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Evidence for differential regulation of lactate metabolic properties in aged and unloaded rat skeletal muscle
Experimental Gerontology 44 (2009) 280–288
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Evidence for differential regulation of lactate metabolic properties in aged and unloaded rat skeletal muscle Shinya Masuda a, Tatsuya Hayashi a,*, Tatsuro Egawa a, Sadayoshi Taguchi b a b
Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto 606-8501, Japan Health and Sport Science Laboratory, Nara Sangyo University, 3-12-1, Tatsuno-kita, Sango-cho, Ikoma-gun, Nara 636-8530, Japan
a r t i c l e
i n f o
Article history: Received 2 September 2008 Received in revised form 4 November 2008 Accepted 5 December 2008 Available online 24 December 2008 Keywords: Aging Fatigue resistance Hindlimb suspension Lactate shuttle Monocarboxylate transporter
a b s t r a c t Skeletal muscles of elderly individuals show fatigue resistance and reduced lactate accumulation compared with those of young subjects during activities that recruit a small amount of muscle mass. To explore the mechanism underlying the functional changes in aged muscle, we focused on lactate metabolic properties, including monocarboxylate transporter (MCT) 1 and MCT4, in muscles from old and young control rats and hindlimb-suspended young rats. MCT1 expression was lower in soleus (SOL) of old rats than in SOL of young control rats, but was similar in young control and hindlimb-suspended rats. MCT4 expression was lower in extensor digitorum longus (EDL) of old rats than in that of young control rats, but did not differ between young control and hindlimb-suspended rats. The ratio of lactate dehydrogenase to citrate synthase activities was higher in SOL of hindlimb-suspended and old rats than in SOL of young control rats, and was lower in EDL of old rats than in those of young control and hindlimb-suspended rats. Our data suggest that aging causes metabolic changes that can reduce lactate accumulation during exercise and increase fatigue resistance in skeletal muscle, and that these changes result from aging rather than from inactivity. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Both aging and muscle unloading induce morphological and metabolic changes in skeletal muscle and lead to attenuated measures of exercise performance, such as maximal workload and maximal oxygen uptake (Capelli et al., 2006; Convertino, 1997; Ferri et al., 2007; Fleg et al., 2005; Mattern et al., 2003). The reduced functional capacity of aged skeletal muscle may be a consequence not only of aging but also of age-related physical inactivity. However, in human studies, older individuals are more fatigue resistant than younger subjects during activities that recruit a relatively small amount of muscle mass (Bilodeau et al., 2001; Ditor and Hicks, 2000; Hunter et al., 2004, 2005; Kent-Braun et al., 2002; Lanza et al., 2004). In humans, there is a greater reduction in intracellular pH in young adults than in older individuals during prolonged muscle contraction (Kent-Braun et al., 2002; Lanza et al., 2005). Furthermore, aging is associated with reduced lactate efflux during muscle contraction (Hepple et al., 2003), and this becomes even more severely impaired between late middle age and senescence than between young adulthood and late middle age (Hepple et al., 2004). In contrast, the intracellular lactate concentration increases more during prolonged muscle contraction in unloaded rat
* Corresponding author. Tel./fax: +81 75 753 6640. E-mail address: [email protected] (T. Hayashi). 0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2008.12.003
muscle than in control muscle (Grichko et al., 2000; Witzmann et al., 1983). These inconsistencies between aged and immobilized muscles suggest that aging, but not age-related inactivity, is associated with reduced lactate accumulation and therefore with increased fatigue resistance in aged skeletal muscle. During contraction, even under the fully aerobic condition, lactate is produced continuously by the glycolytic system in muscle cells. The intracellular system, called intracellular lactate shuttle, transports the produced lactate into mitochondria to be oxidized to reproduce ATP (Brooks, 2000, 2002; Gladden, 2004). Importantly, lactate can be transported across the plasma membrane from glycolytic to oxidative fibers within a working muscle, and from fast-type to slow-type muscles as well. This fiber-to-fiber and muscle-to-muscle transport system, i.e. cell–cell lactate shuttle, facilitates lactate utilization as a respiratory fuel (Brooks, 2000, 2002; Gladden, 2004; Juel and Halestrap, 1999). Lactate flux across the plasma membrane is mediated by specific isoforms of monocarboxylate transporter (MCT), MCT1 and MCT4. MCT1, which is abundant in oxidative muscle fibers, facilitates uptake of lactate into the fibers (Baker et al., 1998; McCullagh et al., 1996, 1997), and in contrast, MCT4 is predominant in glycolytic muscle fibers and is relevant in lactate efflux from fibers (Bonen et al., 2000; Juel and Halestrap, 1999; Wilson et al., 1998). These MCTs have been implicated in enhanced lactate transport by exercise training in rat (Baker et al., 1998) and human (Bonen et al., 1998; Dubouchaud et al., 2000) skeletal muscles. In fact, MCTs pos-
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sess high Km and Vmax values for sarcolemmal lactate transport under physiological lactate concentrations (Brooks, 2000). Based on the increased fatigue resistance and reduced lactate accumulation in aged skeletal muscle, we hypothesized that aging, rather than muscle unloading, is associated with changes in the metabolic properties relevant to lactate shuttle in skeletal muscle, leading to a reduction in lactate accumulation during muscle contraction. To test this hypothesis, we quantified the expression of MCT1 and MCT4 proteins, pyruvate kinase (PK), lactate dehydrogenase (LDH), and citrate synthase (CS) activities in aged skeletal muscles, and compared them with those in young unloaded and young normal skeletal muscles. PK activity is an estimate of anaerobic capacity (Layman et al., 1981). LDH activity is also directly related to the glycolytic capacity in skeletal muscle (Powers et al., 1992). CS, the flux-generating enzyme of the tricarboxylic acid cycle, is a standard marker of oxidative capacity (Bass et al., 1969; Powers et al., 1992).
2. Materials and methods 2.1. Animal care and muscle sampling All experimental procedures were approved by the Animal Research Committee of Graduate School of Human and Environmental Studies at Kyoto University and followed the Guiding Principles for the Care and Use of Animals in the Physiological Society of Japan. Young male Wistar rats aged three months (n = 15) and old male Wistar rats aged 27 months (n = 6) were prepared as experimental animals. Until the experimental periods, they were housed in a temperature-controlled room (22 ± 1 °C), kept on a 12:12 h light–dark cycle, and fed ad libitum. At the beginning of the experimental period, the young rats were randomly divided into the control (n = 7) and suspension (n = 8) groups. In the suspension group, the rats were suspended by their tails to keep their hind legs off the floor. This hindlimb suspension procedure has often been used to induce disuse atrophy in hindlimb muscles (Desplanches et al., 1987; Fitts et al., 1986; Stump et al., 1997). All the rats in the three groups were housed individually for four weeks. After overnight fasting, the rats were anesthetized with pentobarbital sodium (50 mg/kg body weight). Under anesthesia, the unilateral soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from each rat, weighed, immediately frozen in liquid nitrogen, and stored at 80 °C. The contralateral SOL and EDL were also obtained and immediately frozen in 2-methylbutane cooled with solid CO2 (dry ice) and stored at 80 °C for histochemistry. 2.2. Sample preparation for Western blotting Proteins were isolated from muscles for Western blotting by a modified method of McCullagh et al. (1996). Briefly, the muscles were homogenized in buffer A (1:50 w/v) containing 210 mM sucrose, 2 mM EGTA, 40 mM NaCl, 30 mM HEPES, 5 mM EDTA, and 2 mM PMSF, pH 7.4, with a homogenizer (Polytron system PT2100; Kinematica, Littau-Lucerne, Switzerland). Buffer B (1.17 M KCl and 58.3 mM tetrasodium pyrophosphate) was added (37.5 ll/mg of muscle), and the homogenized muscle was mixed briefly and then set on ice for 15 min, after which 1.2 ml of the homogenate was transferred to a microtube and centrifuged at 230,000 g for 75 min at 4 °C. The supernatant fluid was discarded, and the pellet was washed twice with buffer C (200 ll of 10 mM Tris and 1 mM EDTA, pH 7.4). The pellet was resuspended in 206 ll of buffer C and 34 ll of 16% SDS, homogenized, and centrifuged at 1100g for 20 min at room temperature. The supernatant was separated and the total protein concentration of each sample was determined using the DC Protein Assay kit (Bio-Rad Laborato-
