Journal of Applied Physiology
Vol. 83, No. 1, pp. 32-39, July 1997
EXERCISE AND MUSCLE

Oxidation of lactate and acetate in rat skeletal muscle: analysis by ¹³C-nuclear magnetic resonance spectroscopy

Loren A. Bertocci^1,2, John G. Jones², Craig R. Malloy^2,3,4, Ronald G. Victor³, and Gail D. Thomas³

¹ Institute for Exercise and Environmental Medicine, Presbyterian Hospital of Dallas, Dallas 75231; ² Department of Radiology, Mary Nell and Ralph B. Rogers Magnetic Resonance Center, University of Texas Southwestern Medical Center, Dallas 75235-9085; ³ Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8573; and ⁴ Department of Veterans Affairs Medical Center, Dallas, Texas 75216

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Bertocci, Loren A., John G. Jones, Craig R. Malloy, Ronald G. Victor, and Gail D. Thomas. Oxidation of lactate and acetate in rat skeletal muscle: analysis by ¹³C-nuclear magnetic resonance spectroscopy. J. Appl. Physiol. 83(1): 32-39, 1997.The balance between carbohydrate and fatty acid utilization in skeletal muscle previously has been studied in vivo by using a variety of methods such as arteriovenous concentration differences and radioactive isotope tracer techniques. However, these methodologies provide only indirect estimates of substrate oxidation. We used ¹³C-nuclear magnetic resonance (NMR) spectroscopy and non-steady-state isotopomer analysis to directly quantify the relative oxidation of two competing exogenous substrates in rat skeletal muscles. We infused [1,2-¹³C]acetate and [3-¹³C]lactate intravenously in anesthetized rats during the final 30 min of 35 (n = 10) or 95 (n = 10) min of intense, unilateral, rhythmic hindlimb contractions. ¹³C-NMR spectroscopy and isotopomer analysis were performed on extracts of gastrocnemius and soleus muscles from both the contracting and contralateral resting hindlimbs. We found that 1) [¹³C]lactate and [¹³C]acetate were taken up and oxidized by both resting and contracting skeletal muscles; and 2) high-intensity muscle contractions altered the pattern of substrate utilization such that the relative oxidation of acetate decreased while that of lactate remained unchanged or increased. Based on these findings, we propose that ¹³C-NMR spectroscopy in combination with isotopomer analysis can be used to study the general dynamics of substrate competition between carbohydrates and fats in rat skeletal muscle.

citric acid cycle; carbon-13-isotopomer analysis; muscle contraction; lactate; acetate

INTRODUCTION

A COMPLEX MIXTURE OF SUBSTRATES including fatty acids, carbohydrates, ketone bodies, and some amino acids is available for utilization by skeletal muscle in vivo. Selection of one or more of these utilizable substrates for oxidation is typically measured in humans or large experimental animals by arteriovenous concentration differences (2, 6, 7, 15, 18, 21, 27, 43, 47) or respiratory exchange ratios (5, 18, 21) across a muscle bed. However, because of the limited sensitivity and specificity of these methods, most of the specific information that is available concerning substrate oxidation and other metabolic pathways in skeletal muscle has been based on studies of the metabolism of ¹⁴C- or ³H-labeled substrates (2, 5, 11, 12, 14, 19, 23, 32, 40, 43, 47).

A central question in these studies is how the balance between carbohydrate and fatty acid oxidation in skeletal muscle is altered as metabolic demands change, for example, in the transition from rest to high-work states. In part because both glucose and long-chain fatty acids may be taken up and stored as well as oxidized by skeletal muscle, many investigators have examined the oxidation of simpler molecules such as lactate and acetate. Although many studies have provided evidence for lactate production by both resting and contracting skeletal muscle (7, 18, 21, 27), other studies have provided equally compelling evidence for lactate oxidation by skeletal muscle (6, 11, 12, 19, 32, 40, 43). The extent to which acetate is oxidized by skeletal muscle in vivo is similarly unclear. When the infusions of ¹⁴C-enriched acetate were used, for example, both uptake and output have been reported in dogs and humans (2, 42).

