THE ANATOMICAL RECORD 218:267-274 (1987) Physiological Role of Skeletal Muscle Glycogen in Starved Mice MINORU SAKAIDA, JUN WATANABE, SHINSUKE KANAMURA, HIROHIKO TOKUNAGA, AND RYOKEI OGAWA Department of Anatomy and Orthopedics, Kansai Medical University, Fumizonecho 1, Moriguchi, Osaka, 570 Japan ABSTRACT To study the physiological role of skeletal muscle glycogen in starved animals, effects of starvation on glycogen and glycogen phosphorylase (EC 220.127.116.11.) activity were studied in muscle fibers (morphologic study) and in whole muscles (biochemical study) of the rectus femoris muscle of mouse. Glycogen content in the liver of the starved animals was also measured. PAS reaction, strong in muscle fibers of fed animals, became weak predominantly in type IIB fibers after 2 days and almost disappeared after 4 days of starvation. Glycogen particles, numerous in the sarcoplasm between myofibrils of muscle fibers, decreased markedly predominantly in type IIB fibers after 2 days and almost disappeared after 4 days. Phosphorylase a activity, undetected in fibers of fed mice, appeared weak in type IIB fibers and very weak in type IIA fibers after 2 days and became moderate in type IIB fibers and weak in type IIA fibers after 4 days. Muscle glycogen content did not differ by 16 hours from the values of corresponding fed animals. However, liver glycogen content had already decreased after 8 hours and markedly so after 12 hours. The results support our hypothesis--"skeletal muscle glycogen is used for maintaining the blood glucose level in starved mice" (Hirose et al.: Anat. Rec., 216:133-138, 1986)-and show that type IIB fibers play a main role in maintaining the glucose level and that muscle glycogen is utilized after depletion of liver glycogen. Glycogen in skeletal muscle is utilized as the energy source for muscle contraction. However, we recently observed that in the skeletal muscle (rectus femoris muscle) of starved mice glucose 6-phosphatase (GGPase) and glycogen phosphorylase a activities increase and glycogen content decreases, and we supposed that the role of the increased G6Pase in skeletal muscle fibers of starved mice is to release glucose into the blood by hydrolyzing glucose 6-phosphate (G6P) produced through the increased phosphorylase activity from their glycogen (Hirose et al., 1986). In the present study, to obtain further evidence supporting this hypothesis, we examined 1) whether the changes in phosphorylase a activity and glycogen content occur predominantly in any fiber type of the rectus femoris muscle (histochemical and ultrastructural studies) and 2) whether glycogen mobilization in the skeletal muscle occurs before or after the glycogen mobilization in the liver, which is the greatest glucose supplier into the blood during starvation (biochemical study). phorylase activity assay), 8, 12, 16, 24, 48, or 96 hours (glycogen content assay), and 12, 24, 48, and 96 hours (blood glucose assay). Starvation was started at 10 A.M. for morphologic experiments and a t 6 or 10 A.M. for chemical experiments. Experiments of starvation for 5 days or more were not carried out because about a half of the animals died at 5 days of starvation. The animals were killed by cervical dislocation. Light Microscopy Rectus femoris muscles were removed quickly and frozen on dry ice. Serial transverse sections (10 pm in thickness) were cut in a cryostat through the midbelly portion of the muscles. Classification of fiber types Serial fresh frozen sections were incubated for 1)acidstable myofibrilar adenosine triphosphatase (ATPase) activity (Padykula and Herman, 1955; as modified by Brooke and Kaiser, 1970), for 2) alkaline-stable ATPase activity (Guth and Samaha, 19701, and for 3) succinate dehydrogenase (SDH) activity (Barka and Anderson, MATERIALS AND METHODS 1963). Muscle fibers showing dark reaction for acid-staTwo hundred thirty male ddY mice, 2 months old, ble ATPase and light reaction for alkaline-stable ATwere used. The animals were divided into two groups and individually housed in cages. One group of animals had free access to food and water prior to the experiReceived August 12,1986; accepted January 27,1987. ments (fed animals) and the other group of animals Address reprint requests to Dr. Shinsuke Kanamura, Department (starved animals) was given only water for 2 or 4 days of Anatomy, Kansai Medical University, Fumizono-cho 1,Moriguchi, (morphologic experiments), 24, 48, or 96 hours (phos- Osaka, 570 Japan. 