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ries, Richmond, CA, USA). Diaphragm from a young control rat was homogenized similarly, and used as a reference protein for controlling gel-to-gel variation. 2.3. Western blotting for MCT1 and MCT4 Ten micrograms of the sample proteins was added to a 12% SDS–polyacrylamide minigel and electrophoresed at 200 V for 2 h in a chamber controlled at 4 °C. The protein bands were transferred to a polyvinylidene fluoride membrane at 100 V for 1 h in a chamber controlled at 4 °C. The membrane was blocked for 1 h at room temperature in Tris-buffered saline with 1% Tween 20 (TBS-T) containing 5% nonfat dry milk. After washing, the membrane was incubated with rabbit anti-MCT1 polyclonal antibody (AB3540P; Chemicon, Temecula, CA, USA) diluted 1:1000 or rabbit antiMCT4 polyclonal antibody (AB3314P; Chemicon) diluted 1:1000 overnight at 4 °C. The membrane was washed with TBS-T and incubated with anti-rabbit IgG antibody (NA934V; GE Healthcare UK Ltd., England, UK) diluted 1:7700 for 45 min at room temperature. After washing, the signal was detected by using ECL Plus (GE Healthcare). The intensity of the signals was quantified using MultiAnalyst software (Bio-Rad). Every blot was duplicated and the mean value was adopted for each sample. Equal protein loading and transfer was confirmed by Coomassie brilliant blue (CBB) staining after detection of MCT1 or MCT4. 2.4. Measurement of metabolic enzyme activities To measure CS, LDH, and PK activities, part of the muscles was homogenized in homogenate buffer (1:20 w/v) containing 175 mM KCl, 10 mM glutathione (GSH), and 2 mM EDTA, pH 7.4. The homogenates were frozen and thawed three times to disrupt the mitochondrial membranes. CS activity in the supernatant fraction was measured by spectrophotometry as described previously (Hashimoto et al., 2004; Srere, 1972). The homogenate was added to 70 mM Tris buffer (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl-CoA, and 0.5 mM oxaloacetate, and mixed in a cuvette. The absorbance of the sample was measured for 5 min at 412 nm at 30 °C. LDH activity was measured in the pyruvate-to-lactate direction using a modified method of Pesce et al. (1964). The homogenate was added to 100 mM potassium phosphate buffer (pH 7.4), 0.7 mM sodium pyruvate, and 0.12 mM NADH, and mixed in a cuvette. The absorbance of the sample was measured for 5 min at 340 nm at 30 °C. PK activity was measured using a modified method of Suwa et al. (2005). The homogenate was added to 50 mM Tris–HCl buffer (pH 7.6), 0.1 mM KCl, 10 mM MgCl2, 0.28 mM NADH, 1.5 mM ADP, 6 U/ml LDH, and 5 mM phosphoenolpyruvate, and mixed in a cuvette. The absorbance of the sample was measured for 5 min at 340 nm at 30 °C. Because the homogenate buffer contained GSH, a tripeptide, we could not determine the protein content of the homogenate for the metabolic enzyme assay. Therefore, we homogenized part of the muscles in homogenate buffer from which GSH had been removed, and used these to determine the protein content with the DC Protein Assay kit. All enzyme activities were expressed as micromoles of substrate per minute per gram of muscle weight and as nanomoles of substrate per minute per microgram of muscle protein. 2.5. Electrophoresis to assess the composition of MHC isoforms Samples were prepared to assess the myosin heavy chain (MHC) isoforms as follows. The dissected muscle was weighed quickly and homogenized immediately in homogenization buffer (1:40 w/v) containing 5 mM Tris–HCl (pH 6.8), 10% SDS, 1 mM EDTA, 1 mM PMSF, and 1 mM DTT in a homogenizer. The sample was centri-
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fuged for 10 min at 10,000 g, the supernatant was separated, and the total protein concentration of each sample was determined using the DC Protein Assay kit. The samples were mixed with homogenization buffer and loading buffer [75 mM Tris–HCl (pH 6.8), 1 mM EDTA, 20% glycerol, 10% SDS, 5% b-mercaptoethanol, and 0.01% bromophenol blue] at 0.4 lg/ll, and boiled at 95 °C for 2 min. Six micrograms of the sample proteins were separated on an SDS–polyacrylamide (7%) gel, based on the procedure described by Talmadge and Roy (1993). Electrophoresis was carried out at 275 V for 24 h at 4 °C in a temperature-controlled chamber. The gels were stained with CBB and the intensity of the bands was quantified using MultiAnalyst software. 2.6. Histochemistry For histochemical analysis, frozen serial sections (10 lm) were cut from the midbelly of SOL and EDL muscles on a cryostat (Cryocut 1800; Leica Microsystems, Wetzlar, Germany). Immunohistochemical staining of MCT1 and MCT4 was performed essentially as described previously (Hashimoto et al., 2005). The sections were air dried, fixed in acetone at 4 °C for 5 min, and incubated in 0.01 M citrate buffer (pH 6.0) with 0.1% Tween 20 at 95 °C for 30 min. The sections were cooled to room temperature, washed in PBS, blocked with 5% goat serum in PBS for 30 min at room temperature, and incubated at 4 °C overnight with rabbit anti-MCT1 polyclonal antibody (AB3540P; Chemicon) diluted 1:100 or rabbit anti-MCT4 polyclonal antibody (AB3314P; Chemicon) diluted 1:100. The next day, the sections were incubated with secondary antibody (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) for 1 h, incubated with avidin–biotin complex reagent (VECTASTAIN Elite ABC Kit; Vector Laboratories) for 30 min, and stained with a 3,30 -diaminobenzidine substrate kit (SK-4100; Vector Laboratories) for 10 min. In control sections, the primary antibodies were omitted and replaced with PBS. To determine the muscle fiber types, the sections were stained for myosin ATPase, succinate dehydrogenase (SDH), and a-glycerophosphate dehydrogenase (a-GPD) as described previously (Gillespie et al., 1987; Nakatani et al., 1999). For myosin ATPase staining, the sections were air dried for 90 min and preincubated in either acid preincubation solution containing 50 mM sodium acetate and 30 mM sodium barbital (pH 4.4) for 5 min or alkaline preincubation solution containing 75 mM glycine, 50 mM CaCl2, and 75 mM NaCl (pH 10.6) for 15 min at room temperature. The sections were washed and incubated in a solution containing 2.8 mM ATP, 50 mM CaCl2, and 75 mM NaCl (pH 9.4) for 45 min at 37 °C. The sections were then immersed in 1% CaCl2 for 3 min, in 2% CoCl2 for 3 min, and finally in 1% (NH4)2S for 1 min. For SDH staining, the sections were air dried for 90 min and incubated in 0.1 M phosphate buffer (pH 7.6) containing 50 mM sodium succinate and 0.6 mM nitroblue tetrazolium (NBT) for 100 min at 37 °C. For a-GPD staining, the sections were air dried for 90 min and incubated in 0.2 M Tris–HCl buffer (pH 7.4) containing 9.3 mM a-glycerophosphate, 1.2 mM menadione, and 0.24 mM NBT for 150 min at 37 °C. Histochemical analysis was performed using a microscope (Optiphot-2; Nikon, Kyoto, Japan) with a charge-coupled device camera. Classification of the muscle fibers into slow oxidative (SO), fast oxidative glycolytic (FOG), and fast glycolytic (FG) fibers was based on previous studies (Hashimoto et al., 2005; Peter et al., 1972; Taguchi et al., 1985). 2.7. Statistical analysis Values are expressed as means ± SEM. One-way ANOVA was used to estimate the variance between groups and Scheffé’s F-test
was used to examine the significant difference between means. Student’s t-test was used to compare two valuables. 3. Results 3.1. Effect of hindlimb suspension and aging on muscle weight and the composition of MHC isoforms Table 1A shows the mean body weight in the control, suspension, and aging groups before and after 4 weeks of hindlimb suspension period. Body weight did not differ significantly between the control and suspension groups before the hindlimb suspension, but it was significantly lower in the suspension group after hindlimb suspension than in the other two groups. Body weight did not differ significantly between the control and the aging groups after hindlimb suspension. Table 1B shows the muscle wet weight in the control, suspension, and aging groups. SOL wet weight was significantly lower in the suspension group than in the control group. EDL wet weight was significantly lower in the aging group than in the control and suspension groups. Table 1C shows the muscle wet weights relative to body weights. The relative weight of SOL was significantly lower in the suspension and aging groups than in the control group, whereas the relative weight of EDL was significantly lower in the aging group than in the control and suspension groups.
Table 1 Body weight and muscle wet weight. Control
Suspension
Aging
(A) Body weight Before suspension (g) After suspension (g)
509 ± 16 537 ± 18
516 ± 15 445 ± 12**,ààà
– 568 ± 20
(B) Muscle wet weight SOL (mg) EDL (mg)
217 ± 21 252 ± 11
115 ± 11*** 234 ± 8
164 ± 13 120 ± 13***,
0.26 ± 0.02*** 0.52 ± 0.01
0.29 ± 0.02** 0.21 ± 0.03***,
(C) Muscle wet weight per body weight SOL (mg/g) 0.40 ± 0.03 EDL (mg/g) 0.47 ± 0.02
(A) Rat body weight before and after hindlimb suspension. (B) SOL and EDL wet weight in each rat group. (C) SOL and EDL wet weight per body weight in each rat group. Values are means ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). ** P < 0.01, significantly different from control group. *** P < 0.001, significantly different from control group. P < 0.001, significantly different from suspension group. ààà P < 0.001, significantly different from before suspension.
Table 2 Composition of MHC isoforms in SOL and EDL. Control
Suspension
Aging
(A) SOL Type I (%) Type IIa (%) Type IIx (%) Type IIb (%)
99.2 ± 0.5 0.7 ± 0.4 0.1 ± 0.1 ND
96.9 ± 1.3 0.6 ± 0.4 2.5 ± 1.0 ND
93.3 ± 3.0 4.9 ± 2.2 1.8 ± 0.8 ND
(B) EDL Type I (%) Type IIa (%) Type IIx (%) Type IIb (%)
3.0 ± 0.9 14.0 ± 2.8 27.4 ± 4.3 55.6 ± 7.8
2.6 ± 0.7 13.0 ± 2.2 25.4 ± 4.3 59.0 ± 7.0
ND 18.2 ± 3.2 55.9 ± 3.4***, 25.9 ± 3.4*,
Composition of MHC isoforms in (a) SOL and (b) EDL. Values are mean ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). ND, not detected. * P < 0.05, significantly different from control group. *** P < 0.001, significantly different from control group. P < 0.05, significantly different from suspension group. P < 0.001, significantly different from suspension group.
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type IIa
A
type IIx type IIb type I control
suspension
old
diaphragm
B
type IIa type IIx type IIb control
suspension
old
diaphragm
type I
Fig. 1. Representative myosin heavy chain (MHC) bands on electrophoresis gels in (A) SOL and (B) EDL. Diaphragm is used as the control, with which to identify type I, IIa, IIx, and IIb MHC isoforms.
Table 2 shows the composition of the MHC isoforms in SOL and EDL. SOL showed a tendency for slow-to-fast MHC transition in the suspension and aging groups, although this was not significant. In contrast, EDL showed a significant shift from type IIb to type IIx MHC isoforms in the aging group. Representative MHC bands are shown in Fig. 1.
from the aging group was three times higher than the MCT1 expression in the control (P = 0.056) and suspension (P = 0.049) groups (Fig. 2B). MCT4 expression was not significantly different in SOL of the three groups (Fig. 2C), but was significantly lower in EDL from the aging group than in EDL from the control group (Fig. 2D).
