCELO S.; TAVARES, FÁBIO L.; SEELAENDER, MARÍLIA C. L.

Medicine & Science in Sports & Exercise: January 2004 - Volume 36 - Issue 1 - p 60-67
doi: 10.1249/01.MSS.0000106170.26822.55
BASIC SCIENCES: Original Investigations
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BELMONTE, M. A., M. S. AOKI, F. L. TAVARES, and M. C. L. SEELAENDER. Rat Myocellular and Perimysial Intramuscular Triacylglycerol: A Histological Approach. Med. Sci. Sports Exerc., Vol. 36, No. 1, pp. 60–67, 2004.

Purpose There is controversy as to the use of intramuscular triacylglycerol (IMTAG) during exercise and to whether endurance training increases its utilization, despite the various methodologies used to address these questions. We used a histological-morphometrical approach to study the relative contribution of the two compartments of IMTAG storage, intramyocellular, and perimysial adipocytes, during exercise in sedentary and endurance-trained rats.

Methods After osmium impregnation, the soleus (SOL) and gastrocnemius (GAS) were studied under light and electron microscopy. IMTAG content (after Triton WR1339 treatment or not) and 14C-oleate incorporation into the muscles were studied.

Results In GAS, training, but not exercise alone, decreased extramyocellular lipid (P < 0.001 vs sedentary), an effect not found for SOL. Both muscles presented reduced lipid inclusion number (P < 0.001) and area (P < 0.05), immediately after exercise in sedentary and trained rats. For SOL, a greater number (P < 0.001 vs sedentary) of inclusions was found 24 h after exercise in trained rats. Triton WR1339 treatment decreased IMTAG content 12 h after exercise in SOL (but not in GAS), in sedentary (33%), and trained rats (52%).

Conclusions The multi-analytical approach adopted allowed the discernment between the IMTAG compartments and provided evidence for an effect of training upon storage of lipid in perimysial adipocytes in rat gastrocnemius, as well as clearly showed that the IMTAG mobilized during submaximal exercise in sedentary and trained rats derives from intramyocellular lipid, both in SOL and GAS. Moreover, the reposition of these stores 12 h after exercise was shown to be different in GAS and SOL, as plasma triacylglycerol clearly contributed to the process only in the latter, possibly reflecting the differences in lipoprotein lipase activity in the muscles reported by others.

Department of Histology and Embryology, Institute of Biomedical Sciences, University of São Paulo, BRAZIL

Address for correspondence: Marília C. L. Seelaender, Laboratory of Histophysiology, ICB, University of São Paulo: Av. Prof. Lineu Prestes 1524, São Paulo, SP, Brazil 05508-900; E-mail: seelaend@icb.usp.br.

Submitted for publication December 2002.

Accepted for publication August 2003.

Skeletal muscle contains significant amounts of triacylglycerol (TAG), reaching around 20–40 mmol·kg−1 dry muscle in the lean adult man (review in 29). During submaximal-intensity exercise, these stores are thought to contribute with more than 50% of the energy derived from the oxidation of fatty acids (18).

Many authors have suggested that the preferential use of fatty acids deriving from intramuscular triacylglycerol (IMTAG) depots may be advantageous in relation to that of free fatty acids (FFA) arising from peripheral adipose tissue lipolysis (33), on the basis that it would reduce the long diffusion distance required for plasma FFA utilization. Nevertheless, both the relative contribution of IMTAG during exercise, and the effect of training upon its utilization and storage are controversial (25). Although many authors report an increased reliance upon IMTAG in trained subjects, other found no differences (review in 6).

The muscle presents two distinct compartments of TAG accumulation: extramyocellular, in which TAG is stored in adipocytes residing in the connective tissue surrounding the fascicles of muscle fibers, the perimysium; and an intramyocellular pool, comprised of lipid droplets (10). Whereas the first is probably a long-term storage depot, the latter is thought to be a dynamic pool (31), with a similar caloric value to that of muscle glycogen stores (29). In the rat, incidence of extramyocellular stores varies greatly among different muscles, and TAG content within the muscle fiber is dependent on its phenotype (10), with red-type fibers exhibiting higher lipid deposition. Studies concerning IMTAG seldom make a distinction between the different storage sites, and the importance of perimysial adipocytes-related TAG during and after exercise is rarely addressed.