0 1987 ALAN R. LISS, INC 268 M. SAKAIDA ET AL. 269 MUSCLE GLYCOGEN Electron Microscopy Pase were classified as type I, and the fibers showing light reaction for acid-stable ATPase and dark reaction for alkaline-stable ATPase were classified as type 11. Type I1 fibers were subclassified into type IIA if they had intense SDH reaction, into type IIB if they showed weak SDH reaction, and into atypical type 11if they had moderate SDH reaction. Rectus femoris muscles were quickly removed and cut into small pieces. The blocks were immersed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) a t 4°C for 2.5 hours, washed with 8% sucrose in 0.1 M phosphate buffer (pH 7.4) at 4°C for 20 minutes, postfixed with buffered 1% osmium tetroxide at 4°C for 2 hours, dehydrated in graded ethanol, and embedded in Glycogen Spurr's resin. Longitudinal ultrathin sections were Fresh frozen sections were stained with the periodic stained with uranyl acetate and lead citrate and examacid Schiff (PAS) reaction and mounted in glycerin-jelly. ined in a JEM-100s electron microscope. For control, the sections were preincubated with saliva The fiber types at the electron microscope level were a t 37 "C for 30 minutes and stained with PAS reaction. identified on the basis of the size and distribution of mitochondria in the muscle cells (Maxwell et al., 1977; Phosphorylase activity Armstrong, 1980). For histochemical demonstration of total phosphoryBiochemical Methods lase activity, fresh frozen sections were incubated in the reaction medium containing 10.6 mM glucose l-phos- Glycogen content phate (GlP), 2.3 mM adenosine monophosphate (AMP), Rectus femoris muscles or livers were digested with 80 pg/ml glycogen, 3 mM ethylenediaminetetraacetic 20 volumes of 30%KOH at 95°C for 20 minutes. Glycoacid (EDTA), 5 mM NaF, and 0.1 M acetate buffer (pH gen in the digest was purified with ethanol according to 5.7) at 20°C for 1hour (Takeuchi, 1956). For phosphory- the method of Hassid and Abraham (1957), and the glylase a (active form) activity, the sections were incubated cogen amount was estimated by the anthrone reaction in the medium lacking AMP (Godlewski, 1963). The (Hassid and Abraham, 1957). The content of glycogen sections were immersed in 10% Gram solution for 3 was expressed as milligrams glycogen per gram of wet minutes and mounted in iodoglycerin. The sections were tissue. incubated in the medium lacking G1P (for total activity) or lacking G1P and AMP (for phosphorylase a activity) Phosphorylase activity as controls. Rectus femoris muscles were homogenized a t 4°C with 20 volumes of 0.2 M Tris-maleate buffer (pH 6.1) conFigs. 1-12. Serial transverse sections of the rectus femoris muscles. taining 50 mM NaF and 5 mM EDTA for 1 minute at A, type IIA fiber; B, type IIB fiber; X, atypical type I1 fiber. x 120. 2,000 rpm. The homogenate was centrifuged a t 4°C for 10 minutes at 3,OOOg. Phosphorylase activity was asFigs. 1-6. Fed (10 A.M.). sayed by the method of Saheki et al. (1971). An aliquot Fig. 1. Incubated for adenosine triphosphatase (ATPase) activity (0.05 ml) of the supernatant was incubated with 0.15 ml after acid preincubation. of the medium (40 mM GlP, 100 mg/ml glycogen, 13 mM NaF, 1.3 mM EDTA, 50 mM Tris-maleate, pH 6.1) in Fig. 2. Incubated for ATPase activity after alkaline preincubation. the presence or absence of 1 mM AMP a t 30°C for 5 Fig. 3. Incubated for succinate dehydrogenase activity. minutes. The inorganic phosphorus released was determined by Phosphor C-test. The reaction in the presence Fig. 4. PAS reaction. The staining reaction is strong. of AMP shows total phosphorylase activity and the reFig. 