3.2. Effect of hindlimb suspension and aging on MCT1 and MCT4 expression
3.3. Effect of hindlimb suspension and aging on metabolic enzyme activities
In SOL, MCT1 expression was significantly lower in the aging group but did not differ between the suspension and control groups (Fig. 2A). In contrast, the mean MCT1 expression in EDL
The effects of hindlimb suspension and aging on the protein content and metabolic enzyme activities in skeletal muscles are shown in Table 3. The protein content was significantly lower in
100 80 60 40 20 0
MCT4 expression (% of control)
500 400 300 200 100 0
D
C 120 100 80 60 40 20 0
MCT4 expression (% of control)
MCT1 expression (% of control)
120
MCT1 expression (% of control)
B
A
120 100 80 60 40 20 0
Fig. 2. MCT1 expression in (A) SOL and (B) EDL and MCT4 expression in (C) SOL and (D) EDL. Top: Representative immunoblots of MCTs. Bottom: Expression levels of MCTs. White, gray, and black bars indicate the values in the control, suspension, and aging groups, respectively. Values are expressed as a percentage of the value for the muscle in the control group. Values are means ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). Significantly different from control group, *P < 0.05. Significantly different from suspension group, P < 0.05.
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Table 3 Metabolic enzyme activities in SOL and EDL. SOL Control (A) PK (lmol/g muscle/min) LDH (lmol/g muscle/min) CS (lmol/g muscle/min) PK/CS LDH/CS (B) Protein content (lg/ll) PK (nmol/mg protein/min) LDH (nmol/mg protein/min) CS (nmol/mg protein/min)
45 ± 1 45 ± 1 21 ± 1 2.1 ± 0.1 2.1 ± 0.1 3.9 ± 0.1 581 ± 23 581 ± 23 277 ± 12
EDL Suspension 54 ± 5 49 ± 3 18 ± 1 2.9 ± 0.1** 2.7 ± 0.2* 4.1 ± 0.2 665 ± 57 595 ± 35 228 ± 19
Aging 33 ± 2 42 ± 1 15 ± 1** 2.2 ± 0.1 2.8 ± 0.1** 3.5 ± 0.1 472 ± 15 594 ± 19 211 ± 6*
Control
Suspension
Aging
300 ± 10 143 ± 3 23 ± 2 13.7 ± 1.6 6.5 ± 0.6
281 ± 6 137 ± 4 22 ± 1 12.9 ± 0.6 6.3 ± 0.4
132 ± 26***, 93 ± 15**, 28 ± 2 4.7 ± 0.9***, 3.3 ± 0.5**,
4.0 ± 0.2 3834 ± 221 1822 ± 71 291 ± 20
3.7 ± 0.1 3794 ± 98 1845 ± 36 297 ± 14
3.2 ± 0.1**, 2019 ± 394***, 1423 ± 216 430 ± 26***,
(A) Pyruvate kinase (PK), lactate dehydrogenase (LDH), and citrate synthase (CS) activities and glycolytic vs. oxidative activity ratios in SOL and EDL, corrected for muscle weight. (B) Muscle protein contents and metabolic enzyme activities corrected for protein content. Values are mean ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). * P < 0.05, significantly different from control group. ** P < 0.01, significantly different from control group. *** P < 0.001, significantly different from control group. P < 0.05, significantly different from suspension group. P < 0.01, significantly different from suspension group. P < 0.001, significantly different from suspension group.
SOL from the aging group than in SOL from the suspension group, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. When corrected for muscle weight (Table 3A), PK activity was significantly lower in SOL from the aging group than in SOL from the suspension group, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. LDH activity in SOL did not differ between the three groups, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. CS activity was significantly lower in SOL from the aging group than in SOL from the control group, but it was significantly higher in EDL from the aging group than in EDL from the suspension group. PK-to-CS ratio was significantly higher in SOL from the suspension group than in that from the control and aging groups, and it was significantly lower in EDL from the aging group than in that from the control and suspension groups. LDH-to-CS ratio was significantly higher in SOL from the suspension and aging groups than in SOL from the control group, and it was significantly lower in EDL from the aging group than in EDL from the control and suspension groups. When corrected for protein content (Table 3B), PK activity was significantly lower in SOL from the aging group than in SOL from the suspension group, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. LDH activity did not differ between the three groups in SOL or EDL. CS activity was significantly lower in SOL from the aging group than in SOL from the control group, but it was significantly higher in EDL from the aging group than in EDL from the other two groups. 3.4. Histochemical analysis of SOL and EDL We observed marked fiber atrophy in SOL from the suspension group compared with the control group (Fig. 3H–N). Small and angulated fibers, group atrophy, and fiber-type grouping, characteristics of aged muscles, were observed in SOL from the aging group (Fig. 3O–U). Large fiber groups maintained their size, and these were predominantly SO fibers. Small fiber groups, which contained fast-twitch fibers, showed marked atrophy. MCT1 was strongly expressed at the sarcolemma of all fibers in SOL from the control and suspension groups (Fig. 3E and L), but the expression disappeared irregularly in small fibers in SOL from the aging group (Fig. 3S). MCT4 was strongly expressed at the sarcolemma of FOG fibers in SOL from the control and suspension groups
(Fig. 3F and M). In SOL from the aging group, MCT4 expression was not fiber-type specific and was not evident in severely atrophied fibers (Fig. 3T). In EDL, the proportion of FG fibers was markedly lower in the aging group (Fig. 4O–U); this pattern corresponded to the decreased percentage of type IIb MHC isoform shown in Table 2B. EDL in the aging group showed small angulated fibers, group atrophy, and fiber-type grouping. In EDL from the control and suspension groups, MCT1 was strongly expressed at the sarcolemma of SO and FOG fibers (Fig. 4E and L). MCT4 was not expressed at the sarcolemma of SO fibers (Fig. 4F and M). However, the fiber-type-specific expression of MCT1 and MCT4 was not evident in EDL from the aging group (Fig. 4S and T). 4. Discussion In this study, we examined for the first time the effect of aging and unloading on the metabolic properties relevant to the lactate shuttle, and found reduced expression of MCT1 in SOL and MCT4 in EDL with aging, which were not observed in unloaded rats. Along with changes in muscle mass and metabolic enzyme activities, these results suggest that lactate metabolic properties are regulated differently between aging and muscle unloading. The decreased expression of MCT1 in SOL and MCT4 in EDL from the aging group suggests that aging, not disuse atrophy, decreases the expression of MCTs. Wilson et al. (1998) showed that denervation of rat hindlimb muscles decreases MCT1 expression in SOL and MCT4 expression in fast-type muscles, such as white gastrocnemius and white tibialis anterior muscles. In fact, aging induces a reduction in the number of functioning motor units in skeletal muscles (Caccia et al., 1979). Furthermore, angulated and atrophied fibers have been observed in aged skeletal muscle because of loss of motor neurons (Caccia et al., 1979; Einsiedel and Luff, 1992; Larsson, 1995; Snow et al., 2005). Moreover, fiber-type grouping in aged muscles occurs as a result of partial reinnervation by surviving motor neurons (Caccia et al., 1979; Snow et al., 2005). In this study, we found that group atrophy of muscle fibers and grouping of FOG fibers in SOL occurred in the aging group but not in the suspension group (Fig. 3O–U). Similarly, in EDL from the aging group, we found group atrophy of muscle fibers and fiber-type grouping in moderately atrophied fibers (Fig. 4O–U). These observations indicate that loss of motor neurons and partial
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Fig. 3. Serial cross-sections of SOL from (A–G) control, (H–N) suspension, and (O–U) aging groups. The cross-sections are stained for (A, H, and O) myosin ATPase (acid preincubation), (B, I, and P) myosin ATPase (alkaline preincubation), (C, J, and Q) SDH, (D, K, and R) a-GPD, (E, L, and S) MCT1, (F, M, and T) MCT4, and (G, N, and U) no primary antibody (negative control for MCT1 and MCT4). Muscle fibers are classified as SO and FOG fibers as indicated in A, H, and O. MCT1 is strongly expressed in both SO and FOG fibers but MCT4 is strongly expressed only in FOG fibers. Scale bars indicate 100 lm.
reinnervation by surviving motor neurons occurred in the aged muscles. Because our histochemical results show reduced MCT1 and MCT4 expression, particularly in severely atrophied fibers (Figs. 3S and 4T), it is likely that age-induced loss of motor neurons is at least partially involved in the decreased expression of MCTs from the aging group. Given the role of MCTs in lactate transport, the decreased expression of MCT1 in SOL and MCT4 in EDL from the aging group indicates that aging diminishes the potential for lactate uptake into SOL and lactate efflux from EDL. The marked increase of MCT1 expression in EDL from the aging group may result from the age-induced fiber-type shift. FG fibers,
in which MCT1 expression is weak, shift to FOG fibers, in which MCT1 is strongly expressed, with aging, as shown in Fig. 4. However, because the MHC isoform pattern was not different between the control and suspension groups (Table 2B), the age-induced fiber-type shift may be because of aging rather than inactivity. Because MCT1 was expressed in SO and FOG fibers (Fig. 4S), the increase of MCT1 in EDL from the aging group could facilitate lactate uptake into SO and FOG fibers in EDL. Muscles from the aging and suspension groups demonstrated typical changes by aging and suspension, respectively, in muscle mass and morphological characteristics. Although muscle atrophy
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Fig. 4. Serial cross-sections of EDL from (A–G) control, (H–N) suspension, and (O–U) aging groups. The cross-sections are stained for (A, H, and O) myosin ATPase (acid preincubation), (B, I, and P) myosin ATPase (alkaline preincubation), (C, J, and Q) SDH, (D, K, and R) a-GPD, (E, L, and S) MCT1, (F, M, and T) MCT4, and (G, N, and U) no primary antibody (negative control for MCT1 and MCT4). Muscle fibers are classified as SO, FOG, and FG fibers as indicated in A, H, and O. MCT1 is strongly expressed in SO and FOG fibers. MCT4 is expressed strongly in FOG fibers, intermediately in FG fibers, and weakly in SO fibers. Scale bars indicate 100 lm.
