Beef Extract Supplementation Increases Leg Muscle Mass and Modifies Skeletal Muscle Fiber Types in Rats
J Nutr Sci Vitaminol, 52, 183–193, 2006
Beef Extract Supplementation Increases Leg Muscle Mass and Modifies Skeletal Muscle Fiber Types in Rats
Hiroyuki YOSHIHARA1, Jun-ichiro WAKAMATSU1, Fuminori KAWABATA1, Sunao MORI1, Atsushi HARUNO1, Toshiya HAYASHI2, Takeshi SEKIGUCHI3, Wataru MIZUNOYA1, Ryuichi TATSUMI1, Tatsumi ITO1 and Yoshihide IKEUCHI1,*
1Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, Fukuoka 812–8581, Japan 2Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Nagoya 468–8502, Japan 3Ito Life Sciences Inc., Ibaraki 302–0104, Japan (Received August 15, 2005)
Summary The objective of this research was to investigate the effects of beef extract on fat metabolism, muscle mass and muscle fiber types in rats. We also investigated the synergetic effect of endurance exercise. Twenty-four male rats weighing about 270 g were assigned to two diets containing 0 or 6% beef extract (BE). Half the rats fed each diet were subjected to compulsory exercise (CE) for 30 min every other day. After 4 weeks feeding, the blood was collected and various organs were dissected. The muscle fiber type of the soleus and extensor digitorum longus (EDL) muscles were evaluated by histochemical and electrophoretical analyses. Rats supplemented with BE showed a decrease in fat content in liver and abdomen and an increase in the activity of carnitine palmitoyl transferase II in liver. BE as well as exercise increased the relative weights of both soleus and EDL. BE alone and BE plus CE did not affect the distribution of muscle fiber types in soleus. BE without exercise decreased in type IIb of EDL from 54% to 44% with compensatory increase in type IIa from 41% to 49% and type I from 5% to 7% compared with the nonsupplemented, nonexercised control group. No synergetic effect on a fast to slow fiber conversion due to the combination of BE and CE was detected. Thus, BE supplement increased muscle mass and slow type fiber in EDL. The effects of BE supplement on muscle characteristics were similar to those of exercise. Key Words beef extract, fat metabolism, muscle fiber type, muscle mass, L-carnitine
A great deal of research has been done to elucidate ral stimulation. Concretely, endurance exercise re- the general nutritional value of beef on the growth of quiring the consumption of oxygen stimulates a shift to human subjects and the effects of protein quality and the slow twitch, oxidative muscle fibers (8). The fiber exercise on the growth and protein metabolism of ani- types of adult skeletal muscle are closely related to mals and human beings (1–3). However, existing infor- energy metabolic characteristics as described above. mation about the nutritional effects of beef extract on Nutrition-induced regulation in mRNA levels for a muscle metabolism is not sufficient. number of metabolic genes have been well reported (9– Skeletal muscle is composed of at least three fiber 11). Thus, one question concerns whether the transfor- types on the basis of their contractile and energy meta- mation of muscle fiber type can be induced nutrition- bolic properties; type I fibers exhibit an aerobic energy ally. If this would be possible, the diet containing the metabolism (slow-twitch fiber), type IIb fibers are pre- compositions promoting the aerobic metabolism would dominantly glycolytic (fast-twitch fiber) and type IIa be expected to induce fast-slow fiber transformation. fibers are the intermediate type exhibiting both oxida- Beef extract is rich in L-carnitine as well as carnosine, tive and glycolytic metabolisms. Lipids are easily accu- taurine and creatinine. With regard to the relationship mulated in type I fibers (4), while glycogen depletion between carnitine metabolism and muscle fiber type, occurs primarily in the type II (5). It is well known that Constantin-Teodosiu et al. (12) have recently demon- cross-reinnervation, electrical stimulation or exercise strated that accumulation of acetyl-L-carnitine in evokes the fiber type conversion, although the molecu- humans is greater in type I fibers, in which the greater lar mechanisms mediating the transformation are not mitochondria responsible for oxidative metabolism was clearly elucidated (6, 7). This means that skeletal mus- present, than in type II fibers after an exhaustive pro- cle cells possess a capacity to respond and adapt to met- longed one-legged exercise at ~75% VO2max for about abolic changes imposed by physical stimulation or neu- 220 min. However, their report did not describe the relationship between carnitine intake and the fiber type *To whom correspondence should be addressed. transformation. They only suggested the physiological E-mail: [email protected] role of carnitine as a buffer agent neutralizing the