5. Histochemical demonstration of total phosphorylase activity. action in the absence of AMP shows phosphorylase a The staining reaction is strong in type IIA fibers (A) and moderate in activity. The activity was expressed a s micrograms type IIB (B) and atypical type I1 (X) fibers. phosphorus liberated per minute per gram of wet tissue. Fig. 6. Histochemical demonstration of phosphorylase a activity. No staining reaction is seen. Blood glucose Animals were anesthetized with chloroform. Blood was taken from the right atrium by syringe and centrifuged at 4°C for 30 minutes at 3,OOOg. Then, glucose in the Fig. 7. Succinate dehydrogenase activity. serum was assayed by the mutarotase-glucose oxidase Fig. 8. PAS reaction. The staining reaction becomes weak in type method (Miwa et al., 1972). IIB fibers (B) but moderate in type IIA (A) and atypical type I1 (XI Figs. 7, 8. Starved for 2 days (10 A.M.). fibers. Figs. 9-12. Starved for 4 days (10A.M.). Fig. 9. Succinate dehydrogenase activity. Fig. 10. PAS reaction. The staining reaction almost disappears in type IIB (B) and atypical type I1 (X) fibers although it remains very weak in type IIA fibers (A). Fig. 11. Total phosphorylase activity. The staining reaction is strong in type IIB fibers (B), atypical type I1 fibers (X), and few type IIA fibers mostly moderate in type IIA fibers (A). (*) and Fig. 12. Phosphorylase a activity. The staining reaction is moderate in type IIB (B) and atypical type I1 (X) fibers and weak in type IIA fibers (A). Statistical analysis Results were expressed as means k S.E. Student's ttest was used for comparison o€ values between experimental groups. RESULTS The starved mice became gradually less active than the fed mice, and they appeared curled up after 2 or more days of starvation. Average body weight of fed mice was 38.3 k 0.6 g (means + S.E., n = 32). The weight decreased after 2 days (33.9 0.4 g, n = 11)( P < .01) and 4 days of starvation (29.8 0.6 g, n = 11)( P < .01). 270 M. SAKAIDA ET AL. Figs. 13-15. Electron micrographs of muscle fibers in rectus femoris muscles of mice. Fig. 14. Portion of a type IIB fiber from a n animal starved for 2 days. A few glycogen particles are seen in the sarcoplasm between myofibrils at the I bands, whereas the particles are rare or absent in other Fig. 13. Portions of type IIB fibers from fed animals (10 A.M.). A Glycogen particles are abundant in sarcoplasm near the triads and between myofibrils. X30,OOO. B: High-power magnification. Numerous particles, 25-30 nm in diameter, are observed in sarcoplasm near the triad. Some particles are also present between myofilaments (arrows). portions of sarcoplasm. ~ 3 0 , 0 0 0 . ~50,000. Fig. 15. Portions of muscle fibers from animals starved for 4 days. A Portion of a type IIB fiber. Glycogen particles are absent. ~ 3 0 , 0 0 0B: . Portion of a type IIA fiber. A few particles are seen in sarcoplasm between myofibrils (arrows). ~ 3 0 , 0 0 0 . 271 MUSCLE GLYCOGEN Morphologic Results Distribution of fiber types h ?!v) v) .c Rectus femoris muscles contained numerous type IIB fibers (fed: 63.1 rf: 2.5%, starved for 2 days: 65.8 rf: 3.4%, starved for 4 days: 62.9 & 1.8%, n = 4), a few type IIA fibers (fed 25.4 2.3%, starved for 2 days: 26.3 2.3, starved for 4 days: 26.0 & 1.5%), a few atypical type I1 fibers (fed: 11.5 & 1.3%, starved for 2 days: 7.9 rf: 2.7%, starved for 4 days: 11.1 rf: 1.4%), and no type I fibers (Figs. 1-3,7,9). -E" PAS reaction % I - The staining reaction was strong in muscle fibers of fed mice (Fig. 4). The reaction, however, became weak in type IIB fibers and moderate in type IIA and atypical type I1 fibers after 2 days of starvation (Fig. 8).Although very weak reaction was sometimes seen in type IIA fibers, the staining almost disappeared after 4 days of starvation (Fig. 10). -U0x + a, 3 s c 2- C a, * C 0 0 C 0 Glycogen phosphorylase activity In muscle fibers of fed mice, the staining reaction for total phosphorylase activity was strong in type IIA fibers and moderate in type IIB and atypical type I1 fibers (Fig. 5). Phosphorylase a (active form) activity was undetected in muscle fibers of fed mice (Fig. 6). After 2 days of starvation, the reaction for the total activity was somewhat intense, although it was slightly stronger in type IIB and atypical type I1 fibers than in type IIA fibers. The reaction for phosphorylase a activity appeared very weak in type IIA fibers and weak in type IIB and