was observed in SOL and EDL from the aging group, hindlimb suspension induced marked muscle atrophy in SOL; however, this was not evident in EDL (Table 1C), which corresponds with previous studies (Desplanches et al., 1987; Fitts et al., 1986; Stump et al., 1997). Histochemical data showed that aging and hindlimb suspension affected SOL quite differently; for example, disproportional fiber atrophy, which is characteristic in aged SOL, was not observed in the suspended SOL (Fig. 3). In aged rats, a shift from type IIb to type IIx MHC isoforms (Škorjanc et al., 1998) and fast-to-slow fiber-type shifts (Caccia et al., 1979) have been observed in EDL, and slow-to-fast fiber-type
shifts have been observed in SOL (Caccia et al., 1979; Snow et al., 2005). Correspondingly, we found a shift from type IIb to type IIx MHC isoforms in EDL from the aging group (Table 2B), which was not observed in EDL from the suspension group, and a tendency to shift from type I to type II MHC isoforms in SOL from the aging group (Table 2A). In human studies, a marked decline in the fiber area of type II fibers compared with that of type I fibers has also been reported in the vastus lateralis muscle (Lexell, 1995; Vandervoort, 2002) and the population of slow-type fibers decreases with aging in the human masseter muscle, which predominantly contains in slow-type fibers (Monemi et al., 1999).
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We observed no statistically significant slow-to-fast MHC shift in SOL from the suspension group, which seems inconsistent with the results of previous studies, which showed a significant slow-tofast MHC shift after hindlimb unloading (Desplanches et al., 1987; Dubouchaud et al., 1996; Saitoh et al., 1999). It is possible that the extent of the MHC shift depends on age. Saitoh et al. (1999) reported that young rats showed a more marked MHC shift in SOL after hindlimb suspension for four weeks than did adult rats. Dubouchaud et al. (1996) showed a marked slow-to-fast MHC shift in SOL from male Wistar rats, the same strain of rats we used in our experiments, after hindlimb suspension for four weeks. However, the rats used by Dubouchaud et al. were less mature than ours (377 ± 25 g vs. 537 ± 18 g in body weight, respectively). The rats we used were so mature that no statistically significant MHC shift might be observed. The decrease of PK-to-CS ratio and LDH-to-CS ratio in EDL with aging (Table 3A) suggests that there is a relative predominance of lactate oxidation compared with pyruvate and lactate production in aged muscle, resulting in reduced lactate accumulation in EDL during contraction. Our histochemical results also suggest that EDL depends more on oxidative metabolism in the aging group than in the control group. In the control group, SDH staining was weak in FG fibers; however, it was relatively strong in each fiber from the aging group (Fig. 4C and Q). In contrast, in the control group, a-GPD staining was strong in FOG and FG fibers; however, it was weak in the fibers from the aging group (Fig. 4D and R). These changes would be at least partially related to the shift from FG to FOG fibers with aging, and the shift from type IIb to IIx MHC isoforms with aging corresponds to this fiber-type shift. Increased LDH-to-CS ratio in SOL with aging (Table 3A) suggests that slow-type muscles depend more on glycolytic metabolism in aged subjects compared with young subjects. The ratio also increased with hindlimb suspension. However, the PK-to-CS ratio did not change in the aging group, whereas it increased in the suspension group (Table 3A). These findings indicate that the potential for pyruvate production increases with hindlimb suspension, but not with aging, and therefore the potential for lactate production would be lower in SOL from the aging group compared with the suspension group. It should be noted that histochemical data for SOL from the aging group revealed that SDH staining was weak, particularly in SO fibers for which CSA was maintained (Fig. 3Q), whereas the MHC isoform pattern was not significantly changed with aging (Table 2A). Thus, it is likely that the change in metabolic enzyme activities was not because of fiber-type shift but because of decreased oxidative capacities in individual SO fibers for which CSA was maintained. These changes in metabolic properties with aging may be involved in the reduced lactate accumulation and increased fatigue resistance in aged skeletal muscles observed in previous studies (Bilodeau et al., 2001; Ditor and Hicks, 2000; Hepple et al., 2003, 2004; Hunter et al., 2004, 2005; Kent-Braun et al., 2002; Lanza et al., 2004, 2005). The putative effect of aging on lactate metabolism could be described as follows. In fast-type muscles from the aging group, the reduced PK-to-CS and LDH-to-CS ratios increase the capacity for lactate oxidation compared with the capacity for lactate production, reducing the amount of lactate that is transported from the fast-type muscles during contraction. The reduced expression of MCT4 in the fast-type muscles parallels the reduction in lactate during contraction, and thus it does not attenuate lactate efflux from the glycolytic fibers in the fast-type muscles. The increased expression of MCT1 facilitates lactate transport from the glycolytic to the oxidative fibers, to be oxidized within the fasttype muscles. These changes also contribute to a reduction in the amount of lactate that is transported from fast- to slow-type muscles. In slow-type muscles, the increased LDH-to-CS ratio suggests that there is a relative predominance of lactate production from
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pyruvate relative to lactate oxidation. However, the reduction in MCT1 expression may limit lactate uptake and delay lactate accumulation. In contrast, the changes in metabolic properties with muscle unloading may be involved in facilitating lactate accumulation in slow- and fast-type skeletal muscles during contraction, as shown in previous studies (Grichko et al., 2000; Witzmann et al., 1983). The putative effect of muscle unloading on lactate metabolism could be described as follows. In slow-type muscles, the increases in PK-to-CS ratio and LDH-to-CS ratio enhance the capacity for lactate production compared with lactate oxidation. Unchanged MCT1 expression indicates that the lactate uptake rate during contraction is maintained even after unloading. These changes in metabolic properties in slow-type muscles facilitate lactate production and lactate uptake compared with lactate oxidation, leading to earlier elevation in lactate concentration during contraction. In addition, it is notable that hindlimb unloading selectively atrophies slow-type muscles and that unloading does not change metabolic properties in fast-type muscles. Selective atrophy of slow-type muscles may decrease the amount of lactate efflux from fast- to slow-type muscles during contraction and prevents the decrease of lactate concentration in fast-type muscles. In conclusion, aging and muscle unloading regulate the metabolic properties relevant to lactate production, transport, and oxidation in slow- and fast-type muscles in different ways. Our data suggest that aging, not inactivity, contributes to increased fatigue resistance in aged skeletal muscles by reducing lactate production and transport, rather than lactate oxidation in skeletal muscles. Acknowledgements We are grateful to Takeshi Hashimoto for valuable suggestion. This work was supported by a research grant from the Japan Society for the Promotion of Science (#17500424 to Tatsuya Hayashi). References Baker, S.K., McCullagh, K.J.A., Bonen, A., 1998. Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle. J. Appl. Physiol. 84, 987–994. Bass, A., Brdiczka, D., Eyer, P., Hofer, S., Pette, D., 1969. Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10, 198–206. Bilodeau, M., Henderson, T.K., Nolta, B.E., Pursley, P.J., Sandfort, G.L., 2001. Effect of aging on fatigue characteristics of elbow flexor muscles during sustained submaximal contraction. J. Appl. Physiol. 91, 2654–2664. Bonen, A., McCullagh, K.J.A., Putman, C.T., Hultman, E., Jones, N.L., Heigenhauser, G.J., 1998. Short-term training increases human muscle MCT1 and femoral venous lactate in relation to muscle lactate. Am. J. Physiol. 274, E102–E107. Bonen, A., Miskovic, D., Tonouchi, M., Lemieux, K., Wilson, M.C., Marette, A., Halestrap, A.P., 2000. Abundance and subcellular distribution of MCT1 and MCT4 in heart and fast-twitch skeletal muscles. Am. J. Physiol. Endocrinol. Metab. 278, E1067–E1077. Brooks, G.A., 2000. Intra- and extra-cellular lactate shuttles. Med. Sci. Sports Exerc. 32, 790–799. Brooks, G.A., 2002. Lactate shuttles in nature. Biochem. Soc. Trans. 30, 258–264. Caccia, M.R., Harris, J.B., Johnson, M.A., 1979. Morphology and physiology of skeletal muscle in aging rodents. Muscle Nerve 2, 202–212. Capelli, C., Antonutto, G., Kenfack, M.A., Cautero, M., Lador, F., Moia, C., Tam, E., _ 2max decay during Ferretti, G., 2006. Factors determining the time course of VO _ 2max limitation. Eur. J. Appl. Physiol. 98, 152–160. bedrest: implications for VO Convertino, V.A., 1997. Cardiovascular consequences of bed rest: effect on maximal oxygen uptake. Med. Sci. Sports Exerc. 29, 191–196. Desplanches, D., Mayet, M.H., Sempore, B., Flandrois, R., 1987. Structural and functional responses to prolonged hindlimb suspension in rat muscle. J. Appl. Physiol. 63, 558–563. Ditor, D.S., Hicks, A.L., 2000. The effect of age and gender on the relative fatigability of the human adductor pollicis muscle. Can. J. Physiol. Pharmacol. 78, 781–790. Dubouchaud, H., Granier, P., Mercier, J., Le Peuch, C., Préfaut, C., 1996. Lactate uptake by skeletal muscle sarcolemmal vesicles decreases after 4 wk of hindlimb unweighting in rats. J. Appl. Physiol. 80, 416–421. Dubouchaud, H., Butterfield, G.E., Wolfel, E.E., Bergman, B.C., Brooks, G.A., 2000. Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 278, E571–E579.
