Cellular adaptations to endurance training are influenced by the intensity and duration of exercise. To examine the impact of exercise intensity and duration on the acute transcriptional regulation of metabolic genes in red (RG) and white (WG) gastrocnemius muscle, rats completed either low-intensity [∼50% maximal O2 uptake (V̇o2 max)] treadmill exercise (LIE) for 45 min, LIE for 180 min, or high-intensity (∼75% V̇o2 max) exercise (HIE) for 45 min. LIE for 45 min activated (P < 0.05) transcription of the pyruvate dehydrogenase kinase-4 (PDK4), uncoupling protein-3 (UCP3), heme oxygenase-1 (HO-1), and hexokinase II (HK II) genes in RG within 1 h after exercise. In WG, transcription of PDK4, UCP3, HKII, and lipoprotein lipase (LPL) was also induced, whereas transcription of the HO-1 gene did not change. In RG, extending LIE duration from 45 to 180 min elicited a similar activation of PDK4 and UCP3 (∼15-fold) but a far greater increase in HO-1 (>30-fold) and HKII transcription (>25-fold). In WG, extending LIE for 180 min induced a much greater and prolonged (through 2- to 4-h recovery) activation of PDK4, UCP3 (both >200-fold), and HO-1 (>10-fold). HIE elicited a similar pattern of gene activation to LIE in both RG and WG, with the exception that HIE triggered >10-fold activation of HO-1 in WG. These data provide evidence that both the intensity and the duration of exercise affect the transcriptional regulation of metabolic genes in muscle in a fiber type-specific manner, possibly reflecting the relative stress imposed by the exercise bout.
- endurance training
- mitochondrial biogenesis
- gene regulation
endurance exercise training induces a number of adaptations in skeletal muscle, including increased expression of key metabolic enzymes and an overall increase in mitochondrial content (16, 19). At the biochemical level, these adaptations improve the efficiency of substrate utilization during exercise, enhance the sensitivity of the respiratory control system, and increase the overall oxidative capacity of skeletal myofibers (5, 8, 19). The magnitude of change is directly related to the intensity, duration, and frequency of exercise during the training program (7), whereas the total time required to achieve the “trained” state is a function of first-order kinetics, i.e., a function of the turnover rate constants of the specific proteins involved in the adaptive response (43). Thus, under conditions in which all three training parameters are held constant, the training program must be of sufficient length for the cellular proteins to reach their new concentration and the biochemical adaptations to fully develop.
The biochemical adaptations to endurance training however, do not represent a change in the true “steady-state” concentration of proteins, because a continued, regular interval of exercise is required for the adaptive state to be maintained. This implies that the adaptations to exercise training reflect the cellular responses to intermittent periods of contractile activity and inactivity. Indeed, a number of studies have now shown that the transcription and/or mRNA content for several metabolic and stress-related genes is acutely and transiently elevated in skeletal muscle during and/or after a single bout of exercise (10, 21, 23, 25, 27, 33, 35, 38, 44). These findings have led to the hypothesis that the cumulative effects of transient increases in gene expression during recovery from repeated bouts of exercise may represent the underlying basis for the biochemical adaptations induced by endurance training (25, 33, 49).
The purpose of the present study was to more closely examine the acute response to exercise in rats by determining the specific effects of exercise intensity and duration on the transcriptional activation of metabolic genes in both red and white skeletal muscle. On the basis, in part, of previous work (32, 33), we focused on two genes that are particularly sensitive to acute exercise, pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3). PDK4 is expressed in skeletal and cardiac muscle, where it phosphorylates and inactivates the E1α subunit of pyruvate dehydrogenase (PDH), thereby inhibiting the entry of glycolytic products into the mitochondria for oxidation (40). The biological role of UCP3 is not established, although recent evidence suggests that it may function in a feedback mechanism to limit the formation of superoxides in mitochondria under oxidative stress conditions (3, 9). We also examined the oxidative stress gene heme oxygenase-1 (HO-1) and several genes that encode for metabolic enzymes, including hexokinase II (HK II), lipoprotein lipase (LPL), carnitine palmitoyltransferase I (CPT I), and long-chain acyl-CoA dehydrogenase (LCAD). In view of the direct influence of exercise training intensity and duration on mitochondrial biogenesis (7), it was hypothesized that the acute transcriptional activation of each select metabolic gene would be directly affected by the severity of the exercise bout.