atypical type I1 fibers. After 4 days of starvation, the reaction for the total activity became intense, although it was generally stronger in type IIB fibers than in type IIA fibers (Fig. 11). The reaction for phosphorylase a activity became moderate in type IIB and atypical type I1 fibers and weak in type IIA fibers (Fig. 12). Electron microscopy Electron microscopic observations were carried out on typical type IIA and IIB fibers, because atypical type I1 fibers could not be identified under the electron microscope. In fed animals, numerous glycogen particles (beta particles) were observed in the sarcoplasm between myofibrils of all muscle fibers, particularly near the triad (Fig. 13A,B). A few particles were occasionally present in spaces between myofilaments. After 2 days of starvation, the particles decreased markedly, although a few were still seen in the sarcoplasm between myofibrils a t the I-bands (Fig. 14). The remaining particles appeared more numerous in type IIA fibers than in type IIB fibers. After 4 days of starvation, the particles almost disappeared (Fig. 15A), although a few granules were occasionally found in the sarcoplasm between myofibrils of some type IIA fibers (Fig. 15B). Biochemical Results Glycogen content Muscle glycogen content did not differ from values of corresponding fed animals by 12 hours of starvation, although the value decreased to 40% of that of animals at the start of starvation (Fig. 16). After 24, 48, and 96 hours, the values became 23, 18, and 7% of that at the v) 8 12 16 2 2, 3 "I 6 Hours of Starvation Fig. 16. Glycogen content i n rectus femoris muscles of fed (open column) and starved (dotted column) mice. Animals were starved from 6:OO A.M. Each point represents t h e mean of four mice and standard error i s shown as a vertical line. The values of starved animals for 24 hours or longer are different significantly from values of corresponding fed animals ( P < .001,.001,,001).Values at 12,24,48, and 96 hours of starvation were not different from those of animals starved from 1O:OO AM. start of starvation-smaller than those of corresponding fed animals (P < . 001, .001, .001). However, liver glycogen content already decreased after 8 hours to 27% of that at the start of starvation ( P < . 02) and was 33% of that of corresponding fed animals (P <. 02) (Fig. 17). After 12 hours, the value became 3% of that at the start of starvation, the minimal level. There were no differences in values at 12, 24, 48, and 96 hours of starvation between animals starved from 6:OO A.M. and from 1O:OO A.M. Glycogen phosphorylase activity Total glycogen phosphorylase activity did not change during 96 hours of starvation. However, phosphorylase a activity increased between the start and 48 hours ( P < .01)and markedly between 48 and 96 hours of starvation (P <. 05) (Fig. 18). There were no differences in values between animals starved from 6:OO A.M. and from 1O:Oo A.M. Blood glucose The values decreased to 44% of that in fed animals after 24 hours of starvation and then remained unchanged (Table 1). DISCUSSION In the present results, PAS reaction for glycogen and glycogen particles almost disappeared, and histochemical phosphorylase a activity increased in the muscle fibers during 4 days of starvation. These are consistent with our previous biochemical results on glycogen and phosphorylase a activity in the rectus femoris muscle of mice starved for 4 days and support our hypothesis on a physiological role of skeletal muscle glycogen in starved mice (Hirose et al., 1986). 272 M. SAKAIDA ET AL. h a, -2 (I) ffl (I) .c ffl .c c c 3 $ a, -? E" 20 v + C a, + C 0 0 c a, 10 T m 0 n T 0 > U tj o .