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Evidence for differential regulation of lactate metabolic properties in aged and unloaded rat skeletal muscle Shinya Masuda a, Tatsuya Hayashi a,*, Tatsuro Egawa a, Sadayoshi Taguchi b a b
Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto 606-8501, Japan Health and Sport Science Laboratory, Nara Sangyo University, 3-12-1, Tatsuno-kita, Sango-cho, Ikoma-gun, Nara 636-8530, Japan
a r t i c l e
i n f o
Article history: Received 2 September 2008 Received in revised form 4 November 2008 Accepted 5 December 2008 Available online 24 December 2008 Keywords: Aging Fatigue resistance Hindlimb suspension Lactate shuttle Monocarboxylate transporter
a b s t r a c t Skeletal muscles of elderly individuals show fatigue resistance and reduced lactate accumulation compared with those of young subjects during activities that recruit a small amount of muscle mass. To explore the mechanism underlying the functional changes in aged muscle, we focused on lactate metabolic properties, including monocarboxylate transporter (MCT) 1 and MCT4, in muscles from old and young control rats and hindlimb-suspended young rats. MCT1 expression was lower in soleus (SOL) of old rats than in SOL of young control rats, but was similar in young control and hindlimb-suspended rats. MCT4 expression was lower in extensor digitorum longus (EDL) of old rats than in that of young control rats, but did not differ between young control and hindlimb-suspended rats. The ratio of lactate dehydrogenase to citrate synthase activities was higher in SOL of hindlimb-suspended and old rats than in SOL of young control rats, and was lower in EDL of old rats than in those of young control and hindlimb-suspended rats. Our data suggest that aging causes metabolic changes that can reduce lactate accumulation during exercise and increase fatigue resistance in skeletal muscle, and that these changes result from aging rather than from inactivity. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Both aging and muscle unloading induce morphological and metabolic changes in skeletal muscle and lead to attenuated measures of exercise performance, such as maximal workload and maximal oxygen uptake (Capelli et al., 2006; Convertino, 1997; Ferri et al., 2007; Fleg et al., 2005; Mattern et al., 2003). The reduced functional capacity of aged skeletal muscle may be a consequence not only of aging but also of age-related physical inactivity. However, in human studies, older individuals are more fatigue resistant than younger subjects during activities that recruit a relatively small amount of muscle mass (Bilodeau et al., 2001; Ditor and Hicks, 2000; Hunter et al., 2004, 2005; Kent-Braun et al., 2002; Lanza et al., 2004). In humans, there is a greater reduction in intracellular pH in young adults than in older individuals during prolonged muscle contraction (Kent-Braun et al., 2002; Lanza et al., 2005). Furthermore, aging is associated with reduced lactate efflux during muscle contraction (Hepple et al., 2003), and this becomes even more severely impaired between late middle age and senescence than between young adulthood and late middle age (Hepple et al., 2004). In contrast, the intracellular lactate concentration increases more during prolonged muscle contraction in unloaded rat
* Corresponding author. Tel./fax: +81 75 753 6640. E-mail address: [email protected] (T. Hayashi). 0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2008.12.003
muscle than in control muscle (Grichko et al., 2000; Witzmann et al., 1983). These inconsistencies between aged and immobilized muscles suggest that aging, but not age-related inactivity, is associated with reduced lactate accumulation and therefore with increased fatigue resistance in aged skeletal muscle. During contraction, even under the fully aerobic condition, lactate is produced continuously by the glycolytic system in muscle cells. The intracellular system, called intracellular lactate shuttle, transports the produced lactate into mitochondria to be oxidized to reproduce ATP (Brooks, 2000, 2002; Gladden, 2004). Importantly, lactate can be transported across the plasma membrane from glycolytic to oxidative fibers within a working muscle, and from fast-type to slow-type muscles as well. This fiber-to-fiber and muscle-to-muscle transport system, i.e. cell–cell lactate shuttle, facilitates lactate utilization as a respiratory fuel (Brooks, 2000, 2002; Gladden, 2004; Juel and Halestrap, 1999). Lactate flux across the plasma membrane is mediated by specific isoforms of monocarboxylate transporter (MCT), MCT1 and MCT4. MCT1, which is abundant in oxidative muscle fibers, facilitates uptake of lactate into the fibers (Baker et al., 1998; McCullagh et al., 1996, 1997), and in contrast, MCT4 is predominant in glycolytic muscle fibers and is relevant in lactate efflux from fibers (Bonen et al., 2000; Juel and Halestrap, 1999; Wilson et al., 1998). These MCTs have been implicated in enhanced lactate transport by exercise training in rat (Baker et al., 1998) and human (Bonen et al., 1998; Dubouchaud et al., 2000) skeletal muscles. In fact, MCTs pos-
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sess high Km and Vmax values for sarcolemmal lactate transport under physiological lactate concentrations (Brooks, 2000). Based on the increased fatigue resistance and reduced lactate accumulation in aged skeletal muscle, we hypothesized that aging, rather than muscle unloading, is associated with changes in the metabolic properties relevant to lactate shuttle in skeletal muscle, leading to a reduction in lactate accumulation during muscle contraction. To test this hypothesis, we quantified the expression of MCT1 and MCT4 proteins, pyruvate kinase (PK), lactate dehydrogenase (LDH), and citrate synthase (CS) activities in aged skeletal muscles, and compared them with those in young unloaded and young normal skeletal muscles. PK activity is an estimate of anaerobic capacity (Layman et al., 1981). LDH activity is also directly related to the glycolytic capacity in skeletal muscle (Powers et al., 1992). CS, the flux-generating enzyme of the tricarboxylic acid cycle, is a standard marker of oxidative capacity (Bass et al., 1969; Powers et al., 1992).
2. Materials and methods 2.1. Animal care and muscle sampling All experimental procedures were approved by the Animal Research Committee of Graduate School of Human and Environmental Studies at Kyoto University and followed the Guiding Principles for the Care and Use of Animals in the Physiological Society of Japan. Young male Wistar rats aged three months (n = 15) and old male Wistar rats aged 27 months (n = 6) were prepared as experimental animals. Until the experimental periods, they were housed in a temperature-controlled room (22 ± 1 °C), kept on a 12:12 h light–dark cycle, and fed ad libitum. At the beginning of the experimental period, the young rats were randomly divided into the control (n = 7) and suspension (n = 8) groups. In the suspension group, the rats were suspended by their tails to keep their hind legs off the floor. This hindlimb suspension procedure has often been used to induce disuse atrophy in hindlimb muscles (Desplanches et al., 1987; Fitts et al., 1986; Stump et al., 1997). All the rats in the three groups were housed individually for four weeks. After overnight fasting, the rats were anesthetized with pentobarbital sodium (50 mg/kg body weight). Under anesthesia, the unilateral soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from each rat, weighed, immediately frozen in liquid nitrogen, and stored at 80 °C. The contralateral SOL and EDL were also obtained and immediately frozen in 2-methylbutane cooled with solid CO2 (dry ice) and stored at 80 °C for histochemistry. 2.2. Sample preparation for Western blotting Proteins were isolated from muscles for Western blotting by a modified method of McCullagh et al. (1996). Briefly, the muscles were homogenized in buffer A (1:50 w/v) containing 210 mM sucrose, 2 mM EGTA, 40 mM NaCl, 30 mM HEPES, 5 mM EDTA, and 2 mM PMSF, pH 7.4, with a homogenizer (Polytron system PT2100; Kinematica, Littau-Lucerne, Switzerland). Buffer B (1.17 M KCl and 58.3 mM tetrasodium pyrophosphate) was added (37.5 ll/mg of muscle), and the homogenized muscle was mixed briefly and then set on ice for 15 min, after which 1.2 ml of the homogenate was transferred to a microtube and centrifuged at 230,000 g for 75 min at 4 °C. The supernatant fluid was discarded, and the pellet was washed twice with buffer C (200 ll of 10 mM Tris and 1 mM EDTA, pH 7.4). The pellet was resuspended in 206 ll of buffer C and 34 ll of 16% SDS, homogenized, and centrifuged at 1100g for 20 min at room temperature. The supernatant was separated and the total protein concentration of each sample was determined using the DC Protein Assay kit (Bio-Rad Laborato-