Although it is clear that ¹³C-nuclear magnetic resonance (NMR) spectroscopy (NMRS) alone can be used to study skeletal muscle metabolism in vivo (3, 17, 35, 36, 46), ¹³C-NMRS combined with isotopomer analysis of specific ¹³C-containing moieties has been introduced as an alternative method for the evaluation of substrate oxidation (9, 10, 26). This method, which provides a detailed analysis of the ¹³C-labeling pattern of metabolic intermediates (29, 28, 30), has some unique advantages over more conventional approaches used to study substrate utilization, including 1) the greater information content in a single experiment, which permits direct analysis of substrate oxidation even under non-steady-state metabolic or isotopic conditions (30); 2) determination of the relative oxidation of up to as many as three labeled substrates in a single study; and 3) the ability to assess regional variations in metabolism by selective tissue biopsy. Because of the low natural abundance of ¹³C (1.1%), most tissue metabolites normally involved as intermediary metabolites in pathways such as glycolysis or the citric acid cycle must become sufficiently enriched in ¹³C for detection by NMR. This has been accomplished in cardiac and hepatic tissue by supplying physiological concentrations of common substrates labeled with ¹³C, such as glucose, lactate, acetate, or fatty acids (8, 10). Although an early application of ¹³C-NMR isotopomer analysis was used successfully in quiescent rabbit skeletal muscle, a large volume of tissue was available for analysis, and the substrate [2-¹³C]acetate was infused for ~2 h (44). The utility of ¹³C-NMRS coupled with isotopomer analysis methods to study skeletal muscle metabolism in the widely used rat model remains to be demonstrated.

In this study, we simultaneously examined [¹³C]acetate and -lactate oxidation in resting and contracting rat hindlimb skeletal muscles after brief (30-min) infusion of both substrates. This study provides evidence that the glutamate pool of rat gastrocnemius and soleus muscles became sufficiently enriched for ¹³C-NMR isotopomer analysis of tissue extracts after infusion of labeled substrates into whole animals and that, compared with resting muscle, high-intensity hindlimb contractions caused a detectable alteration in the pattern of utilization of the simple fat acetate vs. the simple carbohydrate lactate.

METHODS

Surgical preparation. All experimental procedures were approved by the Institutional Animal Care and Research Advisory Committee. Female Sprague-Dawley rats weighing 230-345 g were anesthetized with ketamine (80 mg im) and -chloralose (60 mg/kg iv, followed by an additional 10 mg · kg¹ · h¹). The right common carotid artery, right internal jugular vein, and cervical trachea were cannulated. Arterial pressure was monitored with the carotid artery catheter connected to a transducer (model 1290A, Hewlett-Packard, Andover, MA). The lungs were mechanically ventilated with room air and supplemental oxygen. Arterial blood gases were measured periodically (model ABL-3, Radiometer, Copenhagen, Denmark) and kept within normal limits. Core temperature was maintained at 37°C with an external heat source. The left sciatic nerve was exposed, covered with warm mineral oil, and affixed to bipolar platinum electrodes connected to an electrical stimulator via an isolation unit (models S88 and S7, Grass Instruments, Quincy, MA). Tension generated by the left triceps surae muscles was measured by connecting the calcaneal tendon to a force-displacement transducer (model FT-10, Grass Instruments). In each experiment, the right hindlimb was left unstimulated to be used as a contralateral control.

Hindlimb muscle stimulation. At the beginning of each experiment, the motor threshold voltage was determined by using a 1-Hz stimulation of the sciatic nerve. To produce intermittent isometric contractions, the left sciatic nerve was electrically stimulated at two times the motor threshold voltage with 100-ms trains of pulses (100 Hz, 0.2-ms duration). Unilateral hindlimb contraction was performed for 35 min at a rate of 30 trains/min (n = 10 rats) or for 95 min at a rate of 60 trains/min for the first 60 min, followed by a rate of 30 trains/min for the final 35 min (n = 10 rats). We have previously demonstrated that the 35-min contraction protocol caused no detectable decrease in glycogen in gastrocnemius or soleus muscles but the 95-min contraction protocol caused an ~75% decrease in muscle glycogen in gastrocnemius, but not soleus, muscles (1). The frequency of sciatic nerve stimulation was selected to recruit all motor nerves but to stay below the threshold frequency that produces progressive failure of neuromuscular transmission. A mixture of sodium L-[3-¹³C]lactate and sodium [1,2-¹³C]acetate in 3.0 ml isotonic saline (final concentrations of 1.67 and 0.83 M, respectively) was infused intravenously during the final 30 min of muscle contraction.