-> -1 a, (I) m - 0 8 12 16 24 36 Hours of Starvation U Fig. 17. Glycogen content in the liver of fed (open column) and starved (dotted column) mice. Animals were starved from 6:OO A.M. Each point represents the mean of four animals and standard error is shown as a vertical line. The values of starved animals for 8 hours or longer are significantly different from values of corresponding fed animals. ( P < .02, .05, ,001, ,001,.001).Values at 12, 24,48,and 96 hours of starvation were not different from those of animals starved from 1O:OO A.M. Phosphorylase a activity was higher and PAS reaction for glycogen decreased more markedly in type IIB fibers than in type IIA fibers after 2 and 4 days of starvation. Type IIA fibers contain numerous mitochondria (Eisenberg and Kuda, 1976; Galvas et al., 1982) and the pathway of the aerobic glycolysis (Jobsis and Duffield, 1967; Burke et al., 1973), whereas type IIB fibers have few mitochondria (Eisenberg and Kuda, 1975; Galvas et al., 1982) and the anaerobic glycolysis occurs in the fibers (Burke et al., 1973). In type IIB fibers, G6P produced via the glycogenolysis is converted into lactate by the anaerobic glycolysis (Conn and Stumpf, 1972; Burke et al., 1973). Lactate thus produced is released into the blood and changed via the Cori-cycle in the liver and kidney into glucose that is released into the blood (Felig et al., 1970; Kusaka and Ui, 1977). On the other hand, in type IIA fibers, G6P is converted into pyruvate by the aerobic glycolysis (Jobsis and Duffield, 1967; Burke et al., 1973). During the process of the aerobic glycolysis, no metabolites that are sources of the blood glucose are released from type IIA fibers (Conn and Stumpf, 1972; Burke et al., 1973). Thus, via the anaerobic glycolysis and Coricycle, some of the glycogen in type IIB fibers is probably used for maintaining the blood glucose level in starved mice. Ichihara and Ui (1974) and Kusaka and Ui (1977) observed a decrease in glucose uptake and increase in lactate production in skeletal muscles of epinephrinetreated rats. The authors noticed these changes to be similar to changes in glucose and lactate in skeletal muscles of starved animals and supposed that epinephrine is the most important factor regulating glucose metabolism in skeletal muscle of starved animals. Further, the authors showed that, in rats injected with epinephrine, most of the lactate released from the skeleta1 muscle into the blood is not derived from its glyco- c a Hours of Starvation Fig. 18. Phosphorylase activity in rectus femoris muscles of fed and starved mice. Animals were starved from 6:OO A.M. Each point represents the mean of five animals and standard error is shown as a vertical line. Total activity (open histogram dotted histogram) is not changed but phosphorylase a activity (dotted histogram) increases between the start and 48 hours (P < ,011and markedly between 48 and 96 hours of starvation ( P < .05).These values were not different from those of animals starved from 1O:OO A.M. + TABLE 1. Blood glucose level (mgldl) of fed and starved mice Group Blood glucose Fed at 10 A.M. at 10 P.M. Starved for: 12 hours (at 10 P.M.) 24 hours (at 10 A.M.) 48 hours (at 10 A.M.) 96 hours (at 10 A.M.) 219.8 k 9.57 192.1 k 27.91 131.4 f 17.04* 96.8 f 2.69** 89.6 k 12.24** 99.3 + - 10.43** + Values are means S.E. for five animals. *P < .05, **P < ,001: Significantly different from the value of corresponding fed animals (Student's t-test). gen but from the blood glucose in spite of a marked decrease in muscle glycogen by increased glycogen phosphorylase activity (Ichihara and Ui, 1974; Kusaka and Ui, 1977). Therefore, if changes in glucose and lactate in skeletal muscles of epinephrine-treated animals are similar to those of starved animals, most of the G6P produced via the glycogenolysis in skeletal muscles of starved animals is possibly metabolized through a pathway other than the anaerobic glycolysis and Cori-cycle mentioned above. We recently found G6Pase activity and its marked increase by starvation in murine skeletal muscle fibers (Watanabe et al., 1986; Hirose et al., 1986). It is therefore likelv that G6P derived from d v cogen in skeletal muscle; of starved mice is largely hydrolyzed by G6Pase activity to produce glucose that is released directly into the blood. U " 273 MUSCLE GLYCOGEN Glycogen is more abundant in the white muscle containing numerous type I1 fibers than in the red muscle including numerous type I fibers (Beatty et al., 1963; Bocek et al., 1966).G6Pase activity is also higher in type I1 fibers than in type I fibers Watanabe et al., 19861, and it increases in both type IIA and type IIB fibers as a result of starvation (Hirose et al., 1986). As