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ries, Richmond, CA, USA). Diaphragm from a young control rat was homogenized similarly, and used as a reference protein for controlling gel-to-gel variation. 2.3. Western blotting for MCT1 and MCT4 Ten micrograms of the sample proteins was added to a 12% SDS–polyacrylamide minigel and electrophoresed at 200 V for 2 h in a chamber controlled at 4 °C. The protein bands were transferred to a polyvinylidene fluoride membrane at 100 V for 1 h in a chamber controlled at 4 °C. The membrane was blocked for 1 h at room temperature in Tris-buffered saline with 1% Tween 20 (TBS-T) containing 5% nonfat dry milk. After washing, the membrane was incubated with rabbit anti-MCT1 polyclonal antibody (AB3540P; Chemicon, Temecula, CA, USA) diluted 1:1000 or rabbit antiMCT4 polyclonal antibody (AB3314P; Chemicon) diluted 1:1000 overnight at 4 °C. The membrane was washed with TBS-T and incubated with anti-rabbit IgG antibody (NA934V; GE Healthcare UK Ltd., England, UK) diluted 1:7700 for 45 min at room temperature. After washing, the signal was detected by using ECL Plus (GE Healthcare). The intensity of the signals was quantified using MultiAnalyst software (Bio-Rad). Every blot was duplicated and the mean value was adopted for each sample. Equal protein loading and transfer was confirmed by Coomassie brilliant blue (CBB) staining after detection of MCT1 or MCT4. 2.4. Measurement of metabolic enzyme activities To measure CS, LDH, and PK activities, part of the muscles was homogenized in homogenate buffer (1:20 w/v) containing 175 mM KCl, 10 mM glutathione (GSH), and 2 mM EDTA, pH 7.4. The homogenates were frozen and thawed three times to disrupt the mitochondrial membranes. CS activity in the supernatant fraction was measured by spectrophotometry as described previously (Hashimoto et al., 2004; Srere, 1972). The homogenate was added to 70 mM Tris buffer (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl-CoA, and 0.5 mM oxaloacetate, and mixed in a cuvette. The absorbance of the sample was measured for 5 min at 412 nm at 30 °C. LDH activity was measured in the pyruvate-to-lactate direction using a modified method of Pesce et al. (1964). The homogenate was added to 100 mM potassium phosphate buffer (pH 7.4), 0.7 mM sodium pyruvate, and 0.12 mM NADH, and mixed in a cuvette. The absorbance of the sample was measured for 5 min at 340 nm at 30 °C. PK activity was measured using a modified method of Suwa et al. (2005). The homogenate was added to 50 mM Tris–HCl buffer (pH 7.6), 0.1 mM KCl, 10 mM MgCl2, 0.28 mM NADH, 1.5 mM ADP, 6 U/ml LDH, and 5 mM phosphoenolpyruvate, and mixed in a cuvette. The absorbance of the sample was measured for 5 min at 340 nm at 30 °C. Because the homogenate buffer contained GSH, a tripeptide, we could not determine the protein content of the homogenate for the metabolic enzyme assay. Therefore, we homogenized part of the muscles in homogenate buffer from which GSH had been removed, and used these to determine the protein content with the DC Protein Assay kit. All enzyme activities were expressed as micromoles of substrate per minute per gram of muscle weight and as nanomoles of substrate per minute per microgram of muscle protein. 2.5. Electrophoresis to assess the composition of MHC isoforms Samples were prepared to assess the myosin heavy chain (MHC) isoforms as follows. The dissected muscle was weighed quickly and homogenized immediately in homogenization buffer (1:40 w/v) containing 5 mM Tris–HCl (pH 6.8), 10% SDS, 1 mM EDTA, 1 mM PMSF, and 1 mM DTT in a homogenizer. The sample was centri-
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fuged for 10 min at 10,000 g, the supernatant was separated, and the total protein concentration of each sample was determined using the DC Protein Assay kit. The samples were mixed with homogenization buffer and loading buffer [75 mM Tris–HCl (pH 6.8), 1 mM EDTA, 20% glycerol, 10% SDS, 5% b-mercaptoethanol, and 0.01% bromophenol blue] at 0.4 lg/ll, and boiled at 95 °C for 2 min. Six micrograms of the sample proteins were separated on an SDS–polyacrylamide (7%) gel, based on the procedure described by Talmadge and Roy (1993). Electrophoresis was carried out at 275 V for 24 h at 4 °C in a temperature-controlled chamber. The gels were stained with CBB and the intensity of the bands was quantified using MultiAnalyst software. 2.6. Histochemistry For histochemical analysis, frozen serial sections (10 lm) were cut from the midbelly of SOL and EDL muscles on a cryostat (Cryocut 1800; Leica Microsystems, Wetzlar, Germany). Immunohistochemical staining of MCT1 and MCT4 was performed essentially as described previously (Hashimoto et al., 2005). The sections were air dried, fixed in acetone at 4 °C for 5 min, and incubated in 0.01 M citrate buffer (pH 6.0) with 0.1% Tween 20 at 95 °C for 30 min. The sections were cooled to room temperature, washed in PBS, blocked with 5% goat serum in PBS for 30 min at room temperature, and incubated at 4 °C overnight with rabbit anti-MCT1 polyclonal antibody (AB3540P; Chemicon) diluted 1:100 or rabbit anti-MCT4 polyclonal antibody (AB3314P; Chemicon) diluted 1:100. The next day, the sections were incubated with secondary antibody (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) for 1 h, incubated with avidin–biotin complex reagent (VECTASTAIN Elite ABC Kit; Vector Laboratories) for 30 min, and stained with a 3,30 -diaminobenzidine substrate kit (SK-4100; Vector Laboratories) for 10 min. In control sections, the primary antibodies were omitted and replaced with PBS. To determine the muscle fiber types, the sections were stained for myosin ATPase, succinate dehydrogenase (SDH), and a-glycerophosphate dehydrogenase (a-GPD) as described previously (Gillespie et al., 1987; Nakatani et al., 1999). For myosin ATPase staining, the sections were air dried for 90 min and preincubated in either acid preincubation solution containing 50 mM sodium acetate and 30 mM sodium barbital (pH 4.4) for 5 min or alkaline preincubation solution containing 75 mM glycine, 50 mM CaCl2, and 75 mM NaCl (pH 10.6) for 15 min at room temperature. The sections were washed and incubated in a solution containing 2.8 mM ATP, 50 mM CaCl2, and 75 mM NaCl (pH 9.4) for 45 min at 37 °C. The sections were then immersed in 1% CaCl2 for 3 min, in 2% CoCl2 for 3 min, and finally in 1% (NH4)2S for 1 min. For SDH staining, the sections were air dried for 90 min and incubated in 0.1 M phosphate buffer (pH 7.6) containing 50 mM sodium succinate and 0.6 mM nitroblue tetrazolium (NBT) for 100 min at 37 °C. For a-GPD staining, the sections were air dried for 90 min and incubated in 0.2 M Tris–HCl buffer (pH 7.4) containing 9.3 mM a-glycerophosphate, 1.2 mM menadione, and 0.24 mM NBT for 150 min at 37 °C. Histochemical analysis was performed using a microscope (Optiphot-2; Nikon, Kyoto, Japan) with a charge-coupled device camera. Classification of the muscle fibers into slow oxidative (SO), fast oxidative glycolytic (FOG), and fast glycolytic (FG) fibers was based on previous studies (Hashimoto et al., 2005; Peter et al., 1972; Taguchi et al., 1985). 2.7. Statistical analysis Values are expressed as means ± SEM. One-way ANOVA was used to estimate the variance between groups and Scheffé’s F-test
was used to examine the significant difference between means. Student’s t-test was used to compare two valuables. 3. Results 3.1. Effect of hindlimb suspension and aging on muscle weight and the composition of MHC isoforms Table 1A shows the mean body weight in the control, suspension, and aging groups before and after 4 weeks of hindlimb suspension period. Body weight did not differ significantly between the control and suspension groups before the hindlimb suspension, but it was significantly lower in the suspension group after hindlimb suspension than in the other two groups. Body weight did not differ significantly between the control and the aging groups after hindlimb suspension. Table 1B shows the muscle wet weight in the control, suspension, and aging groups. SOL wet weight was significantly lower in the suspension group than in the control group. EDL wet weight was significantly lower in the aging group than in the control and suspension groups. Table 1C shows the muscle wet weights relative to body weights. The relative weight of SOL was significantly lower in the suspension and aging groups than in the control group, whereas the relative weight of EDL was significantly lower in the aging group than in the control and suspension groups.
Table 1 Body weight and muscle wet weight. Control
Suspension
Aging
(A) Body weight Before suspension (g) After suspension (g)
509 ± 16 537 ± 18
516 ± 15 445 ± 12**,ààà
– 568 ± 20
(B) Muscle wet weight SOL (mg) EDL (mg)
217 ± 21 252 ± 11
115 ± 11*** 234 ± 8
164 ± 13 120 ± 13***,
0.26 ± 0.02*** 0.52 ± 0.01
0.29 ± 0.02** 0.21 ± 0.03***,
(C) Muscle wet weight per body weight SOL (mg/g) 0.40 ± 0.03 EDL (mg/g) 0.47 ± 0.02
(A) Rat body weight before and after hindlimb suspension. (B) SOL and EDL wet weight in each rat group. (C) SOL and EDL wet weight per body weight in each rat group. Values are means ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). ** P < 0.01, significantly different from control group. *** P < 0.001, significantly different from control group. P < 0.001, significantly different from suspension group. ààà P < 0.001, significantly different from before suspension.
Table 2 Composition of MHC isoforms in SOL and EDL. Control
Suspension
Aging
(A) SOL Type I (%) Type IIa (%) Type IIx (%) Type IIb (%)
99.2 ± 0.5 0.7 ± 0.4 0.1 ± 0.1 ND
96.9 ± 1.3 0.6 ± 0.4 2.5 ± 1.0 ND
93.3 ± 3.0 4.9 ± 2.2 1.8 ± 0.8 ND
(B) EDL Type I (%) Type IIa (%) Type IIx (%) Type IIb (%)
3.0 ± 0.9 14.0 ± 2.8 27.4 ± 4.3 55.6 ± 7.8
2.6 ± 0.7 13.0 ± 2.2 25.4 ± 4.3 59.0 ± 7.0
ND 18.2 ± 3.2 55.9 ± 3.4***, 25.9 ± 3.4*,
Composition of MHC isoforms in (a) SOL and (b) EDL. Values are mean ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). ND, not detected. * P < 0.05, significantly different from control group. *** P < 0.001, significantly different from control group. P < 0.05, significantly different from suspension group. P < 0.001, significantly different from suspension group.
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type IIa
A
type IIx type IIb type I control
suspension
old
diaphragm
B
type IIa type IIx type IIb control
suspension
old
diaphragm
type I
Fig. 1. Representative myosin heavy chain (MHC) bands on electrophoresis gels in (A) SOL and (B) EDL. Diaphragm is used as the control, with which to identify type I, IIa, IIx, and IIb MHC isoforms.
Table 2 shows the composition of the MHC isoforms in SOL and EDL. SOL showed a tendency for slow-to-fast MHC transition in the suspension and aging groups, although this was not significant. In contrast, EDL showed a significant shift from type IIb to type IIx MHC isoforms in the aging group. Representative MHC bands are shown in Fig. 1.
from the aging group was three times higher than the MCT1 expression in the control (P = 0.056) and suspension (P = 0.049) groups (Fig. 2B). MCT4 expression was not significantly different in SOL of the three groups (Fig. 2C), but was significantly lower in EDL from the aging group than in EDL from the control group (Fig. 2D).