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184 YOSHIHARA H et al. excess acetyl group occurring during prolonged exer- Table 1. The composition of two different diets, cise in type I fibers. To our knowledge, few studies have been so far conducted on the effect of nutrients such as Control diet Beef extract diet carnitine on muscle fiber type transformation, al- though undernutrition has been shown to induce an Casein 20 20 Beef extract concentration 0 6 increase in the proportion of slow type fiber (13–15). Olive oil 12 12 Thus, the present experiments were conducted to Sucrose 10 10 confirm the effect of beef extract on fats metabolism and Corn starch 50.9 44.9 anabolic action in rats, and then to examine whether or Cellulose powder 2 2 not the transformation from one type to another in the Mineral mixture 3.5 3.5 soleus and extensor digitorum longus (EDL) occurred in Vitamin mixture 1 1 response to the ingestion of beef extract as occurs with D,L-Methionine 0.3 0.3 exercise. Choline bitartrate 0.2 0.2 Cholesterol 0.1 0.1 MATERIALS AND METHODS Animals, diets and exercise. Wistar (7 wk old) male Total (g) 100 100 rats were purchased from a commercial supplier (Seac Yoshitomi Ltd., Fukuoka, Japan). They were individu- ally housed in stainless-steel wire-mesh cages in an ani- Table 2. The composiiton of beef extract concentration. mal room at 20˚C in 60% humidity under an artificial (%) lighting system of 12 h light and 12 h darkness in the animal raising facility of Biotron Institute of Kyushu Water 3.00 University. Rats were fed a commercial diet (Type MF of Proteins (including nitrogen compounds) 61.40 Lipids 0.00 Oriental Yeast Co. Ltd., Tokyo) and water for 1 wk in Carbohydrate 33.00 order to acclimate them to the environment. After pre- Ash 2.60 feeding, rats were divided into two groups. Twelve rats were assigned to the beef extract-supplemented diet and Total 100 the other twelve rats were given the nonsupplemented diet. Each dietary group was furthermore subdivided Carnitine 5.00 into aerobically exercised and nonexercised groups Anserine 0.45 (total 4 groups); Control: nonexercised, nonsupple- Carnosine 4.95 mented, BE: nonexercised, supplemented, CE: exercised, Taurine 2.82 nonsupplemented, BE CE: exercised, supplemented. Creatine 1.51 Exercised subjects engaged in compulsory exercise for Creatinine 7.29 Phosphoric acid 1.55 30 min every other day with a treadmill running tool Lactic acid 8.14 having 5% grade and 15 m/min speed and this protocol Sodium 0.70 was assumed as low intensity endurance exercise com- pared with previous reports (16). The body weight of each group at the beginning of the experiment (after pre-feeding) was adjusted to be as identical as possible and EDL muscles were harvested, weighed, individually [276.8 9.4 (SD) g in Control, 265.6 17.4 g in BE, quick frozen by placing them in a dry ice-isopenthane 275.1 10.8 g in CE, 265.2 13.7 g in BE CE]. Beef mixture, and then stored at 35˚C for histochemical extract (Ito Life Sciences Inc.) was added to the diets to analysis. achieve a concentration of 6%, which corresponded to All experimental procedures were performed accord- 0.3% L-carnitine. The composition of the two different ing to the guideline for Animal Experiment in the Fac- diets is given in Table 1. Table 2 shows the result of com- ulty of Agriculture and the Graduate Course of Kyushu ponential analysis of beef extract by Ito Life Sciences University and the law [No. 105] and notification [No. Inc. 1] of the Japanese Government. Rats were fed for 4 wk and were given free access to Measurement of fat content and fatty acid analysis. the diets and water supply. Food intake and body weight Lipids were extracted from serum, abdominal fat, liver of each rat were recorded every other day. After 4 weeks and muscle by the method of Folch et al. (17). Fat con- feeding, urine was collected for quantifying carnitine tent was quantified gravimetrically after evaporation of concentration. Food was removed 24 h before killing, the solvent (chloroform-methanol mixture) in the and the rats were killed after anesthetizing them with extracted lipids by freeze-drying. Fatty acid composition ethyl ether. The blood was collected by bleeding from of extracted fat was analyzed by gas chromatography the carotid artery. Sera were collected from the blood by (GC-14B, Gas Chromatograph, Shimazu Co. Ltd., Japan) centrifugation at 3,000 rpm for 10 min, and were kept according to the method described previously (18). at 80˚C until use for assays. Visible abdominal fat, Measurement of carnitine concentrations in serum and liver and other organs were carefully removed from car- urine. L-Carnitine in the serum and urine was deter- cass, weighed and frozen in liquid nitrogen. The soleus mined by the method of a combination of carnitine