shown in the present study, decrease in glycogen and activation of phosphorylase activity during starvation occur predominantly in type IIB fibers. Thus, type IIB fibers possibly play a main role in releasing glucose into the blood in starved mice. ATP is shown to inhibit the activation of phosphorylase in the skeletal muscle of the rabbit (Sanner and Tron, 1975; Kamogawa and Fukui, 1975). ATP production is four times more in aerobic glycolysis than in anaerobic glycolysis (Fairly and Kilgour, 1966). Thus, high ATP concentration is probably a cause of less phosphorylase activation in type IIA fibers than in type IIB fibers. Other factors regulating phosphorylase activation-various hormones, such as insulin, glucagon, and glucocorticoids, and the concentration of G6P, Ca++, phosphorylase b-kinase, and others in the muscle fibers-are known. However, the detailed mechanism of the regulation by these factors is unknown. Liver glycogen decreased to 33%of that of corresponding fed animals after 8 hours and to the minimal level (3%of the value of fed animals at the start of starvation) after 12 hours. However, muscle glycogen content did not differ by 16 hours from values of corresponding fed animals, and it decreased gradually and became smaller than those of corresponding fed animals after 24, 48, and 96 hours. Thus, glycogen decrease in the liver precedes that in the skeletal muscle; glycogen in the muscle is mobilized after its depletion in the liver in starved mice. At the beginning of starvation, liver glycogen maintains the blood glucose level and, after depletion of liver glycogen, muscle glycogen is utilized to maintain the blood glucose level. The blood glucose level decreased to 44% after 24 hours of starvation and then remained unchanged. Some of the blood glucose after 24 or longer hours of starvation is probably derived from muscle glycogen. However, the mechanism of such a combination of the liver and muscle for glucose supply to the blood in starved mice is unknown. The amount of muscle protein decreases during starvation (Li et al., 1979; Hirose et al., 1986). Therefore, protein catabolism in the muscle might be also related t o the maintenance of blood glucose level in starved animals. Felig et al. (1970) reported that alanine, produced by the breakdown of muscle protein and released into the blood, serves as precursor for gluconeogenesis in the liver (glucose-alaninecycle).However, Kusaka and Ui (1977) showed that the amount of glucose produced via the glucose-alanine cycle is very small in starved animals. Further, Garlick et al. (1975) and Li et al. (1979) found that the reduction in muscle protein in starved animals is not due to an increase in the rate of protein catabolism but is the result of a decrease in the rate of protein synthesis. In the present results, rectus femoris muscles of mice contained no type I fibers, although Ariano et al. (1973) reported the presence of at least a small percentage of type I fibers in the muscles of the guinea pig, rat, cat, lesser bushbaby, and slow loris. Moreover, rectus fe- moris muscles of mice contained a few atypical type I1 fibers. Thus, the fiber type composition of the rectus femoris muscle of the mouse is slightly different from that of other species. Histochemical findings for fiber type classification in atypical type I1 fibers were intermediate between those in type IIA and IIB fibers. The fibers are probably “transitional fibers between type IIA and type IIB fibers” as suggested by Wirtz et al. (1983). The starved mice became less active than the fed mice and appeared curled up after 2 or more days of starvation. Therefore, there is probably no necessity to consider the possibility that the glycogen depletion in the muscle observed in the present study is due to high activity of starved mice. LITERATURE CITED Ariano, M.A., R.B. Armstrong, and V.R. Edgerton (1973) Hindlimb muscle fiber populations in five mammals. J. Histochem. Cytochem. 21:51-55. Armstrong, R.B. (1980) Properties and distributions of the fiber types in the locomotory muscles of mammals. In: Comparative Physiology: Primitive Mammals. K. 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