MATERIALS AND METHODS
Animals and materials.
Experimental design. To determine the acute effects of exercise intensity and duration, three studies were conducted. In the first study, rats completed 45 min of treadmill running at a relatively low intensity [20 m/min, 5° incline; ∼50% maximal O2 uptake (V̇o2 max)] (39). In the next study, the same intensity of exercise was performed, but the duration was extended to 3 h. In the final study, rats performed a bout of high-intensity treadmill running (30 m/min, 15° incline) either for 45 min or until exhaustion (inability to maintain pace and support body weight), whichever occurred first. All rats performed ≥41 min of treadmill exercise. In all three studies, rats were anesthetized (35 mg/kg ip pentobarbital sodium) either before exercise (controls), immediately after exercise (0 h recovery), or at various time points during recovery (see figures for recovery times). Food was restricted from rats through 4 h of recovery. Once anesthetized, rats were placed on a heating pad to maintain body temperature, and portions of the red and white gastrocnemius muscle were removed.
Nuclear isolation and nuclear run-on analysis. Nuclei were isolated from red and white muscle and subjected to RT-PCR-based nuclear run-on analysis, as previously described (15). To account for differences in initial nuclear content among samples before the run-on reaction, RT products were diluted with nuclease-free H2O on the basis of the relative genomic DNA content of each nuclear preparation (15). All PCR primer pairs were designed from rat-specific sequence data (Entrez; National Institutes of Health) using DNA analysis computer software (DNASTAR; Lazergene) and, with the exception of PDK4 (forward primer 5′-AGGGCCACGGTCGAGCATCAA-3′ and reverse primer 5′-GGCAAGCCGTAACCAAAACCAG-3′; 224-bp product), have been given previously (15). Annealing temperature, MgCl2 concentration, and PCR cycle number were determined for each primer pair by pretesting to ensure that conditions were optimized and within the linear range for PCR amplification. Control (preexercise) and experimental samples were run in parallel to permit direct relative comparisons. Amplification products were separated by gel (2.5% agarose) electrophoresis, stained with ethidium bromide, visualized, and quantified by ultraviolet exposure using a charge-coupled device integrating camera (Gel Doc; Bio-Rad) and analysis software (Molecular Analyst; Bio-Rad) under nonsaturating conditions (15).
Statistical analysis. Transcription for all metabolic genes was expressed relative to transcription of the β-actin gene. All data within each experimental treatment were expressed relative to data from control rats, with mean data set to 1.0. Statistical analyses were performed using either two-way or one-way ANOVA with all pairwise multiple comparisons among groups performed using the Student-Newman-Keuls method. The level of significance was set at P < 0.05.
To examine the influence of exercise intensity and duration on the acute transcriptional regulation of metabolic genes in red and white skeletal muscle, rats completed a relatively low-intensity (∼50% V̇o2 max) treadmill exercise bout for either 45 or 180 min, or a relatively high-intensity (∼75 V̇o2 max) exercise bout for 45 min (39). A representative composite image of PCR products generated from RT-PCR-based nuclear run-on analysis of nuclei isolated from red and white portions of the gastrocnemius muscle (RG and WG, respectively) of rats after 45 min of low-intensity exercise is shown in Fig. 1. Exercise induced marked transient increases in transcription of the PDK4, UCP3, HO-1, and HK II genes in both RG and WG muscle, particularly during the recovery period after exercise.