Beef Extract Diet and Muscle Fiber Type 185 acetyl transferase and acetyl CoA synthase method conditions. Type II fibers were further divided into type using Roche Kit No. 1242008 (Roche Molecular Bio- IIa and type IIb according to NADH-DH activities; type chemicals, USA). IIa reacted to it strongly and type IIb was unreactive. Measurement of carnitine palmitoyl transferase II (CPT Very few fibers reacted to myosin ATPase under either II) and fatty acid synthase (FAS) activities in liver. Liver pH condition, but these fibers were excluded from the samples (2 g) were gently homogenized with a manual analysis because of their small population. Percentage glass homogenizer in 6 vol/wt of 10 mmol/L Tris-HCl distribution of fiber types was calculated from the num- (pH 7.4) buffer containing 0.25 mol/L sucrose, 1 mmol/ ber of each type counted from a total 600–1,000 fibers L ethylenediamine tetra acetic acid (EDTA) and then in EDL and 500–600 fibers in soleus muscles. centrifuged at 700 g for 10 min. The supernatant was Analysis for myosin heavy chain (MyHC) isoform com- centrifuged at 10,000 g for 10 min, and the pellet position. Frozen soleus and EDL muscle were homoge- containing the mitochondria was removed carefully nized and subjected to high-resolution SDS-polyacryl- from the tube and then resuspended in the same buffer amide gel electrophoresis for the assessment of MyHC described above. This procedure was repeated twice. isoform content as described in detail by Talmadge and The resulting resuspended fraction (mitochondrial frac- Roy (24). Gels were stained with Coomassie Brilliant tion) was used for subsequent determination of CPT II Blue G-250 (Nacalai Tesque Inc., Kyoto, Japan). The activity. On the other hand, the supernatant at second final product was captured on an image scanner and centrifugation was further centrifuged at 125,000 g the relative content of MyHC isoforms was estimated for 60 min to remove microsome (cytosol fraction). Pro- using image analysis software (NIH image 1.61). MyHC tein concentration of each fraction was determined by isoforms were identified according to their apparent the Bradford method with bovine serum albumin as the molecular masses as indicated by migration rate standard (19). These entire procedures were carried out (I IIb IIx IIa). at 4˚C. CPT II activity in mitochondrial fraction and Statistical analysis. Data values are expressed as FAS activity in liver cytosol were determined by the pro- means SD. Statistical analysis was made by two-way cedures of Markwell et al. (20) and Kelley et al. (21), ANOVA to test the effects of exercise, supplement and respectively. their interaction using STATISTICA (Japanese version) Histochemical analysis. Serial frozen sections (8 µm software (Statsoft, USA). The main effects of diet and thick) were cut transversely from the soleus and EDL exercise were tested using linear contrasts, as was the muscles and stained by the histochemical reactions for interaction. Level of significance was set for p 0.05. myosin adenosine triphosphatase (ATPase) activities RESULTS after preincubation of the solutions at pH 4.3 or 10.5 and reduced nicotine amide adenine dinucleotide dehy- Body weight and fat drogenase (NADH-DH) activities (22). On microscopic The body weight gain (%), food intake (g/d) and food photograph ( 50) taken on the same location of the efficiency (g gain/100 g diet) during the 4 weeks feed- specimen, myofibers were categorized into type I, IIa ing are shown in Table 3. Compulsory exercise de- and IIb according to the nomenclature of Brooke and creased body weight gains significantly, but beef extract Kaiser (23). Type I fibers were characterized to stain supplementation did not affected body weight signifi- strongly with NADH-DH, reacted strongly to myosin cantly. Food intake or food efficiency showed a pattern ATPase after acid preincubation and weakly after alka- similar to that of body weight gain. The interactive line preincubation. On the other hand, type II (type IIa effects of supplement and exercise were not significant. and IIb) fibers had weak myosin ATPase activities under Visceral fat weights of exercised rats (CE and BE CE) acidic conditions and strong activities under alkaline and supplemented rats without exercise (BE) were sig-
Table 3. Body weight gain and food efficiency.
Supplement Exercise Body weight gain (%) Food intake (g/d) g gain/100 g diet
Control 155.8 9.0a 25.1 2.1a 25.3 3.0a BE 153.4 12.7a,b 23.2 2.4a,b 25.2 4.4a,b CE 141.9 6.2b 22.9 1.0a,b 21.4 2.1b BE CE 143.0 5.0b 21.6 1.7b 22.0 1.8a,b
Statistical significance Supplement NS1 NS NS Exercise 0.049 0.032 0.013 Interaction NS NS NS
Values are means SD, n 5–6. Statistical significance was calculated from two-way ANOVA, using the factors of supple- mentation and exercise. 1 NS, not significant, p 0.05. Values with different superscripts in the line are significantly different (p 0.05), tested by linear contrasts. BE, beef extract supplemented; CE, compulsory exercise.
186 YOSHIHARA H et al.
Table 4. Liver and visceral fat weights.
Supplement Exercise Liver (g/100 g body weight) Visceral fat (g) Percent fat (%)2
Control 3.28 0.73a 12.05 3.49a 2.75 0.69a BE 2.72 0.26b 7.01 2.09b 1.75 0.34b CE 2.79 0.14a,b 5.63 0.93b 1.47 0.21b BE CE 2.71 0.18b 4.37 1.32b 1.23 0.35b
Statistical significance Supplement NS1 0.006 0.007 Exercise NS 0.001 0.001 Interaction NS NS NS
See the footnote to Table 3. 2 Percent fat (visceral fat weight/the final body weight) 100.
Table 5. Hepatic, muscle and serum lipids.
Liver Muscle Serum Supplement Exercise (g/100 g tissue) (g/100 g tissue) (g/100 mL serum)
Control 8.84 1.89a 1.61 1.80 0.43 0.08 BE 5.87 1.82b 1.95 1.06 0.32 0.11 CE 5.53 1.91b 1.39 1.26 0.36 0.09 BE CE 3.53 1.55b 0.88 0.43 0.36 0.17
Statistical significance Supplement 0.006 NS1 NS Exercise 0.002 NS NS Interaction NS NS NS
See the footnote to Table 3.
Table 6. Concentration of L-carnitine in serum and urine.
