Endurance-oriented exercise training can enhance insulin action through adaptations that occur in skeletal muscle (8,10). One of the adaptations that may play an important role in improving insulin action with training is an increase in the number of glucose transporters (GLUT) that promote the facilitated diffusion of sugar into the cell (9). For example, the mRNA and/or protein content of the insulin-sensitive glucose transporter (GLUT4) is elevated in striated muscle from endurance-trained versus sedentary individuals (11) and increases after endurance-oriented exercise training in previously sedentary subjects (2,4,12-14). The importance of this increase in GLUT4 is evident from transgenic animal studies where whole-body insulin action is enhanced when muscle-specific GLUT4 is overexpressed (26,27). Such findings support the notion that the increase in GLUT4 in skeletal muscle with exercise training contributes, at least in part, to enhanced whole-body insulin action.
Recently, the mRNA of two novel glucose transporters, GLUT8 and GLUT12, has been shown to be expressed in human skeletal muscle (1,5,23). GLUT8 may be relevant to insulin action, as it is insulin-responsive in the murine blastocyst (1) and rat hippocampal neurons (21). GLUT12 is localized to a perinuclear region and can undergo redistribution to the plasma membrane with insulin exposure (23). These observations have led to the suggestion that GLUT8 and/or GLUT12 could contribute to insulin-evoked glucose transport in insulin-responsive tissues such as skeletal muscle (1,15,21,23). In relation to physical activity, increases in GLUT8 and/or GLUT12 with exercise training also could possibly contribute to improving insulin action, similar to GLUT4. However, it is not evident whether exercise training results in an upregulation of GLUT8 and/or GLUT12 gene expression. Therefore, the purpose of this study was to examine the effect of exercise training on GLUT4, GLUT8, and GLUT12 mRNA in human skeletal muscle. We used a cross-sectional design comparing competitive, endurance-trained athletes with their sedentary counterparts in an effort to maximize differences in exposure of the skeletal muscle to contractile activity.
Subjects and experimental protocol.
Glucose transporter mRNA content was compared in skeletal muscle samples from endurance-trained athletes (N = 16) and sedentary subjects (N = 15). Participants were selected and categorized based on reported physical activity and participation as intercollegiate athletes in sports requiring running (track and cross-country). The rationale for selecting intercollegiate athletes was to maximize the exposure of the muscle to contractile activity. Sedentary participants reported no structured exercise for the previous 12 months. Participants were not taking any medications or ergogenic aids known to alter carbohydrate metabolism. Subjects were studied while consuming their free-living diet. Prior to testing, approval was given by the East Carolina University policy and review committee on human research, and participants provided informed consent.
Muscle biopsy and plasma.
Skeletal muscle (50-100 mg) was obtained from the vastus lateralis using the percutaneous needle biopsy technique (6) following a 12-h overnight fast. Trained participants were instructed to perform their regular exercise routine the day prior to the biopsy. A fasting venous blood sample was taken immediately prior to the biopsy. Plasma was separated and frozen at −80°C for subsequent analyses. Plasma was analyzed for glucose (YSI 2300 STAT Plus Glucose and Lactate Analyzer, YSI Inc., Yellow Springs, OH) and by microparticle enzyme immunoassay for insulin (IMx, Abbott Laboratories, Abbott Park, IL). Data were used to determine homeostasis model assessment (HOMA) values [fasting glucose (mg·dL−1) × 0.05551] × fasting insulin (mU·mL−1)/22.5] as an index of insulin action (18).
Maximal oxygen consumption was measured during a graded treadmill test. Initial speed was determined based on training status (8 and 5 mph for the trained and sedentary subjects, respectively). Treadmill grade and/or speed were increased every 2 min until voluntary exhaustion was achieved. The protocol was designed to induce fatigue within 8-12 min. Heart rate and expired gases (True Max 2400, Consentius Technologies, Sandy, UT) were monitored continuously. Criteria for a successful maximal test included achieving at least three of the following: voluntary exhaustion, a rating of perceived exertion of more than 17, a respiratory exchange ratio of > 1.10, or a failure of oxygen uptake or heart rate to increase with an increased workload. Body composition was determined from seven-site skinfolds (16).