PDK4, UCP3 and HO-1 genes. Figure 2 provides a summary of the effects of each of the three exercise protocols on the transcriptional activation of the PDK4, UCP3, and HO-1 genes in both RG and WG muscle. In RG muscle, low-intensity exercise induced a nearly identical degree and pattern of activation of the PDK4 and UCP3 genes regardless of the duration of exercise (45 or 180 min). Transcription of both genes increased 5- to 15-fold (P < 0.05) immediately after exercise (0 h recovery), remained at ∼10- to 15-fold over control levels through ≥4 h of recovery, but returned to near baseline levels within 24 h after exercise. High-intensity exercise also elicited a significant (P < 0.05) increase in PDK4 and UCP3 transcription, although the magnitude of activation was slightly less (∼4- to 9-fold) and somewhat delayed (no increase evident immediately after exercise). Exercise also activated transcription of the HO-1 gene, a response that was sensitive to the duration of exercise, increasing ∼30-fold immediately after 180 min of low-intensity exercise compared with only an ∼5-fold increase after 45 min of either low- or high-intensity exercise.
In WG muscle, activation of the PDK4 and UCP3 genes was influenced by both the intensity and the duration of exercise. Low-intensity exercise performed for 45 min activated transcription of PDK4 and UCP3 40- to 80-fold, a response that persisted through ≥4 h of recovery (Fig. 2). Extending exercise duration to 180 min elicited a further activation of PDK4 and UCP3 to >200-fold over baseline. Again, activation of both genes persisted (>150-fold) for ≥4 h after exercise but returned to baseline within 24 h. Interestingly, 45 min of exercise performed at the higher intensity resulted in a somewhat delayed (no increase immediately after exercise) and less dramatic (∼15-fold) activation of PDK4 and UCP3. HO-1 transcription increased ∼15-fold in response to both 45 min of high-intensity exercise and 180 min of low-intensity exercise but was not significantly increased in response to 45 min of low-intensity exercise.
HK II, LPL, CPT I, and LCAD. Exercise performed for 45 min elicited a three- to fivefold increase in transcription of the HK II gene in both RG and WG, irrespective of the intensity of the exercise (Fig. 3). Interestingly, however, extending exercise duration to 180 min elicited a >25-fold increase in HK II transcription, a response that occurred specifically during exercise (i.e., immediately after exercise and not during recovery) and only in the RG muscle (Fig. 3). Somewhat surprisingly, exercise failed to elicit marked changes in the transcription of oxidative metabolism genes in either RG or WG muscle. In general, transcription of LPL, CPT I (Fig. 3), and LCAD (data not shown) increased approximately two- to threefold (P < 0.05) in response to low-intensity exercise. High-intensity exercise did not induce any signficant increase in transcription of these genes.
The results of the present study demonstrate that the intensity and duration of a single bout of exercise dramatically affect the transcriptional regulation of select genes associated with metabolism and oxidative stress in both red and white skeletal muscle of rats. The influence of exercise intensity/duration was particularly evident in white muscle, where transcription of the PDK4 and UCP3 genes increased ∼10-fold after 45 min of high-intensity exercise, 40- to 80-fold after 45 min of low-intensity exercise, and >200-fold when the duration of low-intensity exercise was extended to 180 min. Interestingly, transcription of the oxidative stress gene HO-1 was not activated in response to 45 min of low-intensity exercise but increased >10-fold in red muscle when the duration of exercise was increased and in white muscle when either the intensity or the duration of exercise was increased. Regardless of the exercise protocol, transcriptional activation of all responsive genes peaked within the first several hours after exercise and returned to preexercise control levels within 24 h of recovery. Although calcium transients have recently been implicated in the control of mitochondrial biogenesis associated with endurance training (50), the clear disparity in the pattern and degree of transcriptional activation among the different genes expressed in mitochondria (PDK4, UCP3, CPT I, and LCAD) and between the different types of muscle (red vs. white) suggests that the molecular response to exercise is likely mediated by multiple signaling/control mechanisms.