Most discrepancies concerning the physiology of IMTAG arise from methodological limitations, compromising the discrimination between the depots represented by perimysial adipocytes and intramyocellular TAG (31). In the present study, we evaluated the effect of exercise and endurance training upon rat soleus (SOL) and gastrocnemius (GAS) TAG deposition, in an attempt to assess the relative contribution to exercise fueling of these two compartments through a histological approach, including light and electron microscopy after osmium impregnation. This method allows full preservation of lipid and maintenance of inclusions shape and size. The number and the area of perimysial adipocytes were assessed, as well as various morphometric parameters of intramyocellular lipid droplets (number, area, maximum and minimum diameters, shape factor, and contact area between lipid droplets and mitochondria) in the muscles of sedentary or endurance-trained rats, before, immediately after, and 24 h after an exercise bout. To our knowledge, this is the first study to approach the dynamic histology of IMTAG deposition in such detail. Concomitantly, biochemical determination of muscle TAG content and muscle exogenous oleate incorporation was carried out as a control for the histological results.

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METHODS

In all experiments, all ACSM guidelines for animal care were followed, and the local animal care facility board authorized all protocols.

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Experimental design.

Sedentary and endurance-trained rats, at rest, or after a single bout of long-duration, submaximal-intensity exercise were studied for the determination of IMTAG stores through histological-morphometrical and biochemical analysis. The morphometric analysis was performed to allow the recognition of the relative importance and contribution of each the intramyocellular and the perimysial sites of TAG storage to exercise fueling. The biochemical parameters studied, which served as a control, rendered the results of total IMTAG recruitment comparable with the results obtained by others who adopted biochemical approaches to the problem. The determinations were carried out before, immediately after, and 12 h and 24 h after the exercise bout in order to assess the dynamic aspect of IMTAG utilization and reposition in each of the compartments. In the histological approach, a recovery period of at least 24 h after the exercise bout was required to allow the observation of significant differences in morphometric aspects, as determined in preliminary experiments.

The dependent variables used in the present study were: body weight variation; glycemia; muscle weight/body weight ratio; muscle TAG and glycogen content; muscle oleate incorporation; percentage of total section occupied by perimisyal adipocytes; number of lipid droplets in microscopic muscle fiber preparations; and area, maximum and minimum diameters, as well as the shape factor of intracellular lipid inclusions. The independent variables were time and exercise and, for TAG content determinations, Triton WR 1339 treatment.

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Animals.

Sixty male Wistar rats (200–300 g) of equivalent age, were kept under controlled temperature (23 ± 1°C) and photoperiod (12-h light, 12-h dark, lights on at 7:00 a.m.), and received water and food ad libitum. For the histological/morphometric studies, rats were divided into five groups of five animals each (sedentary rats (S); sedentary rats studied immediately after a single exercise bout (E0); sedentary rats studied 24 h after a single exercise bout (E24); trained rats, studied immediately after exercise (T0); trained rats, studied 24 h after exercise (T24)). For the biochemical determinations, seven groups of five animals each were studied (S; E0; E24; T0; T24; and sedentary or trained rats studied 12 h after a single exercise bout, E12 and T12, respectively). Another 84 animals were divided in the same groups (12 animals/each) for determination of the effect of Triton WR 1339 treatment on IMTAG content.

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Training protocol.