Tissue preparation. Immediately on completion of the experimental protocol, the gastrocnemius and soleus muscles of the contracting hindlimb and the contralateral resting hindlimb were dissected free and separately freeze clamped (<5 s to excise and freeze each muscle in order) with aluminum tongs at liquid nitrogen temperatures and later stored at 80°C. Because of the small volume of individual muscles (gastrocnemius weight ~1.5 g each, soleus ~150 mg each), the muscles obtained from two rats were pooled before extraction. This resulted in a total of five extracts each of resting and contracting muscles for both the 35- and 95-min contraction protocols. Muscles were homogenized in frozen 4% perchloric acid, centrifuged, neutralized with potassium hydroxide, and centrifuged again, and the supernatant was lyophilized. For NMR analysis, the tissue extracts were brought to a volume of ~500 µl in ²H₂O and placed in 5-mm glass NMR tubes for analysis.

NMRS. ¹³C-NMR was performed on a 9.4 T Omega spectrometer with a standard 5-mm broadband probe operating at 400 MHz for ¹H (Bruker Instruments, Billerica, MA). ¹H-decoupled ¹³C-NMR spectra were acquired in blocks of 32-K bits, over a spectral width of ±20,000 Hz, using a pulse repetition rate of 2.5 s, a pulse width of 8 µs (a 45° flip angle), and ¹H decoupling with WALTZ 16. Between 10 and 25 k of scans were acquired. ¹H spectra of selected muscle extracts were acquired with the same probe as the ¹³C spectra. Data from one acquisition were collected into 8-k-bit data files, over a spectral width of ±6,000 Hz and a pulse width of 8 µs (a 90° flip angle). Solvent signal was suppressed with a narrow-band low-power presaturatation pulse of 2-s duration.

NMR data were processed and analyzed by using NMR1 data-analysis software (Tripos, St. Louis, MO) on a SUN Sparcstation GX10 (SUN Computers, Mountain View, CA). The free induction decays were baseline corrected and multiplied by an exponential function (0.5-Hz line broadening) before Fourier transformation. Areas of each resonance were determined by fitting to a Lorentzian curve using a Levenberg-Marquadt minimization routine with limits of ±5 line widths.

Metabolic analysis. Oxidation of any compound in the citric acid cycle first requires metabolism to acetyl-CoA. Carbons of the acetyl group enter the citric acid cycle where they enrich all of the intermediates, including -ketoglutarate, which is in rapid exchange with glutamate. Under these experimental conditions, glutamate is present in high concentration and is easily detected by ¹³C-NMR. Thus glutamate was used to assess ¹³C-labeling in the citric acid cycle.

With the substrates used in this study, the acetyl group of acetyl-CoA was labeled in three possible patterns: enriched in C2 (derived only from [3-¹³C]lactate), enriched in both C1 and C2 (derived only from [1,2-¹³C]acetate), or unenriched (derived from endogenous substrates such as glycogen, triglycerides, or blood glucose) (Fig. 1). When incorporated into the citric acid cycle, each of these acetyl-CoA labeling patterns is reflected in the enrichment of C3, C4, and C5 of glutamate, which are easily distinguished as separate resonances in the ¹³C-NMR spectrum. The underlying principle of ¹³C-isotopomer analysis is that the resonance of each glutamate carbon is modulated in a predictable way by the labeling pattern of the immediately adjacent carbon atoms (Fig. 1). Thus, when glutamate is labeled in the C4 position only, the ¹³C-NMR spectrum shows a single resonance. However, when ¹³C is present also in the adjacent C3 position, the C4 resonance is split into a doublet. Similarly, labeling in the C4 and C5 positions produces another doublet. Simultaneous labeling of C3, C4, and C5 produces a doublet of doublets, or a quartet. Therefore, a mixture of [4-¹³C], [3,4-¹³C], [4,5-¹³C], and [3,4,5-¹³C]glutamate produces a characteristic nine-peak multiplet at the C4 resonance position.