Supplement Exercise Serum (mg/L serum) Urine (mg/d)
Control 5.79 2.23a 0.08 0.10a BE 10.25 0.79b 2.00 0.63b CE 7.66 1.12a 0.04 0.01a BE CE 7.84 1.77a 1.75 0.47b
Statistical significance Supplement 0.004 0.001 Exercise NS1 NS Interaction 0.008 NS
See the footnote to Table 3. nificantly reduced from that of the control. Liver partition in rat bodies was not influenced by beef weights of supplemented rats with or without exercise extract intake (data not shown). (BE and BE CE) were significantly lower than for the Free carnitine concentrations in the serum and urine control (Table 4). The interactive effects of the supple- In order to confirm the absorption of carnitine in beef mentation and exercise on visceral fat weights were not extract in the rat body, free carnitine concentrations in observed. serum and urine were spectrophotometrically mea- Supplementation significantly reduced lipid content sured (Table 6). In nonexercised groups, serum car- in the liver and this was obviously further enhanced by nitine concentrations were significantly higher in the exercise (Table 5). On the other hand, lipid content in supplemented rats compared to control rats (Control vs. muscle was not significantly influenced by supplemen- BE), whereas in exercised groups they were not appre- tation but a slight decrease was observed in the exer- ciably affected by supplementation (CE vs. BE CE). Lev- cised groups. els of urinary excretion of carnitine were significantly Fatty acid composition in each organ (visceral fat, higher in supplemented rats than in nonsupplemented muscle, liver, serum) was not changed by exercise or rats with and without exercise. These results were in beef extract supplement, indicating that fatty acid re- agreement with those of Siliprandi et al. (25). Nuesch et Beef Extract Diet and Muscle Fiber Type 187
Fig. 1. Carnitine palmitoyl transferase II (A) and fatty acid synthase (B) activities in the liver of male rats fed the nonsup- plemented diet or supplemented diet with beef extract. Values are means SD (n 5–6). Bars with different superscripts are significantly different (p 0.05). Con, control; BE, beef extract supplemented; CE, compulsory exercise.
Table 7. Muscle weights of extensor digitorum longus (EDL) and soleus.
EDL Soleus EDL Soleus Supplement Exercise (mg/100 g body weight) (mg/100 g body weight) (mg) (mg)
Control 44.21 2.95a 40.04 3.60a 191.08 9.42 173.43 18.92a BE 48.25 3.74b 49.44 5.33b 190.36 19.39 194.30 14.77b CE 50.48 1.69b,c 53.33 3.60b 190.12 7.91 201.00 16.38b BE CE 52.61 3.57c 50.24 2.08b 185.38 10.35 177.20 8.68a
Statistical significance Supplement 0.035 NS1 NS NS Exercise 0.001 0.001 NS NS Interaction NS 0.002 NS 0.004
See the footnote to Table 3. al. also observed an increase of free carnitine concen- increased the relative weights (mg/100 g body weight) trations in the plasma and urine with and without exer- of EDL and soleus, while the muscle weights were also cise after taking L-carnitine orally (26). increased by exercise alone compared to the control CPT II and FAS activities in the liver group. However, muscle weights of the exercised rats Since CPT I is easy to denature by freeze-thawing, a were not significantly influenced by beef extract supple- precise measurement of the activity is generally difficult ment in either EDL or soleus. The absolute weight of (27). Therefore, the activity of CPT II in mitochondria soleus was significantly increased in the nonexercised isolated from rat liver, which is resistant to freezing, was supplemented rats compared with the control group measured in this experiment (Fig. 1A). Supplementa- (p 0.05). The absolute weight of EDL were not signifi- tion, exercise or both showed a trend of elevating the cantly changed among all groups (Table 7). CPT II activity, but there were not significant differences Myofibrils with a negative reaction for alkaline in the activity among these three treatments. ATPase and a positive for acid ATPase were designated The activity of FAS in mitochondria isolated from as type I and those with the reverse reactions as type II. liver, which is responsible for fatty acid synthesis, was Figure 2 shows enzyme activities in serial transverse also measured. Exercise significantly reduced the FAS sections of EDL in rats without exercise. A large number activity, as evidenced by Fig. 1B. The most pronounced of myofibrils showing a positive reaction for acid ATPase reduction was in the case of the nonsupplemented-exer- (pH 4.3) was observed in supplemented rats (B-1). The cised group. On the other hand, adding beef extract strong NADH-DH activity in EDL was indicated by type tended to diminish the effect of exercise on the FAS I or IIa fiber that contained many mitochondria. This activity. type was observed frequently in the specimen of nonex- Muscle weight and muscle fiber type analysis ercised, supplemented rats (B-2). Type IIb fibers showed Table 7 shows the combined effect of exercise and weak NADH-DH activity. In the specimen of control rats beef extract supplement on the relative muscle weights (nonexercised, nonsupplemented), type I fibers were of soleus (slow-twitch, oxidative) and EDL (fast-twitch, observed at lower frequency (A-1), but type IIb were glycolytic) after 4 weeks feeding. In nonexercised abundant (A-2). groups, beef extract supplementation significantly On the basis of histochemical observations in Fig. 2, 188 YOSHIHARA H et al.