Rats ran on a treadmill (FUNBEC ESB-01) for 8 wk at 60% O2max (3), 5 d·wk−1, with increasing time and speed, until reaching the final speed of 20 m·min−1 and 1 h of continuous exercise. Muscle (SOL) citrate synthase activity (measured as described in 1 and 3) showed a twofold increase in trained rats (from 18.52 ± 0.04 nmol·min−1·mg−1 of protein in the sedentary group to 36.69 ± 0.21 nmol·min−1·mg−1 of protein in trained rats), indicating an increased oxidative capacity of the muscle induced by endurance training. Plasma lactate measurement (23), which was carried out for 4 nonconsecutive weeks of the training program, was shown not to be different in the animals before (2.2 ± 0.1 mmol·mL−1, 2.2 ± 0.1 mmol·mL−1, 2.0 ± 0.2 mmol·mL−1, 1.9 ± 0.1 mmol·mL−1 for the 2nd, 4th, 6th, and 8th week of training, respectively), during (1.6 ± 0.1 mmol·mL−1, 1.7 ± 0.1 mmol·mL−1, 1.6 ± 0.2 mmol·mL−1, 1.5 ± 0.1 mmol·mL−1 for the 2nd, 4th, 6th, and 8th week of training, respectively), and after the exercise bout (1.8 ± 0.1 mmol·mL−1, 2.2 ± 0.3 mmol·mL−1, 1.4 ± 0.2 mmol·mL−1, 1.6 ± 0.2 mmol·mL−1 for the 2nd, 4th, 6th, and 8th week of training, respectively), proving the adopted exercise intensity to be submaximal.

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Morphological analysis.

After anesthesia with a 10% aqueous solution of chloral hydrate (0.3 mL·100 g−1 corporal weight), animals were perfused with cooled 0.9% NaCl solution for removal of the blood and then, for 30 min, with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. After overnight postfixation of SOL and GAS samples with a solution consisting of 1% formaldehyde, 1% calcium chloride, 1% cadmium chloride diluted in 0.1 M sodium cacodylate buffer, pH 7.2 (13), followed by washing and 2 h of postfixation with 1% osmium tetroxide, the samples were infiltrated into 1:1 (v/v) hydroxy-ethyl-metacrylate (Historesin 18500, Leica)/distilled water for 2 h and then overnight with Historesin. Samples were included into gelatin capsules (48 h at 37°C). Microtome sections of 10–12 μm were then counter-stained with methyl green-pironin for 15 min and in hematoxylin for 10 min at 37°C. Counting and morphometric analysis were carried for adipocytes present in the perimysium, but not for those in the epimysium, the connective tissue surrounding the whole muscle.

For the ultrastructural studies, 2 mm3 of the muscles, obtained after perfusion, were postfixed with a 1% paraformaldehyde, 2.5% glutaraldehyde, 2.5 mM CaCl2 solution in 0.1 M sodium cacodylate buffer (pH 7.2–7.4), and prepared for electron microscopy analysis as in (2,12). After inclusion in Spurr, the 250-nm semithin sections were obtained with an ultra-microtome (MT1-Sorvall). Ultra-thin sections (70 nm) were collected onto copper grids (200-mesh) and contrasted with 2% uranyl acetate and lead citrate (24).

Sigma Scan Pro (Jandel Co.) software was used for image analysis of optical microscopy preparations. Quantitative analysis was performed with 10 nonserial sections (50-μm interval) of each the SOL and GAS. The area of the section and that of present osmium-impregnated adipocytes were determined in the captured image after careful manual delimitation of the cells. Results are expressed as the percentage of total section area occupied by adipocytes. For the ultrastructural analysis of the osmium-impregnated lipid inclusions within the muscle fiber (manually delimitated), 30 fields from 30 grids/muscle/animal were examined. The number, area, and percentage of the area of lipid droplets that were in contact with adjacent mitochondria, as well as the shape factor (the ratio between the area and perimeter: SF = 4π·(lipid image area)/(cell perimeter)2 (32), were evaluated. A shape factor of one (1.0) corresponds to a perfectly round shape (implying greater TAG content), whereas the closer the value to zero, the more polygonal the droplet.

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Metabolic analysis.