The most striking finding of the present study was the large induction of the PDK4, UCP3, HO-1, and HK II genes that occurred in response to exercise. Given the stark contrast in the magnitude of activation of these genes relative to the lipid metabolism genes, it seems reasonable to speculate that the strategy employed by myofibers to regulate the transcription of a specific gene reflects the need for that gene product relative to the demand imposed by the exercise bout itself. For example, HK II may represent a metabolic enzyme whose basal concentration is not sufficient to meet the contracting myofibers' need for glucose during prolonged low-intensity exercise (particularly in red muscle) and thus is subject to signaling/regulatory mechanisms that acutely activate transcription (present study) (27). On the other hand, it appears that most enzymes required for oxidative metabolism, such as CPT I and LCAD, are present at such a sufficient level under normal conditions that a large increase in expression is not required to support the immediate metabolic needs of the myofiber either during or after exercise. Although the increase in oxidative enzyme activity that is generated after several weeks of training clearly improves the efficiency and capacity of substrate utilization (17, 19), such a response may be viewed as a beneficial but not necessarily a required adaptation for acute survival of the cell. In contrast, activation of HO-1, a gene that encodes for an antioxidant enzyme that is nearly undetectable in muscle under basal conditions (10, 45), occurred in a pattern consistent with the level of oxidative stress predicted in red and white muscle for the three different exercise bouts, suggesting that HO-1 was required to meet a specific need in the myofibers. Likewise, the fact that both PDK4 and UCP3, two genes that are also expressed at low levels under basal conditions, displayed such an acute and dramatic increase in transcription implies that the products of these two genes serve an important function in skeletal muscle during and/or after exercise.
PDK4 is a member of a family of protein kinases that phosphorylate and inactivate the PDH complex, inhibiting the conversion of pyruvate to acetyl-CoA and thus preventing products of glycolysis from entering the mitochondria for oxidation (14, 40). In skeletal muscle, PDK4 is induced in response to fasting, high-fat feeding, and streptozotocin-induced diabetes (18, 30, 31, 34, 41, 51), all of which represent metabolic states in which there is either a real or at least a perceived (i.e., insulin resistance) deficit in whole body glucose availability. Prolonged endurance exercise also represents a situation in which there is a gradual transition from carbohydrate to nonesterified fatty acid (NEFA) metabolism (1). The traditional view, based on the Randle hypothesis (36), is that this shift in substrate utilization in exercising muscle is driven by the increased delivery and oxidation of NEFAs, eliciting a progressive rise in acetyl-CoA that allosterically inhibits PDH activity. The dramatic induction of PDK4 found in the present study as well as in our previous work in humans (32, 33) suggests that PDK4-mediated inhibition of PDH in muscle may represent a mechanism for conserving whole body carbohydrate content during prolonged exercise by gradually limiting the entry of glycolytic products into the mitochondria for oxidation and thereby conserving three-carbon compounds for gluconeogenesis. This function may be particularly important in muscle fibers that rely heavily on glycolytic metabolism, as evidenced by the direct influence of exercise duration on the magnitude of PDK4 induction in WG muscle. It is also tempting to speculate that a persistent elevation in PDK4 expression during recovery from exercise may ensure that glucose entering the cell is preferentially used for muscle glycogen resynthesis, reflecting the high metabolic priority given to the replenishment of energy reserves. Further studies will be required to more thoroughly examine the fiber type-specific effects of exercise on PDK4 expression, PDH phosphorylation, PDH activity, and glucose/glycogen metabolism.