Fig. 2. Histochemical staining photographs of extensor digitorum longus (EDL) muscle. Fed with control diet (A) and experimental diet including beef extract (B) at nonexercised group. A-1, B-1: ATPase (pH 4.3); A-2, B-2: nicotine amide adenine dinucleotide dehydrogenase (NADH-DH). the distributions of three fiber types in each muscle fiber types was less than the additive of their individual were estimated by two observers who were blinded to effects, indicating the lack of synergetic effect between this experimental aim. In soleus muscle of nonexer- them. In the case of soleus, no treatment differences in cised, nonsupplemented rats (control), type I and type the fiber type compositions were detected at the end of IIa fibers accounted for approximately 93% and 7%, the feeding experiment (Fig. 3B). This result was unex- respectively (Fig. 3B). In EDL, the population was 5, 41 pected, but might be due to a physiological characteris- and 54% for type I, IIa and IIb, respectively (Fig. 3A). tic of soleus itself that specializes in oxidative metabo- Beef extract supplement increased the type I and type lism. IIa fibers in EDL muscle of nonexercised rats with a SDS-polyacrylamide gel electrophoresis, which is compensatory decrease in type IIb fibers (Fig. 3C–E). As based on the differences in distributions of MyHC iso- a result, the mean percent of fibers classified as type I forms is a commonly used procedure for determining and type IIa was increased from 5.0% (control) to 7.0% fiber types in mammalian skeletal muscles. Figure 4 and from 41% (control) to 49% in nonexcercised, sup- illustrates the typical MyHC electrophoresis pattern in plemented rats. The combination of exercise and sup- the EDL. The mean percent of type IIb MyHC distribu- plementation induced an increase in type IIa with a tion tended to decrease from 56.4 to 50.1%, while type compensatory decrease in type IIb as well in the case of IIa and IIx were slightly increased and type I increased supplemented rats without exercise. However, the significantly from 2.0 to 4.6% in nonexcercised, supple- increase in the percentage of type I was small (from mented rats. No synergetic effect between beef extract 5.0% to 6.4%). The effect of the combination of beef and exercise was observed (Fig. 5). Regardless of treat- extract supplement and exercise on the distributions of ments, diffrences in MyHC compositions of soleus were Beef Extract Diet and Muscle Fiber Type 189
Fig. 3. Percentage distribution of histochemical fiber types in the EDL (A) and soleus (B) in male rats after feeding. Type I, solid bars; type IIa, hatched bars; type IIb, dotted bars. Percentage distributions of histochemical fiber type I (C), type IIa (D) and type IIb (E) in extensor digitorum longus (EDL) in male rats after feeding. Bars with different superscripts are sig- nificantly different (p 0.05). Values are means SD (n 6). Percentage distribution of each fiber type was calculated from the number of each type counted from a total 600–1,000 fibers in muscle (n 4–5). Con, control; BE, beef extract supple- mented; CE, compulsory exercise.
DISCUSSION Present results indicate that beef extract supplement caused an increase in leg muscle weight and modified the muscle fiber type, particularly in EDL (Table 7, Figs. 2–5). It was concluded that the former finding was not due to the accumulation of lipids in the tissue, but rather due to the hypertrophy of muscle, judging from Fig. 4. Myosin heavy chain (MyHC) region of Coo- the fact that neither the supplement nor exercise massie-blue stained SDS-PAGE gel of EDL muscle. Con, control; BE, beef extract supplemented; CE, compulsory affected lipid content of those muscles (Table 5). Exer- exercise. cise or beef extract supplementation may promote the growth of muscle by inducing an increase in serum concentrations of growth factors such as insulin-like not detected as in the case of histochemical observa- growth factor (IGF)-I and S-myotrophin, which stimu- tions (data not shown). late the synthesis of proteins and nucleic acids (28, 29). However, this idea does not provide a clear answer to 190 YOSHIHARA H et al.
Based on the daily food consumption by the rats in the present experiment (Table 3), carnitine intake was estimated to be about 200 mg/kg rat body weight, which did not seem to be a toxic dosage (32). Analysis of Table 6 showed an increase in carnitine in the serum and urine with beef extract supplement, indicating that the dietary carnitine was absorbed by rats to some extent. Serum carnitine concentration in BE CE were not appreciably affected by the supplement. However, the previous reports showed that carnitine in serum was less susceptible than in urine (25, 33). Beef extract supplement alone decreased liver and visceral fat weights of rats, reflecting the promotion of fat metabolism, although the effect was not as large as that of exercise. This might be due to the effect of car- nitine in beef extract. Hongu and Sachan (32) reported that fat pad weights and total lipids of epididymal, inguinal and perirenal regions were significantly reduced by the supplement as well as by exercise although they used caffeine and choline as a supple- ment besides carnitine. Shimura and Hasegawa (34) reported that levels of cholesterol and triglycerides in liver of rats fed a high fat diet decreased with adminis- tration of carnitine. The slightly lower food intake in BE than Control (92.4%) might affect body fat level to some