Total RNA was extracted from the vastus lateralis samples using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Reverse transcription was performed using an oligo-dT primer and SuperscriptII (Invitrogen).
Human GLUT4, GLUT8, GLUT12, and GAPDH mRNA expression were measured with the use of gene-specific double-fluorescent-labeled probes, TaqMan universal PCR Mix, and a 7700 sequence detector (Applied Biosystems, Foster, CA). FAM was used as the 5-fluorescent reporter while TAMRA was added to the 3 end as a quencher. Primer and probe sequences are shown in Table 1. Primers and probes were designed using ABI Prism 7000 SDS Software. PCR was performed under the following conditions: 95°C × 10 min, 40 cycles (95°C × 30 s, 60°C × 1 min). Relative quantification was calculated by normalizing the data to TaqMan GAPDH Control Reagent (Human) (Applied Biosystems).
Data was analyzed using a Student's t-test. Significance was accepted at P < 0.05. Results are presented as mean ± SE.
Subject characteristics are presented in Table 2. The exercise-trained group consisted of 11 men and 5 women, whereas the sedentary group comprised 11 men and 4 women. Results were not statistically different between sexes; combined data are thus presented. The exercised-trained subjects possessed a higher aerobic capacity (V̇O2max) and had a lower percentage of body fat. Plasma insulin (~40%, P = 0.19) and HOMA (~35% P = 0.22) tended to be lower in the trained subjects, although there was no statistically significant difference between groups. Others (3,20,25) have reported a similar or a less pronounced difference in fasting plasma insulin concentration in endurance-trained athletes compared with their sedentary counterparts while insulin action as assessed by a glucose clamp or another method was markedly enhanced in the athletes. There was no statistically significant difference in fasting plasma glucose concentration between the groups.
mRNA expression of GLUT4, GLUT8, and GLUT12.
GLUT8 mRNA content did not differ between the groups (Fig. 1). GLUT12 mRNA levels were actually 40 ± 14% (P < 0.05) lower in the exercise-trained compared with the sedentary subjects (Fig. 2). GLUT4 mRNA levels were significantly (P < 0.05) higher (78 ± 27%) in the skeletal muscle of the exercise-trained compared with the sedentary subjects (Fig. 3). GLUT4 mRNA were significantly correlated with percent fat (r = −0.46, P = 0.01), V̇O2max (r = 0.57, P = 0.001), insulin (r = −0.455, P = 0.01), and HOMA (r = −0.44, P = 0.015). No statistically significant correlations were observed with GLUT8 and GLUT12 mRNA.
Relatively little is known concerning the response of GLUT8 and GLUT12 to contractile activity, particularly in human skeletal muscle. Using qualitative immunostaining, Gaster et al. (7) examined GLUT8 and GLUT12 protein expression in the vastus lateralis of obese individuals, type 2 diabetics, sedentary and endurance-trained subjects, and in muscle from several pathological states. They reported no immunoreactivity with their antibody under any of the conditions and concluded that the GLUT8 and GLUT12 protein are not expressed in adult human skeletal muscle (7). However, others have detected the mRNA for GLUT8 (5) and GLUT12 (23) and the GLUT12 protein (23) in human skeletal muscle. These findings are relevant to the current study because they indicate 1) the sparseness and conflicting nature of the information available on GLUT8 and GLUT12 in human skeletal muscle, 2) that the effect of endurance-oriented exercise training on GLUT8 and GLUT12 is not readily evident, and 3) the utility of measuring mRNA as an index of alterations in the regulation/expression for the GLUT8 and GLUT12 transporters. In an attempt to further the current level of understanding, we performed a cross-sectional study measuring the mRNA of GLUT8 and GLUT12 in skeletal muscle from endurance-trained and sedentary individuals to help discern whether these transporters are influenced by repeated days of contractile activity. We did not measure GLUT protein content but rather focused on mRNA because of the consistent finding of GLUT8 and GLUT12 mRNA in human skeletal muscle (5,23)
GLUT12 has been identified as a member of the extended glucose transporter gene family expressed in skeletal muscle (17,23). The main finding of the current study was that GLUT12 mRNA content in the skeletal muscle of exercise-trained subjects was actually depressed compared with sedentary individuals (Fig. 2). This finding suggests that GLUT12 mRNA is regulated in the opposite direction of GLUT4 mRNA (Fig. 3) in endurance-trained individuals, perhaps in response to the contractile stimulus. This is a new observation in human skeletal muscle. Unfortunately, we cannot conjecture upon a mechanism(s) explaining this observation because relatively little is known concerning stimuli that influence GLUT12 gene regulation in response to muscle contractile activity and/or exercise.