The plasma of the overnight fasted animals was collected, always between 9 and 10 a.m., for the determination of the concentration of glucose (with the Advantage® monitor). Muscle glycogen content was determined as in (11): after incubation of muscle for 30 min with 5.5 M KOH (in boiling bath), extraction was carried out with 70% ethanol, and sample glycogen content was assessed after adding a 1 M anthrone solution in sulfuric acid. The reaction product was determined at 540 nm (Hitachi U-2002 spectrophotometer).

For determination of muscle fat content and its capacity to incorporate exogenous oleate, the animals received 0.5 mL of [14C]-triolein (Amersham, approximately 2.5 μCi) intragastrically, as in (21). After 5 h, the SOL, GAS, and digestive tract were removed and submitted to the method described in (30): duplicate samples were digested with 30% KOH (w/v) for 15 min and then, after absolute ethanol was added, incubated for 2 h at 70°C. The free fatty acids from the saponifiable lipid fraction were extracted three times with petroleum ether. After evaporation, the mass of the lipid present in the sample was assessed. Scintillation fluid was added to samples, whose radioactivity was determined in a scintillation counter (Packard, TRICARB 2100). To assess the total radioactivity in the tissue, 0.5 mL of NaOH (1N) was added to 300 mg of either liver or digestive tract and the samples incubated for 30 min at 70°C. An aliquot of 100 μL was transferred to a vial containing scintillation fluid, 100 μL of HCl and some drops of hydrogen peroxide (130 V). The total radioactivity present in the tissue was measured to provide a control value for comparison with the amount of radiolabeled oleate incorporated in the form of lipid. To allow an empirical estimation of the total radioactivity absorbed by the intestines, the digestive tract and the feces were homogenized and submitted to the same procedures. The encountered value of radioactivity was subtracted from that present in the bolus given intragastrically, so that tissue radioactivity could be expressed as a percentage of the total radioactivity absorbed. The same procedures were adopted after Triton WR 1339 (Sigma Chemical Co.) treatment (16): rats received an IV injection (600 mg·kg−1 in 0.9% NaCl) of the detergent, and arterial blood (80 μL) was collected every 30 min, for 2 h, for measurement of plasma TAG content, a control of the effect of the detergent.

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Statistical analysis.

Variance homogeneity was assessed, and data transformation was employed when necessary. One-factor ANOVA, followed by multiple comparisons with Tukey’s posttest, was adopted when comparing S, T0, and E0 in relation to the following dependent variables: body weight variation; glycemia; muscle weight/body weight ratio; muscle TAG and glycogen content; muscle oleate incorporation; percentage of total section occupied by perimisyal adipocytes; number of lipid droplets in microscopic muscle fiber preparations; and area, maximum, and minimum diameters, as well as the shape factor of intracellular lipid inclusions. The same dependent variables were analyzed by ANOVA with two factors, exercise (exercise and training) and time (0, 12, and 24 h), or three factors (exercise, time, and Triton WR 1339 treatment), followed by Tukey’s posttest. The significance level adopted was P < 0.05, at least. The observed power (using alpha = 0.05) for each interaction studied was of at least 0.998.

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RESULTS

Morphological analysis.

Figures 1 and 2 illustrate the aspect of the GAS in sedentary and trained (T0) rats in the preparations for light and electron microscopy, respectively. Although no difference concerning the total area occupied by perimysial adipocytes per section (Table 1) was found for the SOL among the groups, a reduction of this parameter was observed for the GAS in T0 compared with S (51%) and E0 (48%), and in T24 in relation to E24 (46%). The number of lipid droplets in each section of SOL and GAS (Table 1) was reduced (P < 0.001) in E0 in relation to the preexercise condition (S). After 24 h, however, an equivalent number of lipid inclusions was found in these groups. For T0, a decreased number of lipid droplets (46% and 62% for GAS and SOL, respectively) as compared with S was found; after 24 h, however, a difference from T0 was observed (P < 0.05 for GAS and P < 0.01 for SOL). It is interesting to note that for SOL, the number of droplets 24 after exercise (T24) was higher than in S (P < 0.01).