extent. However, its effect would be little because Ji et al. reported that diet restricted to 95% for 9 wk did not affect the percentage of body fat in rats (35). To further account for the effect of carnitine on fat metabolism, we measured CPT II and FAS activities in the liver. CPT complex is composed of CPT I, located on the outer surface of the outer mitochondrial membrane, acylcarnitine translocase and CPT II, located on the Fig. 5. The percentage composition of adult myosin inner mitochondrial membrane, and plays a part in the heavy chain (MyHC) isoform from EDL muscle in male transfer of long-chain fatty acids in the mitochondria rats after feeding. (A) MyHC I, (B) MyHC IIa and IIx, for subsequent -oxidation. As a result, CPT II activity and (C) MyHC IIb. Values are means SD (n 4–6). was found to increase significantly with supplement Bars with different superscripts are significantly differ- alone or with exercise alone as shown in Fig. 1A. ent (p 0.05). Recently, Starritt et al. (27) measured CPT I activity in human skeletal muscle, and reported that the maximal CPT I activity was higher in the exercised group than in the result in Table 7. the nonexercised group. Since their results suggested It is difficult to immediately explain what substances that the increase in CPT I activity might explain the contained in beef extracts effect physiological change in promotion of the oxidation of fatty acids, the observed this experiment. Beef extract contains a large number of increase of CPT II activity with the supplement alone physiologically active substances, L-carnitine, carnos- may also explain the contribution of carnitine to the ine, taurine and so on (Table 2). Each of these sub- loss in fat weight of rats (Table 4). In contrast, exercise stances might affect skeletal muscle property separately significantly decreased FAS activity. This result was in or cooperatively, but it is possible that unknown minor agreement with the report of Griffiths et al. (36) that substances specific to beef extract affected it. prolonged exercise suppressed induction of both FAS L-Carnitine, which is highly present in beef, may be mRNA and enzyme activity of rats fed a high carbohy- one of the candidates for nutritional muscle fiber type drate diet. However, in the present experiment the sup- transformation. L-Carnitine plays an important role in plement showed a trend to diminish the effect of exer- fat metabolism because it promotes the mitochondrial cise on the FAS activity. The reason is unclear, but uptake of long-chain fatty acids for -oxidation coupled carnitine seems to affect only the oxidation of fatty with ATP production (30). Therefore, carnitine supple- acids, not their synthesis. mentation is clinically used to improve muscle function As described above, the diet with beef extract evi- during endurance exercise (31). However, whether or dently increased the carnitine content in the serum and not L-carnitine intake has an effect on these perfor- urine, and mitochondrial CPT II activity in the liver. mances is not fully understood. Furthermore, fat pad mass in rats was surely decreased Beef Extract Diet and Muscle Fiber Type 191 by beef extract intake. Thus, it was appreciated that the white fiber (probably type IIb rich fiber) was affected these results were due to the effect of carnitine as a more greatly by starvation than the red fiber (probably facilitator of fatty acid oxidation. However, the combi- type I or type IIa rich fiber). This report suggested the nation effect of carnitine and other nutrients in beef possibility that the decrease in glycogen content in the extract could not be ruled out as suggested by Askew et fibers during starvation led to conversion of muscle al. (37), which needs to be examined in future studies. fiber type in response to the extreme nutritional envi- For example, it is reported that taurine also decreased ronment. Recently Higashiura et al. (14) reported that body fat (38). high fructose-fed rats (insulin-resistance diabetogenic Individual muscle contains fiber types in different diet) increased the percentage of type IIa at the expense combinations. For example, the adult soleus muscle of a decrease in type I in soleus, suggesting the alter- contains type I fibers in addition to relatively small ation of muscle fiber composition linkage to insulin amounts of type IIa and type IIb fibers, while the adult resistance and hypertension. Kikuzawa et al. reported EDL muscle contains only a few type I fibers, the others that ingestion of L-arabinose increased type I fibers in being type II fibers. Histochemical observation and the rectus femoris muscle in rats (40). However, these MyHC composition of the soleus and EDL muscles facts were not always useful as an explanation of our showed the obvious evidence that the muscle fiber type present results because we studied the effect of beef could be changeable in response to the nutrition envi- extract as a supplement but not as the energy source. ronment (Figs. 2–5). In particular, it was quite reason- Our understanding of fiber type-specific genes and the able that the dietary carnitine participating the aerobic molecular mechanism regulating adult skeletal muscle ATP production in muscle contributed to conversion of fiber transformation has recently been advanced by the fast-glycolytic EDL into slow-oxidative EDL. How- experiments in which cross-innervation or electrical ever, unexpectedly no synergistic effect of dietary