Non-steady-state analysis was used to determine the fractional contribution of acetyl-CoA derived from each labeled substrate to the total acetyl-CoA pool. These relationships are independent of the number of citric acid cycle turns, changing pool sizes, or other variables and are described by the following equations (30) where C4/C3 is the ratio of total resonance areas for C4 and C3 of glutamate; C4Q is the area of the quartet produced by enrichment in C3, C4, and C5; and C4D34 is the area of the doublet produced by enrichment in C3 and C4 (28-30). The fraction of acetyl-CoA derived from unlabeled sources was determined by subtracting the fraction of acetyl-CoA from labeled sources from 1.

Statistics. All results are expressed as means ± SE. For the physiological data, repeated-measures analyses of variance with Dunnet's post hoc tests were used to compare responses with baseline values within each group. For the ¹³C-isotopomer analyses, each data point resulted from analysis on two animals for gastrocnemius muscles. Analyses of variance with Tukey's post hoc tests were used to make pairwise comparisons of means. Differences were considered statistically significant when P < 0.05.

RESULTS

Physiological responses. Arterial pressure and force outputs are presented in Table 1. In the chloralose-anesthetized rat, unilateral hindlimb contraction elicited by electrical stimulation of the sciatic nerve had no effect on mean arterial pressure. Infusion of labeled lactate and acetate resulted in equivalent decreases in mean arterial pressure of ~20 mmHg in both groups of rats. Initial forces produced by the contracting hindlimbs were not different between groups. During the 35-min contraction protocol, there was a moderate decrease in force output of 22% during the first 5 min of hindlimb contraction. By the end of the 35 min of contraction and 30 min of substrate infusion, force had decreased by 65%. During the 95-min contraction protocol, there was a marked decrease in force output of 45% during the first 65 min of hindlimb contraction. During the last 30 min of this protocol, in which substrate infusion was superimposed on hindlimb contraction, force had decreased by 57% at the midpoint of infusion and by 68% at the end of infusion. The forces produced by the contracting hindlimbs during the 30 min of substrate infusion were not different between the two groups of rats.

Table  1.   Summary of physiological responses of rats to unilateral hindlimb contraction for 35 or 95 min plus infusion of [1,2-13C]acetate and [3-13C]lactate Time,  min: 35-min Contraction 95-min Contraction 0 5 20 35 0 5 65 80 95 MAP, mmHg 91 ± 6  89 ± 6  66 ± 4* 69 ± 5* 98 ± 6  93 ± 7  89 ± 8  70 ± 6* 68 ± 4* Force, kg 1.59 ± 0.12  1.19 ± 0.10* 0.71 ± 0.14* 0.56 ± 0.13* 1.52 ± 0.17  0.79 ± 0.06* 0.76 ± 0.06* 0.59 ± 0.08* 0.42 ± 0.08* Force, %initial 100 79 ± 6* 46 ± 8* 35 ± 7* 100 56 ± 4* 55 ± 6* 43 ± 6* 32 ± 7* Values are means ± SE. 13C substrates were infused during final 30 min of each contraction period ( ; n = 10 rats per contraction protocol). MAP, mean arterial pressure. * P < 0.05 vs. baseline values at 0 min.

Table  2.   Multiplets in the 13C-NMR spectra of C4 and C3 resonances of glutamate 35-min Contraction 95-min Contraction Contracting muscle Resting muscle Contracting muscle Resting muscle C4S 0.236 ± 0.011  0.114 ± 0.012  0.280 ± 0.059* 0.129 ± 0.010  C4D34 0.194 ± 0.012* 0.104 ± 0.013  0.185 ± 0.033  0.139 ± 0.024  C4D45 0.273 ± 0.011* 0.427 ± 0.025  0.304 ± 0.031  0.374 ± 0.025  C4Q 0.297 ± 0.026  0.356 ± 0.018  0.231 ± 0.053  0.358 ± 0.013  C3S 0.158 ± 0.003  0.127 ± 0.026  0.094 ± 0.025  0.102 ± 0.025  C3D 0.417 ± 0.010* 0.482 ± 0.012  0.412 ± 0.010* 0.469 ± 0.009  C3T 0.425 ± 0.010  0.391 ± 0.036  0.494 ± 0.034  0.430 ± 0.034  C4/C3 1.443 ± 0.049  1.849 ± 0.157  1.261 ± 0.098  1.812 ± 0.070 Values are means ± SE. NMR, nuclear magnetic resonance. Extracts of gastrocnemius muscles were obtained after infusion of [1,2-13C]acetate and [3-13C]lactate during final 30 min of each contraction period (n = 5 for each data point). S, singlet; D, doublet; Q, quartet; T, triplet. * P < 0.05 vs. contralateral resting muscle.