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

TABLE 1

TABLE 1

Not only the number but also the morphometric parameters of lipid inclusions were changed in response to exercise and training (Table 2). For GAS, mean lipid droplet area was decreased immediately after the exercise bout (32% for E0 and 27% for T0) compared with S. The area of T24 lipid inclusions was 15% smaller than that of E24. Similar differences in the maximum and minimum diameters accompanied the area alterations (P < 0.05). The shape factor of lipid droplets was decreased in GAS of E0, as they were more polygonal in these samples. The mean area of SOL intramyocellular lipid inclusions was also reduced in E0 (25%) and T0 (30%) as compared with S, but, after 24 h, it increased (P < 0.05) to values similar to S. The same pattern of variation was observed in regard to maximum and minimum diameters. The shape factor was slightly (5–7%) decreased (P < 0.05) in SOL for E0 in relation to S and E24. Contact area between droplets and mitochondria (Table 3) was augmented (60%) in T0 (which showed the smallest number of lipid inclusions of all groups) compared with E0 in the SOL.

TABLE 2

TABLE 2

TABLE 3

TABLE 3

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Metabolic analysis.

No differences in the final weight of animals (304.3 ± 7.4 g, 303.3 ± 7.7 g, and 296.1 ± 5.6 g for S, E, and T, respectively), and in plasma glycemia (S: 102 ± 4 mg·dL−1; E0: 112 ± 11 mg·dL−1; E12: 94 ± 6 mg·dL−1; E24: 109 ± 3 mg·dL−1; T0: 111 ± 16 mg·dL−1; T12: 93 ± 3 mg·dL−1; and T24: 97 ± 8 mg·dL−1) were observed (N = 12 animals per group). Table 4 shows that GAS relative weight was decreased in T0 as compared with all other groups (P < 0.001), and a reduced TAG content as compared with S (48%) and E0 (50%) was also observed. Along with the reduction of IMTAG, glycogen concentration in GAS was also significantly reduced (50%) in T0 in relation to S (0.3 ± 0.1 mg·100 mg−1 tissue and 0.6 ± 0.1 mg·100 mg−1 tissue, respectively). No other significant differences in glycogen content were found among the studied groups. The fat content of GAS in T12 and T24 was significantly decreased in relation to E12 and E24 (P < 0.01 in both cases). An enhancement of TAG content (34%) was observed in T12 compared with T0. The incorporation of radiolabeled oleate in GAS was also enhanced in T12 compared with T0, in which a marked decrease of this parameter was obtained in relation to S (10-fold, P < 0.01), and E0 (P < 0.001). After 12 h, 14C-oleate incorporation in the GAS of trained animals increased (4.6-fold, P < 0.01) and, at 24 h, was similar to that of S. Gastrocnemius E0 oleate incorporation was diminished (60%) as compared with S. In these experiments, the percentage of incorporation of 14C-oleate in the muscle, from the estimated radioactivity present in the amount of oleate that was absorbed by the intestines (minus that remaining in the digestive tract and feces), was assessed. The actual amount of oleate absorbed by the gut was affected by training but not by exercise alone. In the GAS, a 24% increase for T0 (84.2 ± 0.87% from the total amount administered intragastrically) compared with S (67.8 ± 2.2%) was observed, whereas T12 (75.3 ± 1.9%) and T24 (82.2 ± 1.7%) increased 16% and 27% in relation to E12 (65.0 ± 2.3%) and E24 (64.9 ± 2.1%), respectively.

TABLE 4

TABLE 4

Training induced a reduction of the relative weight of SOL in all groups as compared with S (45%, P < 0.001, for T0 vs S) and with each of the corresponding E groups (P < 0.01) (Table 4). The results show increased TAG content (68%) and oleate incorporation (P < 0.001) in T12 as compared with T0. After 24 h, trained rat SOL showed diminished TAG content and oleate incorporation in relation to T12 (P < 0.01 for both parameters). Twelve hours after a single bout of exercise, sedentary rats showed increased TAG content in SOL (P < 0.01).