sup- stimulation can convert one myofiber subtype to plement and exercise was observed. This result may another. At present, it is proposed that fiber-type-spe- appear surprising since exercise leads to the transfor- cific gene expression is regulated by a signaling path- mation of muscle fiber types (6, 7), but it might be inter- way that involves calcineurin (calcium-regulated preted that EDL muscle itself restrained over response serine/threonine phosphatase) which controls the func- during prolonged exercise by limiting uptake of fatty tional activity of the nuclear family of activated T cell acids. It was also conceivable that the intensity or (NFAT) (41, 42). This model is attractive for an under- period of exercise in this experiment was not so strong standing of our finding, that is nutrition-induced fiber as to cause notable change of skeletal muscle fiber transformation, although the mechanisms of molecular types. Sullivan et al. showed that exercise training, response may be different. which was 18–20 m/min treadmill running (60 min) Our present results showed strong evidence that the 5 d/wk for 8 wk, resulted in a decrease in the percent- muscle cell has a capacity to respond and adapt to the age of type IIb MyHC (untrained, 43.0%; trained, nutritional state as well as physical stimulation. Further 29.9%) and an increase in the percentage of type IIa studies are required to identify the nutritional constitu- MyHC (untrained, 14.0%; trained, 17.5%) in plantaris ent(s) in beef extract that converts fast type fiber to slow muscle in rats (16). Therefore, it is necessary to exam- type fiber, and then to study the mechanisms of molec- ine high intensity or long term effects of exercise train- ular response for the cellular adaptations associated ing in order to check the synergistic effect of beef with the nutrition. To accomplish these aims, experi- extract and exercise. Interestingly, neither supplement ments are now in progress in our laboratory. nor exercise affected the proportion of fiber types in the soleus muscle. Brass et al. (31) reported that EDL and Acknowledgments soleus muscles took up carnitine from the blood to the We thank Dr. T. P. Carr, University of Nebraska, for same extent, but in only soleus muscle was the his critical reading of this manuscript. increased accumulation of glycogen by carnitine This work was supported by Ito Memorial Founda- administration observed. This suggested that soleus tion, Tokyo, Japan. muscle could effectively use the energy of ATP produced REFERENCES by -oxidation of fatty acids, resulting in saving the consumption of stored glycogen. The soleus muscle is 1)Jansen GR. 1984. Assessment of the need for regulating intrinsically a typical slow-oxidative red muscle, so that the protein quality of meat and poultry products. Am J it may not be required for the change to oxidative state Clin Nutr 40: 685–703. 2)Willis PE, Chadan S, Baracos V, Parkhouse WS. 1997. any more. Acute exercise attenuates age-associated resistance to It is important to discuss how nutrients (i.e. beef insulin-like growth factor I. Am J Physiol 272: E397– extract including carnitine) might specifically induce 404. the transformation from type IIb to type I and type IIa 3) Omoto K, Nakagawa Y, Iwashita K, Hayashi T, Johno K, in EDL without exercise. There are few reports describ- Tuchida N, Fotovati A, Ito T. 1999. Combination effect ing the relationship between nutrition (diet) and the of protein nutrition and treadmill exercise on the con- fiber type characteristics of skeletal muscle. Dietary centration of serum IGF-1 and transferrin in growing restriction failed to change the fiber types of soleus and male rats. Proceedings of the 45th International Con- EDL in rats or pigs (15, 39). Suzuki (13) reported that gress of Meat Science and Technology, p 706–707. 192 YOSHIHARA H et al.
4) Essen-Gustavsson B, Karlsson A, Lundström K, Enfält nutritional status on the activity of lipogenic enzymes in AC. 1994. Intramuscular fat and muscle fibre lipid con- primary monolayer cultures of rat hepatocytes. Biochem tents in halothane-gene-free pigs fed high or low protein J 235: 87–90. diets and its relation to meat quality. Meat Sci 38: 269– 22) Morita S, Iwamoto H, Fukumitsu Y, Gotoh T, Nishimura 277. S, Ono Y. 1999. Heterogeneous composition of his- 5) Greenhaff PL, Nevill ME, Soderlund K, Bodin K, Boobis tochemical fibre types in the different parts of M. longis- LH, Williams C, Hultman E. 1994. The metabolic simus thoracis from Mishima (Japanese native) steers. responses of human type I and II muscle fibres during Meat Sci 54: 59–63. maximal treadmill sprinting. J Physiol 478 (Pt 1): 149– 23) Brooke MH, Kaiser KK. 1970. Muscle fiber types: how 155. many and what kind? Arch Neurol 23: 369–379. 6) Close R. 1969. Dynamic properties of fast and slow skel- 24)Talmadge RJ, Roy RR. 1993. Electrophoretic separation etal muscles of the rat after nerve cross-union. J Physiol of rat skeletal muscle myosin heavy-chain isoforms. J 204: 331–346. Appl Physiol 75: 2337–2340. 