Table  3.   Sources of acetyl-CoA in the gastrocnemius muscles as determined by non-steady-state analysis after infusion of [3-13C]lactate and [1,2-13C]acetate during final 30 min of each contraction period 35-min Contraction 95-min Contraction Contracting muscle Resting muscle Contracting muscle Resting muscle [3-13C]Lactate 0.280 ± 0.019  0.189 ± 0.020  0.225 ± 0.033  0.254 ± 0.049  [1,2-13C]Acetate 0.427 ± 0.037* 0.650 ± 0.037  0.304 ± 0.084* 0.648 ± 0.032  Unlabeled 0.293 ± 0.028  0.162 ± 0.036  0.472 ± 0.094* 0.099 ± 0.055 Values are means ± SE; n = 5 for each data point. * P < 0.05 vs. contralateral resting muscle.

DISCUSSION

To our knowledge, this study represents the first use of ¹³C-NMRS and ¹³C-isotopomer analysis to simultaneously monitor lactate and acetate oxidation in resting and contracting rat skeletal muscles. Although this method has been applied previously to heart (9), liver (10), and brain (25), it was not known whether an adequate signal-to-noise ratio would be present in rat skeletal muscle because of a combination of small tissue mass and uncertainty about achieving sufficient enrichment of skeletal muscle citric acid cycle intermediates after a brief infusion of ¹³C-labeled substrates. It is clear from our results that this methodology can, indeed, be used successfully to study skeletal muscle metabolism in a small-animal model. We chose lactate and acetate as model substrates with which to monitor the competition between the two primary pathways for acetyl-CoA production in vivo, carbohydrate, and fatty acid oxidation. We found that exogenous [¹³C]lactate and [¹³C]acetate were oxidized by both resting and contracting rat skeletal muscles. Furthermore, high-intensity muscle contractions decreased the relative oxidation of acetate but not of lactate. On the basis of these findings, we believe that ¹³C-NMRS in combination with isotopomer analysis provides a powerful technique to study the general dynamics of substrate competition between carbohydrates and fats in skeletal muscle over a wide range of metabolic conditions.

The question remains, however, about the utility of these magnetic resonance methods vs. the more traditional methods for the study of skeletal muscle metabolism, particularly in the study of the relative flux through competing metabolic pathways into and out of the citric acid cycle. Generally, the ¹⁴C methods used to quantify substrate oxidation, although very sensitive, are restricted by factors such as the requirement for metabolic and isotopic steady state, the limitation to study only one labeled compound in a single experiment, and the constraints imposed by radiation containment that simply preclude the use of ¹⁴C in certain situations. The present study illustrates that each of these problems can be overcome by using our combination of ¹³C-NMR methods. Furthermore, ¹³C-isotopomer analysis directly detects the labeling of metabolic intermediates present in the tissue of interest, as opposed to more indirect methods such as the measurement of respiratory quotients, arteriovenous differences, or specific-activity changes in the labeled substrate. Two of the major advantages of this ¹³C-NMR method, the analysis of substrate oxidation under non-steady-state conditions and the ability to measure the metabolism of two substrates simultaneously, have recently been claimed by investigators using other methods. Jensen et al. (20) reported that ¹⁴C-enriched fatty acid turnover can be measured under non-steady-state conditions; however, this study required a complex computer-controlled infusion pump and assumptions about the volumes of fatty acid distribution. Peronnet and colleagues (34) have demonstrated that the oxidation of two ¹³C-enriched substrates ingested simultaneously may be quantified but this method required more than one experiment.

In our experiments, intravenous infusion of ¹³C-labeled substrates was performed in combination with unilateral hindlimb muscle contraction. This allowed each animal to serve as its own control, since the contracting and contralateral resting hindlimb muscles were exposed to the same pool of exogenous substrates. Shortly after the infusion of acetate and lactate began, mean arterial pressure decreased by ~20 mmHg (Table 1). We attribute this hypotensive effect mainly to the infusion of acetate, which is a well-known vasodilator (24, 48). However, we do not believe that this decrease in arterial pressure complicates the interpretation of our results, because in each animal the contracting and contralateral resting hindlimbs were exposed to the same perfusion pressures.