To assess the importance of plasma TAG to the reposition of IMTAG, we treated 12 animals of each group with Triton WR 1339 (an inhibitor of LPL activity) at the same studied intervals. A marked difference in IMTAG content (39.1 ± 2.2 mg fat·g−1 tissue before and 26.3 ± 1.0 mg fat·g−1 tissue after Triton treatment for E12; and 47.4 ± 4.3 mg fat·g−1 tissue before and 22.6 ± 1.1 mg fat·g−1 tissue after Triton treatment for T12) was found between the treated and nontreated groups in the soleus. In the gastrocnemius, however, Triton treatment did not induce differences (14.4 ± 0.5 mg fat·g−1 tissue before and 9.5 ± 0.3 mg fat·g−1 tissue after Triton treatment for E12; and 9.4 ± 0.3 mg fat·g−1 tissue before and 11.0 ± 0.2 mg fat·g−1 tissue, after Triton treatment for T12)of IMTAG content.

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DISCUSSION

There is controversy as to the use of IMTAG during exercise (26,29) and to whether endurance training increases its utilization (27). Biochemical determination of IMTAG is, however, considered problematic (27). Different methodologies have been used in the attempt to investigate the role of IMTAG in fueling endurance exercise (29), but its deposition is very variable, depending upon the type of fiber (8), diet (4), degree of training (18), intensity of the exercise bout, and on the interval between the exercise bout and the experimental trial (15). 1H magnetic resonance spectroscopy studies face difficulties with absolute calibration (5) and provide but a qualitative analysis of the muscle, being expensive and technically demanding (19). Muscle biopsy techniques are subject to variations regarding great fiber heterogeneity in humans (31), a problem that can be overcome by the adoption of multiple sampling sites. The methodology presently adopted could be of use in human studies, provided that the biopsies are taken from multiple sites (at least three) and further sampling after the different time intervals, made from sites adjacent to the first ones. The common Bergström technique, which allows the obtainment of 20–30 mg of tissue and takes 3–8 s to be performed, would certainly provide more than the 2 mm3 of tissue required for this histological/morphometrical analysis.

Many of the discrepancies in the assessment of IMTAG originate from the lack of discrimination between intramyocellular lipid and fat stored in the perimysium, the connective tissue that surrounds small groups of fibers. In the present study, we used a rodent model, in which the entire muscle can be studied. We have assessed both perimysial and intramyocellular TAG stores, through ultrastructural, light microscopy, and biochemical analysis immediately after, and 12 or 24 h after exertion at 65% O2max. Guo et al. (9), who used nuclear magnetic resonance proton spectroscopy and microcomputed tomography for IMTAG analysis of Sprague-Dawley rats, did not find adipocytes between muscle fiber bundles. In the present study, the existence of perimysial adipocytes was demonstrated by both light and electron microscopy. This discrepancy of results could be accounted for by the osmium impregnation of samples we adopted. This method warrants perfect lipid fixation and preservation of droplet shape and size, whereas in the study conducted by Guo et al., the samples were frozen and/or fixed by formalin and then dehydrated with ethanol, procedures known to cause total or great loss of lipid (13).

The effects of a single exercise bout and of endurance training were studied. Because different muscle fiber types present different TAG content and composition (10), we studied both the soleus (rich in slow-twitch oxidative fibers) and the gastrocnemius (with a more mixed proportion of oxidative and fast-twitch glycolytic fibers).

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Effect of exercise.