The genomic analyses of mouse GLUT12 indicate there are multiple sequences with high homology to known insulin-response elements that mediate the effects of insulin on gene transcription (28). The finding of lower GLUT12 mRNA in endurance-trained subjects (Fig. 2) and a lack of a statistically significant correlation between GLUT12 mRNA and HOMA suggest that GLUT12 does not contribute to the improvement in insulin action seen in such individuals (3,11,20,25). This interpretation is speculative because the mRNA measurements obtained in this study may not extend to the protein level. However, when considering that GLUT4 expression is increased via transcription in response to contractile activity (19), the mRNA data obtained in the present study may provide at least some insight into how GLUT12 gene regulation is affected with physical activity in human tissue. Also worth considering are the potential shortcomings inherent with the cross-sectional study design we utilized, because inherent traits predisposing an individual towards endurance training may also influence muscle gene expression. For example, successful endurance-trained athletes may have a higher percentage of type I muscle fibers that could influence glucose transporter content (7). The reduced adiposity evident in the endurance-trained individuals (Table 2) or differing dietary habits (i.e., carbohydrate intake) may have also influenced glucose transporter expression. However, in relation to the GLUT family, cross-sectional findings indicating an increase in GLUT4 with exercise training using endurance-trained athletes (11) have been verified with longitudinal designs studying sedentary subjects before and after an exercise program (2,12-14). The same may be true with GLUT12, although this can only be verified by a prospective exercise training study. Regardless of these caveats, the current data do indicate a differential regulation of GLUT4 and GLUT12 in the skeletal muscle of endurance-trained versus sedentary individuals, which may be a result of the pronounced difference in contractile activity exposure with chronic exercise training; the cellular mechanism responsible remains to be discerned.
GLUT8 has also been recently identified as a member of the extended glucose transporter family (1,5,17,22). Although the boundaries of the critical promoter elements in the GLUT8 gene are roughly identified, exercise- or hormonal-responsive regulatory elements have yet to be described (24). In this study, GLUT8 mRNA levels were similar in the endurance-trained compared with the sedentary subjects (Fig. 1), which suggests a lack of an exercise-responsive element in the human GLUT8 promoter. The finding of similar mRNA levels in our two groups also implies that GLUT8 does not play a role in skeletal muscle with respect to the enhanced insulin action seen with chronic exercise training. However, the same constraints evident with our GLUT12 findings must also be applied to this hypothesis because of the measurement of mRNA and the cross-sectional study design. Ultimately, studies including immunolocalization and protein quantification are necessary to clarify the role of GLUT8 and GLUT12 in relation to endurance training.
Our finding of an elevation in GLUT4 mRNA in the trained subjects (Fig. 3) is in agreement with other cross-sectional and longitudinal findings where exercise training increased GLUT4 protein content and/or mRNA in human skeletal muscle (2,4,10-14). We specifically measured GLUT4 mRNA as GLUT4 content increases with exercise via increased transcription (19). GLUT4 mRNA thus essentially served as a positive control in our study for an exercise training effect.
In conclusion, the mRNA of the glucose transporter isoforms GLUT4, GLUT8, and GLUT12 were compared in skeletal muscle from endurance-trained and sedentary individuals. GLUT12 mRNA was depressed in the physically active subjects, whereas there was no difference in GLUT8 mRNA. In contrast, GLUT4 mRNA content was elevated in the exercise-trained individuals. These findings suggest that skeletal muscle GLUT4, GLUT8, and GLUT12 are differentially regulated in human skeletal muscle in response to repeated days of exercise training.
This work was supported by grants from the National Institutes of Health DK47425, HL73163, HL5811 (M. J. Charron) and DK56112 (J. A. Houmard), and the American Diabetes Association to M. J. Charron.
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