Under the experimental conditions present in this study, ~65% of the acetyl-CoA incorporated into the citric acid cycle in resting gastrocnemius muscles was derived from [¹³C]acetate. Although acetate metabolism has not been studied extensively, the available evidence indicates that the affinity of skeletal muscle for acetate in the blood is high and that acetate extraction by resting skeletal muscles is proportional to arterial concentration (23, 42). The clearance rate and fractional extraction of blood acetate by dog skeletal muscles (2) are ~20- and 30-fold higher, respectively, than the clearance rate and fractional extraction of free fatty acids (31, 45). Whereas these studies by other investigators have demonstrated that skeletal muscle readily extracts acetate from the peripheral circulation, our study provides direct evidence for the oxidation of acetate by resting skeletal muscle. Although acetate is a major source of energy in ruminant animals, it is of less importance as a metabolic fuel in nonruminants. Nevertheless, the ability of skeletal muscles to readily oxidize acetate will assume greater importance during conditions in which plasma acetate levels are elevated, such as during ethanol metabolism (21, 27) or hemodialysis with acetate-containing media (48).

In contrast to acetate, there is an abundance of information concerning lactate utilization by resting skeletal muscles. Although it is generally agreed that the extraction of lactate by skeletal muscle increases in proportion to blood lactate concentration (6, 13, 14, 19, 43), the fate of this extracted lactate remains controversial. In isolated rabbit skeletal muscles perfused with [¹⁴C]lactate, 30-56% of ¹⁴C was recovered in CO₂, with the remainder recovered in amino acids, glycogen, and pyruvate (32). Studies using tracer techniques in a variety of experimental models, including rats (12, 40), dogs (11, 19), and humans (6, 43), have reported that skeletal muscle extracts and oxidizes blood lactate. In contrast, a recent study in humans indicated that resting arm muscles took up, but did not metabolize, the lactate produced by exercising leg muscles (7). Whereas studies such as these and others (33) have provided important kinetic information with regard to lactate disposal, none of the techniques used specifically documents lactate oxidation. This is because once lactate is converted to pyruvate, pyruvate is generally metabolized either through a carboxylation pathway to form oxaloacetate or through a decarboxylation pathway to form acetyl-CoA. Measurement of arteriovenous lactate differences or ¹⁴CO₂ release cannot distinguish between the carboxylation and decarboxylation pathways. In contrast, the ¹³C-NMR method specifically documents lactate oxidation, since the only way the ¹³C label can be incorporated into the C4 of glutamate is by first passing through the acetyl-CoA pool. In the present study, we used this ¹³C-NMR methodology to provide direct evidence for lactate oxidation by resting skeletal muscles in vivo. In fact, oxidation of [¹³C] lactate was apparent in 10 out of 10 gastrocnemius muscle extracts, providing 12-42% of the total acetyl-CoA incorporated into the citric acid cycle.

Although both fatty acids and carbohydrates are important substrates for skeletal muscles, the relative oxidation of these fuels depends on a variety of factors, such as metabolic rate, substrate availability, and muscle fiber type. Resting skeletal muscles generally meet most of their metabolic needs by the oxidation of fatty acids. In exercising skeletal muscles, the relative contribution of fatty acids tends to decrease as exercise intensity increases. Previous studies in humans have demonstrated that acetate is extracted from the blood by exercising skeletal muscles (27, 37, 41); however, it is not known whether the utilization of acetate is different in exercising compared with resting muscles. In the present study, we found that the relative oxidation of acetate decreased by 31-53% in contracting gastrocnemius muscles compared with the contralateral resting muscles. This decrease might be explained in part by acetate's effect on the activity of pyruvate dehydrogenase (PDH), a key regulatory enzyme complex in the glycolytic pathway that catalyzes the conversion of pyruvate to acetyl-CoA. A recent study in humans demonstrated that acetate infusion decreased PDH activity in resting skeletal muscle, presumably by elevating muscle citrate, acetyl-CoA, and the ratio of acetyl-CoA to free CoA (37). However, acetate infusion did not prevent PDH activity from increasing normally in exercising muscle (37). An increase in PDH activity would result in an increased supply of acetyl-CoA derived from pyruvate and a subsequent decrease in the oxidation of acetyl-CoA derived from acetate.