IMTAG has been reported to decrease, to increase, or to be unaffected after exercise (26). The present results clearly show that the area occupied by perimysial adipocytes in the sections was not changed by exercise alone in the muscles studied, as would be expected if any considerable amount of TAG had been removed from this compartment to provide substrate to the contracting muscles. Indeed, TAG stored within muscle adipocytes is believed to function as a long-term storage depot (31). The number of intrafibrillar lipid inclusions, nevertheless, was decreased by a single bout of exercise in both muscles (P < 0.001). The average area, and the maximum and minimum diameters of lipid droplets were also decreased in SOL and GAS, and the shape of the inclusions in both muscles was more polygonal immediately after the exercise bout, an indication of diminished storage. A reduction of the size of lipid droplets after exercise was previously reported for human muscle (20). According to Sacchetti et al. (26), in the human muscle, IMTAG is simultaneously synthesized and degraded, and muscle contraction decreases the rate of TAG re-esterification, but not of hydrolysis, resulting in a decrease of 30% of the substrate in the last 4 h of exercise, whereas, after 1-h intramuscular palmitate content was found by the authors to be already significantly lower in the exercising vastus lateralis compared with the resting leg muscle. Similarly, a significant degradation of IMTAG was reported for contracting rat SOL (6), concomitant to enhanced endogenous lipid oxidation and to overall ATP provision in the fiber.

Twenty-four hours after exercise, these parameters were similar to the preexercise condition (S). Contact area between the droplets and mitochondria did not vary. The biochemical analysis showed that GAS and SOL weight and TAG content were not changed by exercise. However, after 12 h, SOL TAG content increased (1.6-fold) in relation to the preexercise condition, accompanied by increasing (P < 0.01) oleate incorporation until 24 h after the bout. Sacchetti et al. (26) reported a fourfold increase of intracellular palmitate concentration 5 h after exercise in human vastus lateralis, as compared with the level found after 1 h. In GAS, decreased oleate incorporation (from plasma TAG), compared with S (P < 0.001), was noticed immediately after exercise, and reached control values already after 12 h. It is important to mention, nevertheless, that the decreased incorporation herein reported could be due to diminished uptake, or augmented oxidation of exogenous oleate, or both. The increase of this parameter during rest may, similarly reflect augmented uptake, decreased oxidation or both.

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Effect of endurance training.

The effect of endurance training on the relative contribution of intramuscular lipid during contraction is poorly understood. Although various studies point to augmented reliance upon IMTAG during exercise in trained humans, others found no differences (29). In the SOL, no changes in the area occupied by perimysial adipocytes were observed postexercise in trained animals, but in GAS, there was a clear decrease of this parameter as compared with both S (50.8%) and E0 (48%) in the trained group. To our knowledge, this is the first study to report a reduction of the size of perimysial adipocytes in trained rats. After 24 h, there was no change in relation to T0. Results from studies with humans (31) have demonstrated that trained subjects are less prone to storing fat between muscle fibers than sedentary counterparts. It seems possible, therefore, to affirm that these stores do not provide the substrate for exercise either in the SOL or in the GAS but are reduced in the latter as a response to endurance training.

The number of intrafibrillar lipid inclusions was decreased after exercise in both the SOL and GAS of trained rats. After 24 h, however, GAS and SOL responded differently. For both muscles, there was an increased number of lipid droplets as compared with T0, but for SOL, the final number was higher than that found in S (P < 0.01). The area and the maximum and minimum diameters of the inclusions were decreased for SOL and GAS in T0. However, after 24 h, only SOL presented values similar to the preexercise condition. Lipid droplets within the fibers were not arranged uniformly as reported for human muscle (7) but rather showed a widespread deposition, with inclusions in the intermyofibrillar space prevailing.