3.2. Effect of hindlimb suspension and aging on MCT1 and MCT4 expression
3.3. Effect of hindlimb suspension and aging on metabolic enzyme activities
In SOL, MCT1 expression was significantly lower in the aging group but did not differ between the suspension and control groups (Fig. 2A). In contrast, the mean MCT1 expression in EDL
The effects of hindlimb suspension and aging on the protein content and metabolic enzyme activities in skeletal muscles are shown in Table 3. The protein content was significantly lower in
100 80 60 40 20 0
MCT4 expression (% of control)
500 400 300 200 100 0
D
C 120 100 80 60 40 20 0
MCT4 expression (% of control)
MCT1 expression (% of control)
120
MCT1 expression (% of control)
B
A
120 100 80 60 40 20 0
Fig. 2. MCT1 expression in (A) SOL and (B) EDL and MCT4 expression in (C) SOL and (D) EDL. Top: Representative immunoblots of MCTs. Bottom: Expression levels of MCTs. White, gray, and black bars indicate the values in the control, suspension, and aging groups, respectively. Values are expressed as a percentage of the value for the muscle in the control group. Values are means ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). Significantly different from control group, *P < 0.05. Significantly different from suspension group, P < 0.05.
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Table 3 Metabolic enzyme activities in SOL and EDL. SOL Control (A) PK (lmol/g muscle/min) LDH (lmol/g muscle/min) CS (lmol/g muscle/min) PK/CS LDH/CS (B) Protein content (lg/ll) PK (nmol/mg protein/min) LDH (nmol/mg protein/min) CS (nmol/mg protein/min)
45 ± 1 45 ± 1 21 ± 1 2.1 ± 0.1 2.1 ± 0.1 3.9 ± 0.1 581 ± 23 581 ± 23 277 ± 12
EDL Suspension 54 ± 5 49 ± 3 18 ± 1 2.9 ± 0.1** 2.7 ± 0.2* 4.1 ± 0.2 665 ± 57 595 ± 35 228 ± 19
Aging 33 ± 2 42 ± 1 15 ± 1** 2.2 ± 0.1 2.8 ± 0.1** 3.5 ± 0.1 472 ± 15 594 ± 19 211 ± 6*
Control
Suspension
Aging
300 ± 10 143 ± 3 23 ± 2 13.7 ± 1.6 6.5 ± 0.6
281 ± 6 137 ± 4 22 ± 1 12.9 ± 0.6 6.3 ± 0.4
132 ± 26***, 93 ± 15**, 28 ± 2 4.7 ± 0.9***, 3.3 ± 0.5**,
4.0 ± 0.2 3834 ± 221 1822 ± 71 291 ± 20
3.7 ± 0.1 3794 ± 98 1845 ± 36 297 ± 14
3.2 ± 0.1**, 2019 ± 394***, 1423 ± 216 430 ± 26***,
(A) Pyruvate kinase (PK), lactate dehydrogenase (LDH), and citrate synthase (CS) activities and glycolytic vs. oxidative activity ratios in SOL and EDL, corrected for muscle weight. (B) Muscle protein contents and metabolic enzyme activities corrected for protein content. Values are mean ± SEM (control, n = 7; suspension, n = 8; aging, n = 6). * P < 0.05, significantly different from control group. ** P < 0.01, significantly different from control group. *** P < 0.001, significantly different from control group. P < 0.05, significantly different from suspension group. P < 0.01, significantly different from suspension group. P < 0.001, significantly different from suspension group.
SOL from the aging group than in SOL from the suspension group, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. When corrected for muscle weight (Table 3A), PK activity was significantly lower in SOL from the aging group than in SOL from the suspension group, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. LDH activity in SOL did not differ between the three groups, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. CS activity was significantly lower in SOL from the aging group than in SOL from the control group, but it was significantly higher in EDL from the aging group than in EDL from the suspension group. PK-to-CS ratio was significantly higher in SOL from the suspension group than in that from the control and aging groups, and it was significantly lower in EDL from the aging group than in that from the control and suspension groups. LDH-to-CS ratio was significantly higher in SOL from the suspension and aging groups than in SOL from the control group, and it was significantly lower in EDL from the aging group than in EDL from the control and suspension groups. When corrected for protein content (Table 3B), PK activity was significantly lower in SOL from the aging group than in SOL from the suspension group, and it was significantly lower in EDL from the aging group than in EDL from the other two groups. LDH activity did not differ between the three groups in SOL or EDL. CS activity was significantly lower in SOL from the aging group than in SOL from the control group, but it was significantly higher in EDL from the aging group than in EDL from the other two groups. 3.4. Histochemical analysis of SOL and EDL We observed marked fiber atrophy in SOL from the suspension group compared with the control group (Fig. 3H–N). Small and angulated fibers, group atrophy, and fiber-type grouping, characteristics of aged muscles, were observed in SOL from the aging group (Fig. 3O–U). Large fiber groups maintained their size, and these were predominantly SO fibers. Small fiber groups, which contained fast-twitch fibers, showed marked atrophy. MCT1 was strongly expressed at the sarcolemma of all fibers in SOL from the control and suspension groups (Fig. 3E and L), but the expression disappeared irregularly in small fibers in SOL from the aging group (Fig. 3S). MCT4 was strongly expressed at the sarcolemma of FOG fibers in SOL from the control and suspension groups
(Fig. 3F and M). In SOL from the aging group, MCT4 expression was not fiber-type specific and was not evident in severely atrophied fibers (Fig. 3T). In EDL, the proportion of FG fibers was markedly lower in the aging group (Fig. 4O–U); this pattern corresponded to the decreased percentage of type IIb MHC isoform shown in Table 2B. EDL in the aging group showed small angulated fibers, group atrophy, and fiber-type grouping. In EDL from the control and suspension groups, MCT1 was strongly expressed at the sarcolemma of SO and FOG fibers (Fig. 4E and L). MCT4 was not expressed at the sarcolemma of SO fibers (Fig. 4F and M). However, the fiber-type-specific expression of MCT1 and MCT4 was not evident in EDL from the aging group (Fig. 4S and T). 4. Discussion In this study, we examined for the first time the effect of aging and unloading on the metabolic properties relevant to the lactate shuttle, and found reduced expression of MCT1 in SOL and MCT4 in EDL with aging, which were not observed in unloaded rats. Along with changes in muscle mass and metabolic enzyme activities, these results suggest that lactate metabolic properties are regulated differently between aging and muscle unloading. The decreased expression of MCT1 in SOL and MCT4 in EDL from the aging group suggests that aging, not disuse atrophy, decreases the expression of MCTs. Wilson et al. (1998) showed that denervation of rat hindlimb muscles decreases MCT1 expression in SOL and MCT4 expression in fast-type muscles, such as white gastrocnemius and white tibialis anterior muscles. In fact, aging induces a reduction in the number of functioning motor units in skeletal muscles (Caccia et al., 1979). Furthermore, angulated and atrophied fibers have been observed in aged skeletal muscle because of loss of motor neurons (Caccia et al., 1979; Einsiedel and Luff, 1992; Larsson, 1995; Snow et al., 2005). Moreover, fiber-type grouping in aged muscles occurs as a result of partial reinnervation by surviving motor neurons (Caccia et al., 1979; Snow et al., 2005). In this study, we found that group atrophy of muscle fibers and grouping of FOG fibers in SOL occurred in the aging group but not in the suspension group (Fig. 3O–U). Similarly, in EDL from the aging group, we found group atrophy of muscle fibers and fiber-type grouping in moderately atrophied fibers (Fig. 4O–U). These observations indicate that loss of motor neurons and partial
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Fig. 3. Serial cross-sections of SOL from (A–G) control, (H–N) suspension, and (O–U) aging groups. The cross-sections are stained for (A, H, and O) myosin ATPase (acid preincubation), (B, I, and P) myosin ATPase (alkaline preincubation), (C, J, and Q) SDH, (D, K, and R) a-GPD, (E, L, and S) MCT1, (F, M, and T) MCT4, and (G, N, and U) no primary antibody (negative control for MCT1 and MCT4). Muscle fibers are classified as SO and FOG fibers as indicated in A, H, and O. MCT1 is strongly expressed in both SO and FOG fibers but MCT4 is strongly expressed only in FOG fibers. Scale bars indicate 100 lm.
reinnervation by surviving motor neurons occurred in the aged muscles. Because our histochemical results show reduced MCT1 and MCT4 expression, particularly in severely atrophied fibers (Figs. 3S and 4T), it is likely that age-induced loss of motor neurons is at least partially involved in the decreased expression of MCTs from the aging group. Given the role of MCTs in lactate transport, the decreased expression of MCT1 in SOL and MCT4 in EDL from the aging group indicates that aging diminishes the potential for lactate uptake into SOL and lactate efflux from EDL. The marked increase of MCT1 expression in EDL from the aging group may result from the age-induced fiber-type shift. FG fibers,
in which MCT1 expression is weak, shift to FOG fibers, in which MCT1 is strongly expressed, with aging, as shown in Fig. 4. However, because the MHC isoform pattern was not different between the control and suspension groups (Table 2B), the age-induced fiber-type shift may be because of aging rather than inactivity. Because MCT1 was expressed in SO and FOG fibers (Fig. 4S), the increase of MCT1 in EDL from the aging group could facilitate lactate uptake into SO and FOG fibers in EDL. Muscles from the aging and suspension groups demonstrated typical changes by aging and suspension, respectively, in muscle mass and morphological characteristics. Although muscle atrophy
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Fig. 4. Serial cross-sections of EDL from (A–G) control, (H–N) suspension, and (O–U) aging groups. The cross-sections are stained for (A, H, and O) myosin ATPase (acid preincubation), (B, I, and P) myosin ATPase (alkaline preincubation), (C, J, and Q) SDH, (D, K, and R) a-GPD, (E, L, and S) MCT1, (F, M, and T) MCT4, and (G, N, and U) no primary antibody (negative control for MCT1 and MCT4). Muscle fibers are classified as SO, FOG, and FG fibers as indicated in A, H, and O. MCT1 is strongly expressed in SO and FOG fibers. MCT4 is expressed strongly in FOG fibers, intermediately in FG fibers, and weakly in SO fibers. Scale bars indicate 100 lm.