It is generally agreed that glycogen is the preferred substrate of contracting skeletal muscle (38). During intense exercise, glycogen metabolism leads to the accumulation and release into the blood of lactate, which often is considered to be a metabolic waste product (16). However, studies have demonstrated that contracting muscles extract blood lactate even during conditions when the muscles themselves are producing and releasing lactate (6, 15, 19, 43). Lactate extraction by exercising muscles occurs in proportion to arterial lactate concentration (6, 43) and is directly correlated with the muscle's metabolic rate (12, 15, 19, 43). Previous studies have reported that the fate of this lactate extracted by exercising skeletal muscles is oxidation (6, 15), but a recent study has disputed this conclusion (7). With the use of ¹³C-NMR to detect lactate oxidation directly, our finding that the relative incorporation of labeled lactate into acetyl-CoA was well maintained in contracting gastrocnemius muscles supports the hypothesis that lactate is not simply a metabolic by-product of intense muscle contraction but that it serves as an oxidizable energy source in contracting as well as resting skeletal muscles.

Although our data were obtained from a heterogenous mix of muscle fiber types (39) and, therefore, do not permit us to detect the behavior of individual muscle cells, our data are consistent with the hypothesis that lactate produced by one muscle fiber (such as a contracting or a fast-twitch cell) is utilized by an adjacent muscle fiber (such as a resting or a slow-twitch cell) (4).

One of the advantages of the methodology used in this study is that ¹³C- and ¹H-NMR spectra can be obtained from the same muscle extracts. Both ¹³C-labeled and unlabeled substrates are visible in the ¹H spectra providing a means by which the ratio of ¹³C (exogenous labeled) to ¹²C (endogenous unlabeled) substrate can be estimated. To demonstrate this, we collected ¹H spectra from one set of gastrocnemius extracts. By visual inspection of these spectra it can be seen that the [¹³C]acetate resonances appear much larger than the [¹²C]acetate resonance (Fig. 4) in resting gastrocnemius muscle. This difference appeared to become even larger in the spectra from contracting muscle. Thus the significant decrease in the fractional incorporation of labeled acetate that we observed in contracting gastrocnemius muscles (Table 3) is inconsistent with the explanation of a decrease in the availability of [¹³C]acetate in the muscles. In contrast, the [¹³C]lactate resonances appeared much smaller than the [¹²C]lactate resonances in resting gastrocnemius muscle. This difference appeared to become even greater in contracted muscle, probably because of the large increase in endogenous unlabeled lactate produced by the intensely contracting muscles. Thus, despite a large increase in the availability of endogenous [¹²C]lactate in contracting gastrocnemius muscles, our representative spectra were inconsistent with a decrease in the fractional incorporation of exogenous [¹³C]lactate.

In summary, we have demonstrated that both resting and contracting rat skeletal muscles can extract sufficient quantities of exogenous lactate and acetate as to be detectable via ¹³C-NMRS. Thus the powerful technique of ¹³C-isotopomer analysis, previously applied to cardiac and hepatic tissues, can also be used to study metabolic pathways in mammalian skeletal muscle. Using this technique, we demonstrated that intense, unilateral hindlimb contractions caused a detectable alteration in the pattern of substrate utilization in the gastrocnemius and soleus muscles such that the contribution of acetyl-CoA units derived from the simple fat acetate decreased while those derived from the simple carbohydrate lactate remained unchanged or increased. Among the limitations to extending this method to studies of skeletal muscle metabolism in humans is the fundamentally poor sensitivity of ¹³C-NMR observations. Although this limitation is not necessarily a complete preclusion, it is more likely that this combination of ¹³C-NMR and isotopomer analysis will be more valuable for the analysis of metabolism in the numerous animal models of human myopathies (22), particularly those disorders that have an impact on substrate oxidation or mitochondrial function such as nutritional diseases, chronic ethanol use, and drug-induced myopathies.

ACKNOWLEDGEMENTS

We acknowledge technical support by Paul R. Anderson and David Earnest.

FOOTNOTES

Address for reprint requests: L. A. Bertocci, Institute for Exercise and Environmental Medicine, Presbyterian Hospital of Dallas, 7232 Greenville Ave., Dallas, TX 75231 (E-mail: bertocl{at}wpmail.phscare.org).

Received 16 August 1996; accepted in final form 20 February 1997.

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