Muscle weight, TAG incorporation, and content and glycogen concentration in the GAS were reduced in T0 but recovered in 24 h. In SOL we found increased TAG incorporation resulting in higher TAG content already after 12 h. The increase in LPL activity induced by endurance training could account for the replenishment of the TAG accumulated in the droplets (22), occurring probably during rest, as the priority in the first hours of recovery would be further utilization of these depots as an energetic source ensuring glycogen resynthesis (17). Dyck et al. (6) showed that trained rat SOL and GAS presented increased palmitate uptake both during rest and contraction: palmitate was preferentially diverted toward esterification, with no alteration of oxidation rate, whereas during contraction, both esterification and oxidation of exogenous palmitate were found to be increased, along with decreased hydrolysis of IMTAG. An IMTAG-sparing effect was thus observed in the trained muscle. However, the results presented by these authors were obtained under unrestricted availability of exogenous fatty acid, whereas in the present condition, the limits imposed by NEFA delivery in the trained state in vivo (35) could account for the enhancement in endogenous TAG utilization during exercise.

The contact area between lipid inclusions and mitochondria was found to be increased in the SOL in T0 (P < 0.05) and returned to values equivalent to those presented by S after 24 h. Vock et al. (34) reported similar increased contact area between lipid droplets and mitochondria in the muscles of trained pigmy goats and dogs. Hoppeler et al. (14) found that muscle lipid inclusions were located adjacent to mitochondria in trained men, whereas in sedentary subjects the same was not observed.

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CONCLUSIONS

Taken together, the results indicate that the osmium impregnation microscopy technique herein adopted permits the distinction of intramyocellular and extramyocellular compartments of TAG deposition within the muscle and allows the comparison of the relative contribution of these stores as a source of substrate for exercise. The results clearly demonstrate that the two compartments have different roles and that the intrafibrillar lipid is the source of IMTAG mobilized during exercise, both in sedentary and trained rats. Furthermore, the results point out fiber-type variations: in GAS (mixed slow-twitch and fast-twitch fibers), training induced the reduction of TAG storage in perimysial adipocytes, in contrast to slow-twitch–rich SOL, in which one such effect was absent. The multi-technique approach adopted allows us to speculate that the lack of variation of TAG content in GAS from trained animals in the studied interval is related to the preservation of IMTAG stored in the extramyocellular compartment during exercise. The amount of IMTAG in this compartment is, on the other hand, reduced in this muscle (and hence, contributes to decreased relative weight as compared with S and E0), as a function of training.

The morphometric approach was similarly validated by the biochemical analysis when the intramyocellular lipid stores were studied, emphasizing the differences between the muscles studied. Whereas in both SOL and GAS a single bout of exercise caused the reduction in the number and size of lipid droplets, which returned to values close to the control (S) after 24 h, concomitant to increases of TAG content and oleate incorporation appearing already after 12 h, for the trained animals, the differences were more pronounced: the exercise bout provoked a more evident alteration of all the morphometric and biochemical parameters in SOL, compared with GAS, and the recovery of the stores was also more conspicuous in the former. As recovery of IMTAG is thought to depend on plasma TAG (19), variation of LPL activity could account for the differences observed between SOL and GAS. Smol and colleagues (28) reported marked differences of LPL activity for rat gastrocnemius and soleus. The activity of LPL was higher in SOL than in the red portion of GAS, whereas in the white portion no activity was detected. The results obtained after Triton WR 1339 treatment showed differences in IMTAG reposition patterns between the muscles 12 h after the exercise bout. SOL seemed to rely on plasma TAG for the replenishment of intramyocellular TAG, and when this source was not available (after Triton WR 1339 treatment), there was a reduction of IM TAG content. The same was not observed for GAS. The fact that in GAS LPL activity is much lower than in SOL (28) could be related to the decreased importance of plasma TAG as a source of IMTAG reposition for this muscle.

We would like to thank Prof. Paulo A. Abrahamsohn for helping with the morphological analysis, Ms. Cleusa M. R. Pellegrini for technical assistance, and Ms. Rosana Duarte Prisco for the statistical analysis.

M. A. Belmonte received a scholarship from CAPES.

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Keywords:

MUSCLE; LIPID INCLUSIONS; ADIPOCYTES; MICROSCOPY; EXERCISE; TRAINING; MORPHOLOGY

©2004The American College of Sports Medicine