Like PDK4, the remarkable sensitivity of the UCP3 gene to exercise strongly suggests that the product of this gene serves an important function in skeletal muscle during and after exercise. On the basis of its homology with the well-characterized uncoupling protein in brown adipose tissue (UCP1), UCP3 was originally thought to mediate basal proton leak in skeletal muscle mitochondria (12, 24, 46), a process that accounts for ∼20% of the basal metabolic rate in rats (37). Surprisingly though, UCP3 knockout mice (UCP3–/–) display no overt phenotype with respect to body weight gain, exercise tolerance, cold-induced thermogenesis, or whole body energy metabolism (4, 13, 47), providing evidence that UCP3 is not responsible for catalyzing the basal proton leak in skeletal muscle mitochondria. Knockout of the UCP3 gene however, does lead to increased production of reactive oxygen species (ROS) and increased oxidative damage to mitochondrial proteins in muscle (3, 47). Echtay et al. (9) have further shown that addition of superoxide to in vitro mitochondrial preparations activates proton conductance in muscle mitochondria isolated from wild-type but not UCP3–/– mice, raising the alternative hypothesis that UCP3-mediated uncoupling activity is stimulated in an effort to limit damage by endogenous superoxide. As proposed by Brand et al. (3), such a defense mechanism may represent a feedback system to limit further generation of superoxide under conditions in which mitochondrial membrane potential is high relative to the demand for ATP. Whether the induction of UCP3 in skeletal muscle is also linked to transient increases in oxidative stress, particularly in white myofibers, is not known. Interestingly however, oxygen consumption by skeletal muscle remains elevated for several hours after exercise. Thus an attractive hypothesis is that the recovery period after exercise may represent a metabolic state in which respiratory chain activity and mitochondrial membrane potential are elevated in the face of low ATP demand, creating an environment favoring the formation of superoxide, which, in turn, triggers an increase in the expression and activity of UCP3 as a protective mechanism to lower membrane potential and minimize reactive oxygen species formation.
This study firmly establishes that an acute, transient increase in transcription of select genes is a component of the adaptive response to a single bout of exercise in both red and white skeletal muscle, both confirming and extending previous work from our laboratory (25, 32, 33, 35) as well as from others (6, 21, 23, 26, 28, 38, 44). Our initial hypothesis was that the level of induction of those genes that encode for proteins involved in lipid/oxidative metabolism (e.g., LPL, CPT I, LCAD) would be directly related to the intensity and duration of the exercise bout, consistent with the well-established effects of exercise intensity and duration on the adaptive increases in mitochondrial enzyme content in muscle with endurance training (7). Surprisingly, however, acute changes in transcription of the LPL, CPT I, and LCAD genes were difficult to detect in the present study, reaching significance only in response to the low-intensity, prolonged exercise bout (Figs. 2 and 3). Although exercise has been reported to increase LPL mRNA and protein expression in skeletal muscle of humans (38), acute increases in either the transcription and/or mRNA content of mitochondrial enzymes have been difficult to detect (33, 35). Although it may be argued from these data that increases in the expression of oxidative metabolism proteins with endurance training may not involve regulation at the level of transcription, two factors are important to keep in mind in this regard. First, the turnover rate of cytochrome c protein in skeletal muscle is estimated to be on the order of 6–10 days (2, 42), suggesting that mitochondrial enzymes are fairly stable relative to other metabolic proteins such as GLUT4 or HK II (half-lives ∼12–48 h) (20, 48). Second, endurance training, even for several months, typically elicits no more than a twofold increase in muscle oxidative enzyme content (17, 43). Therefore, it is possible that even a small, transient increase in transcription of genes such as LPL, CPT I, or LCAD, when repeated daily, may be sufficient to generate a cumulative increase in mRNA and thus, presumably, protein content after several weeks to months of training (49). Of course, increases in mRNA (11) and/or protein stability (29) or changes in translational efficiency (22) may also contribute to the regulation of these genes in response to endurance exercise. Clearly, further work will be required to determine the relative contribution of transcriptional and posttranscriptional control mechanisms regulating the expression of oxidative enzymes with endurance training.
In summary, the results from the present study demonstrate the acute and transient nature of the transcriptional activation of several metabolic genes in skeletal muscle in response to exercise. Moreover, these data provide evidence that both the intensity and the duration of exercise influence the regulation of specific genes in a fiber type-specific manner, possibly reflecting the need for specific gene products relative to the metabolic and/or related stresses imposed by the exercise bout. Our findings also provide evidence that recovery from exercise is associated with the persistent activation of select metabolic genes, likely reflecting the priority given to the reestablishment of metabolic homeostasis in muscle. The acute and dramatic activation of the PDK4 and UCP3 genes, particularly in response to prolonged exercise, suggests that the products of these two genes play a critical role in muscle metabolism. Further work will be required to define the functional role of these two gene products during recovery from exercise.
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45372.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2003 by American Physiological Society