7) Salmons S, Sreter FA. 1976. Significance of impulse 25) Siliprandi N, Di Lisa F, Pieralisi G, Ripari P, Maccari F, activity in the transformation of skeletal muscle type. Menabo R, Giamberardino MA, Vecchiet L. 1990. Meta- Nature 263: 30–34. bolic changes induced by maximal exercise in human 8) Schantz P, Henriksson J. 1983. Increases in myofibrillar subjects following L-carnitine administration. Biochim ATPase intermediate human skeletal muscle fibers in Biophys Acta 1034: 17–21. response to endurance training. Muscle Nerve 6: 553– 26) Nuesch R, Rossetto M, Martina B. 1999. Plasma and 556. urine carnitine concentrations in well-trained athletes 9) Emler CA, Schalch DS. 1987. Nutritionally-induced at rest and after exercise. Influence of L-carnitine intake. changes in hepatic insulin-like growth factor I (IGF-I) Drugs Exp Clin Res 25: 167–171. gene expression in rats. Endocrinology 120: 832–834. 27) Starritt EC, Howlett RA, Heigenhauser GJ, Spriet LL. 10) Goodridge AG. 1987. Dietary regulation of gene expres- 2000. Sensitivity of CPT I to malonyl-CoA in trained sion: enzymes involved in carbohydrate and lipid metab- and untrained human skeletal muscle. Am J Physiol olism. Annu Rev Nutr 7: 157–185. Endocrinol Metab 278: E462–468. 11) Hillgartner FB, Salati LM, Goodridge AG. 1995. Physio- 28) Musaro A, McCullagh KJ, Naya FJ, Olson EN, Rosenthal logical and molecular mechanisms involved in nutri- N. 1999. IGF-1 induces skeletal myocyte hypertrophy tional regulation of fatty acid synthesis. Physiol Rev 75: through calcineurin in association with GATA-2 and 47–76. NF-ATc1. Nature 400: 581–585. 12) Constantin-Teodosiu D, Howell S, Greenhaff PL. 1996. 29)Hayashi T, Ogawa T, Sato M, Tsuchida N, Fotovati A, Carnitine metabolism in human muscle fiber types dur- Iwamoto H, Ikeuchi Y, Cassens RG, Ito T. 2001. S-Myo- ing submaximal dynamic exercise. J Appl Physiol 80: trophin promotes the hypertrophy of myotube as insu- 1061–1064. lin-like growth factor-I does. Int J Biochem Cell Biol 33: 13) Suzuki A. 1973. Histochemical observations of individ- 831–838. ual skeletal muscle fibers in starved sheep. Jpn J Zootech 30)Rebouche CJ, Seim H. 1998. Carnitine metabolism and Sci 44: 50–58. its regulation in microorganisms and mammals. Annu 14) Higashiura K, Ura N, Takada T, Agata J, Yoshida H, Rev Nutr 18: 39–61. Miyazaki Y, Shimamoto K. 1999. Alteration of muscle 31)Brass EP, Scarrow AM, Ruff LJ, Masterson KA, Van Lun- fiber composition linking to insulin resistance and teren E. 1993. Carnitine delays rat skeletal muscle hypertension in fructose-fed rats. Am J Hypertens 12: fatigue in vitro. J Appl Physiol 75: 1595–1600. 596–602. 32) Hongu N, Sachan DS. 2000. Caffeine, carnitine and 15) Harrison AP, Rowlerson AM, Dauncey MJ. 1996. Selec- choline supplementation of rats decreases body fat and tive regulation of myofiber differentiation by energy sta- serum leptin concentration as does exercise. J Nutr 130: tus during postnatal development. Am J Physiol 270: 152–157. R667–674. 33) Hongu N, Sachan DS. 2003. Carnitine and choline sup- 16) Sullivan VK, Powers SK, Criswell DS, Tumer N, Laro- plementation with exercise alter carnitine profiles, bio- chelle JS, Lowenthal D. 1995. Myosin heavy chain com- chemical markers of fat metabolism and serum leptin position in young and old rat skeletal muscle: effects of concentration in healthy women. J Nutr 133: 84–89. endurance exercise. J Appl Physiol 78: 2115–2120. 34) Shimura S, Hasegawa T. 1993. Changes of lipid concen- 17)Folch J, Lees M, Sloane Stanley GH. 1957. A simple trations in liver and serum by administration of car- method for the isolation and purification of total lipides nitine added diets in rats. J Vet Med Sci 55: 845–847. from animal tissues. J Biol Chem 226: 497–509. 35) Ji LL, Doan TD, Lennon DL, Nagle FJ, Lardy HA. 1987. 18)Kawahara S, Inoue K, Hayashi T, Masuda Y, Ito T. 1997. The effects of beta-adrenergic blockade on body compo- Lipids easy to accumulate in the liver and lipids hard to sition in free-fed and diet-restricted rats. J Am Coll Nutr accumulate in it. Nutr Res 17: 1013–1023. 6: 175–177. 19)Bradford MM. 1976. A rapid and sensitive method for 36) Griffiths MA, Fiebig R, Gore MT, Baker DH, Esser K, the quantitation of microgram quantities of protein uti- Oscai L, Ji LL. 1996. Exercise down-regulates hepatic lizing the principle of protein-dye binding. Anal Biochem lipogenic enzymes in food-deprived and refed rats. J Nutr 72: 248–254. 126: 1959–1971. 20) Markwell MA, McGroarty EJ, Bieber LL, Tolbert NE. 37) Askew EW, Hecker AL, Wise WR Jr. 1977. Dietary car- 1973. The subcellular distribution of carnitine acyl- nitine and adipose tissue turnover rate in exercise transferases in mammalian liver and kidney. A new per- trained rats. J Nutr 107: 132–142. oxisomal enzyme. J Biol Chem 248: 3426–3432. 38) Harada N, Ninomiya C, Osako Y, Morishima M, Mawa- 21)Kelley DS, Nelson GJ, Hunt JE. 1986. Effect of prior tari K, Takahashi A, Nakaya Y. 2004. Taurine alters res- Beef Extract Diet and Muscle Fiber Type 193
piratory gas exchange and nutrient metabolism in type C, Shelton JM, Wu H, Zhu W, Bassel-Duby R, Williams 2 diabetic rats. Obes Res 12: 1077–1084. RS. 1998. A calcineurin-dependent transcriptional 39)Yamaguchi A, Horio Y, Sakuma K, Katsuta S. 1993. The pathway controls skeletal muscle fiber type. Genes Dev effect of nutrition on the size and proportion of muscle 12: 2499–2509. fibre types during growth. J Anat 182 (Pt 1): 29–36. 42) Serrano AL, Murgia M, Pallafacchina G, Calabria E, 40) Kikuzawa A, Tanaka N, Ichikawa M, Miyasaka K. 2005. Coniglio P, Lomo T, Schiaffino S. 2001. Calcineurin con- Effects of L-arabinose on skeletal muscle fiber composi- trols nerve activity-dependent specification ofslow skele- tion. J Jpn Soc Nutr Food Sci 58: 51–57. tal muscle fibers but not muscle growth. Proc Natl Acad 41) Chin ER, Olson EN, Richardson JA, Yang Q, Humphries Sci USA 98: 13108–13113.