was observed in SOL and EDL from the aging group, hindlimb suspension induced marked muscle atrophy in SOL; however, this was not evident in EDL (Table 1C), which corresponds with previous studies (Desplanches et al., 1987; Fitts et al., 1986; Stump et al., 1997). Histochemical data showed that aging and hindlimb suspension affected SOL quite differently; for example, disproportional fiber atrophy, which is characteristic in aged SOL, was not observed in the suspended SOL (Fig. 3). In aged rats, a shift from type IIb to type IIx MHC isoforms (Škorjanc et al., 1998) and fast-to-slow fiber-type shifts (Caccia et al., 1979) have been observed in EDL, and slow-to-fast fiber-type
shifts have been observed in SOL (Caccia et al., 1979; Snow et al., 2005). Correspondingly, we found a shift from type IIb to type IIx MHC isoforms in EDL from the aging group (Table 2B), which was not observed in EDL from the suspension group, and a tendency to shift from type I to type II MHC isoforms in SOL from the aging group (Table 2A). In human studies, a marked decline in the fiber area of type II fibers compared with that of type I fibers has also been reported in the vastus lateralis muscle (Lexell, 1995; Vandervoort, 2002) and the population of slow-type fibers decreases with aging in the human masseter muscle, which predominantly contains in slow-type fibers (Monemi et al., 1999).
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We observed no statistically significant slow-to-fast MHC shift in SOL from the suspension group, which seems inconsistent with the results of previous studies, which showed a significant slow-tofast MHC shift after hindlimb unloading (Desplanches et al., 1987; Dubouchaud et al., 1996; Saitoh et al., 1999). It is possible that the extent of the MHC shift depends on age. Saitoh et al. (1999) reported that young rats showed a more marked MHC shift in SOL after hindlimb suspension for four weeks than did adult rats. Dubouchaud et al. (1996) showed a marked slow-to-fast MHC shift in SOL from male Wistar rats, the same strain of rats we used in our experiments, after hindlimb suspension for four weeks. However, the rats used by Dubouchaud et al. were less mature than ours (377 ± 25 g vs. 537 ± 18 g in body weight, respectively). The rats we used were so mature that no statistically significant MHC shift might be observed. The decrease of PK-to-CS ratio and LDH-to-CS ratio in EDL with aging (Table 3A) suggests that there is a relative predominance of lactate oxidation compared with pyruvate and lactate production in aged muscle, resulting in reduced lactate accumulation in EDL during contraction. Our histochemical results also suggest that EDL depends more on oxidative metabolism in the aging group than in the control group. In the control group, SDH staining was weak in FG fibers; however, it was relatively strong in each fiber from the aging group (Fig. 4C and Q). In contrast, in the control group, a-GPD staining was strong in FOG and FG fibers; however, it was weak in the fibers from the aging group (Fig. 4D and R). These changes would be at least partially related to the shift from FG to FOG fibers with aging, and the shift from type IIb to IIx MHC isoforms with aging corresponds to this fiber-type shift. Increased LDH-to-CS ratio in SOL with aging (Table 3A) suggests that slow-type muscles depend more on glycolytic metabolism in aged subjects compared with young subjects. The ratio also increased with hindlimb suspension. However, the PK-to-CS ratio did not change in the aging group, whereas it increased in the suspension group (Table 3A). These findings indicate that the potential for pyruvate production increases with hindlimb suspension, but not with aging, and therefore the potential for lactate production would be lower in SOL from the aging group compared with the suspension group. It should be noted that histochemical data for SOL from the aging group revealed that SDH staining was weak, particularly in SO fibers for which CSA was maintained (Fig. 3Q), whereas the MHC isoform pattern was not significantly changed with aging (Table 2A). Thus, it is likely that the change in metabolic enzyme activities was not because of fiber-type shift but because of decreased oxidative capacities in individual SO fibers for which CSA was maintained. These changes in metabolic properties with aging may be involved in the reduced lactate accumulation and increased fatigue resistance in aged skeletal muscles observed in previous studies (Bilodeau et al., 2001; Ditor and Hicks, 2000; Hepple et al., 2003, 2004; Hunter et al., 2004, 2005; Kent-Braun et al., 2002; Lanza et al., 2004, 2005). The putative effect of aging on lactate metabolism could be described as follows. In fast-type muscles from the aging group, the reduced PK-to-CS and LDH-to-CS ratios increase the capacity for lactate oxidation compared with the capacity for lactate production, reducing the amount of lactate that is transported from the fast-type muscles during contraction. The reduced expression of MCT4 in the fast-type muscles parallels the reduction in lactate during contraction, and thus it does not attenuate lactate efflux from the glycolytic fibers in the fast-type muscles. The increased expression of MCT1 facilitates lactate transport from the glycolytic to the oxidative fibers, to be oxidized within the fasttype muscles. These changes also contribute to a reduction in the amount of lactate that is transported from fast- to slow-type muscles. In slow-type muscles, the increased LDH-to-CS ratio suggests that there is a relative predominance of lactate production from
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pyruvate relative to lactate oxidation. However, the reduction in MCT1 expression may limit lactate uptake and delay lactate accumulation. In contrast, the changes in metabolic properties with muscle unloading may be involved in facilitating lactate accumulation in slow- and fast-type skeletal muscles during contraction, as shown in previous studies (Grichko et al., 2000; Witzmann et al., 1983). The putative effect of muscle unloading on lactate metabolism could be described as follows. In slow-type muscles, the increases in PK-to-CS ratio and LDH-to-CS ratio enhance the capacity for lactate production compared with lactate oxidation. Unchanged MCT1 expression indicates that the lactate uptake rate during contraction is maintained even after unloading. These changes in metabolic properties in slow-type muscles facilitate lactate production and lactate uptake compared with lactate oxidation, leading to earlier elevation in lactate concentration during contraction. In addition, it is notable that hindlimb unloading selectively atrophies slow-type muscles and that unloading does not change metabolic properties in fast-type muscles. Selective atrophy of slow-type muscles may decrease the amount of lactate efflux from fast- to slow-type muscles during contraction and prevents the decrease of lactate concentration in fast-type muscles. In conclusion, aging and muscle unloading regulate the metabolic properties relevant to lactate production, transport, and oxidation in slow- and fast-type muscles in different ways. Our data suggest that aging, not inactivity, contributes to increased fatigue resistance in aged skeletal muscles by reducing lactate production and transport, rather than lactate oxidation in skeletal muscles. Acknowledgements We are grateful to Takeshi Hashimoto for valuable suggestion. This work was supported by a research grant from the Japan Society for the Promotion of Science (#17500424 to Tatsuya Hayashi). References Baker, S.K., McCullagh, K.J.A., Bonen, A., 1998. Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle. J. Appl. Physiol. 84, 987–994. Bass, A., Brdiczka, D., Eyer, P., Hofer, S., Pette, D., 1969. Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10, 198–206. Bilodeau, M., Henderson, T.K., Nolta, B.E., Pursley, P.J., Sandfort, G.L., 2001. Effect of aging on fatigue characteristics of elbow flexor muscles during sustained submaximal contraction. J. Appl. Physiol. 91, 2654–2664. Bonen, A., McCullagh, K.J.A., Putman, C.T., Hultman, E., Jones, N.L., Heigenhauser, G.J., 1998. Short-term training increases human muscle MCT1 and femoral venous lactate in relation to muscle lactate. Am. J. Physiol. 274, E102–E107. Bonen, A., Miskovic, D., Tonouchi, M., Lemieux, K., Wilson, M.C., Marette, A., Halestrap, A.P., 2000. Abundance and subcellular distribution of MCT1 and MCT4 in heart and fast-twitch skeletal muscles. Am. J. Physiol. Endocrinol. Metab. 278, E1067–E1077. Brooks, G.A., 2000. Intra- and extra-cellular lactate shuttles. Med. Sci. Sports Exerc. 32, 790–799. Brooks, G.A., 2002. Lactate shuttles in nature. Biochem. Soc. Trans. 30, 258–264. Caccia, M.R., Harris, J.B., Johnson, M.A., 1979. Morphology and physiology of skeletal muscle in aging rodents. Muscle Nerve 2, 202–212. Capelli, C., Antonutto, G., Kenfack, M.A., Cautero, M., Lador, F., Moia, C., Tam, E., _ 2max decay during Ferretti, G., 2006. Factors determining the time course of VO _ 2max limitation. Eur. J. Appl. Physiol. 98, 152–160. bedrest: implications for VO Convertino, V.A., 1997. Cardiovascular consequences of bed rest: effect on maximal oxygen uptake. Med. Sci. Sports Exerc. 29, 191–196. Desplanches, D., Mayet, M.H., Sempore, B., Flandrois, R., 1987. Structural and functional responses to prolonged hindlimb suspension in rat muscle. J. Appl. Physiol. 63, 558–563. Ditor, D.S., Hicks, A.L., 2000. The effect of age and gender on the relative fatigability of the human adductor pollicis muscle. Can. J. Physiol. Pharmacol. 78, 781–790. Dubouchaud, H., Granier, P., Mercier, J., Le Peuch, C., Préfaut, C., 1996. Lactate uptake by skeletal muscle sarcolemmal vesicles decreases after 4 wk of hindlimb unweighting in rats. J. Appl. Physiol. 80, 416–421. Dubouchaud, H., Butterfield, G.E., Wolfel, E.E., Bergman, B.C., Brooks, G.A., 2000. Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 278, E571–E579.
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