Human Skeletal Muscle Glycogen Branching Enzyme ...

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Human Skeletal Muscle Glycogen Branching Enzyme Activities with Exercise and Training

Received July 3, 1973 TAYLOR, A. W., STOTHART, J., BOOTH,M. A., THAYER, R., and RAO,S. 1974. Human skeletal muscle glycogen branching enzyme activities with exercise and training. Can. J . Physiol. Pharmacol. 52, B B 9- 122. Sixteen healthy male subjects classified as sedentary (8) or active (8), exercised to exhaustion on a bicycle ergometer at a load requiring 70% of their maximal aerobic capacity. Biopsy samples of the vastus lateralis muscle were taken at rest and at the time of fatigue. A 12 week training program increased skeletal muscle glycagen content and branching enzyme activities twofold. The exhaustive submaximal exercise reduced the glycogen levels of the trained group to values similar to the fatigue levels of the norm-trained sub-jects. Skeletal muscle glycogen branching enzyme activities decreased with submaximal exercise to fatigue in all graups. Maximal exercise to fatigue resulted in snsall increases in the activities of the enzyme. The results of the present stildy and a previous study (Taylor et al. 1972. Can. J . Physiol. Pharmacol. 50, 41 1-415) indicate that the activities of the glycogen synthcsizing enzymes are highly correlated with the skeletal muscle resting glycogen concentration and the relative fitness of the subjects.

Introduction Glycogen is synthesized by the actions of two enzymes. Glycogen synthetase (EC 2.4.1.1 1, UDPglucose : glycogen hY-4-glucosyltransferasc) catalyzes the synthesis of a-( 1+4) -glucosyl chains from uridiilc diphosphate glucose (UBPC) to thc terminal nonreducing chain ends of preformed glycogen (Leloir and Cardini 1957; Leloir 1964). These extended outer chains are then joined together by a- ( 1-6) glucosidic interchain linkages by glycogen branching enzyme (EC 2.4.1.1 8, a- 1,4-glucan : a- 1,4-glucan 6-glycosyltransfcr3se) (Verhue and Hers 1966). Smith (1968) has suggested that the relative activities of thc two enzymcs would be expected to be of primary importance in controlling the structure and thus the synthesis of the glycogen molecule. In a previous study we (Taylor et al. 1972b) demonstrated that exercise training produced a twofold increase in the resting activities of glycogen synthetase, both the T and D forms. Smith (1966, 1968) has observed that the average chain length of glycogen synthesized is relatively constant over a wide range of concentrations of branching enzyme and the reason for the apparent inherent regulation of the chain length may be due to the stability of the phosphorylase b - glycogen complex in which the enzyme is held at the surface of the glycogen molecule. We have previously noted (Taylor et ul. 1972h) that exercise training

produces a twofold increase in phosphoryiase a activity but does not affect the activity of phosphorylase tp. Since Manners (1968) has suggested that in most glycogen-synthesizing tissues there is a fairly constant ratio of activity between the branching enzyme and glycogen synthetase it was deemed important to investigate the effects of exercise and training upon tissue glycogen branching enzyme responses. This is the first investigation to study the training effects on the activities of this enzyme in human skeletal muscle. A group sf 16 male subjects ranging in age from 21 to 26 ( T - 24) years old was divided equally into a sedentary group, which followed no regular exercise regimen, and an active group, which consisted of "out-of-season athletes" who had not participated in a regular exercise program for at least 6 months. The subjects were placed into the sedentary o r pctive groups on the basis of maximal 0, .uptake MVo,, nsaximal 0, uptake per kilogram MVo,/kg, and work time to fatigue ('I'able I ) . The active group was tested before and upon the completion of a 12 week training regimen of running 2 miles, three times per week at a pace desigilecl and previously shown to increase MVo,, MVoz/kg. and skeletal muscle glycogen levels. On the day of the testing session, each subject reported to the laboratory after a light meal. No effort was made to control the diet and activity of the subjects before testing except that each subject was asked to refrain from strenuous exercise, alcohol, and abnormal diets for 24 h before testing. r\ resting


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C A N . J . PHYS1C)I.. P l I A R M A C O L . V O I . . 5 2 , 1974

TABLE1. -

-

-

Age (years)

Group -

-

- -

-

Anthropometric data for sedentary, active, and trained groups

---

-

Height (cm )

- - - - - - - -- -

23

-

- -

-

Weight (kg) -

--

- -

- - - - - -

Maximal O2 uptake ( I jmin)

- - - - -

- -- --

Maximal O2 uptake (ml/min/kg) --

- --

-

- - -

- - - - . - - - - - - - - - A - p - -

- - - -

.-

--- -

Work time to fatigue (mi n) -

-

-

+ 3.91

-

-

-

75.2 (65-79)

3 . 1 6 -t 0.29

(21-25)

176.8 (191-179)

Active, pretraining

24 (22-26)

177.2 (175-181)

73.6 (69-76)

3.52

k 0.37"

47.$3 -t 4.63"

74" (58-87)

Active, trained

25 (23-26)

177.8 ( 1 76-1 8%)

43.3 (48-76)

3.91

$-

0.42b

53.34 f 5.12b

93 (64-1 17)

Sedentary

42.02

- - - - - - - -- - - - - - - - - - -- - OActtvt: pretralncd group differs significantly from the sedentary group (P i 0.05). bTra~nedgroup dlffers s~gnrficantl)from the other groups ( P < 0.05). N O I F :\slues are nleans k S.E.M. for elght subjects per group. Values in parentheses are the range. -

muscle sample 435-70 rng, F = 48 nng) was taken from the vastus lateralis using ehc needle biopsy technique described by Hultnlan ( f 967a ) . The subject proceeded to exercise on a Monark bicycle ergor.stetea at a pedal frequency of 60 r.p.m. and a work load that required approximately 70% s f the saibject's aerobic capacity. Exercise was continlied until the subject was unable to maintain the pedal freq:~cncy. M~rscle samples were again collected at the time of fatigue with successive muscle sarnples being taken from the same incision; the biopsy needle was moved in an inferior direction, but the depth of sanapling remained consistent. Seven days later, each subject followed a similar procedure witin the cxception that a rnaximal test of 3-5 min was performed. 'Tissuc samples were quichly frozen (within 15 s of ren~ovctl)with licpid nitrogen (77 in 1 m%/100 rng nlusc%e of buffer solution containing 20 rnM sodium rrmercapte~etlnanol. I ~ a i M ED'I'A, 100 rnlW NaF. and 20 n-nM sodiuna glycerophosphate, pH 6.10 k 0.05. The frozcn bufTer and s a n ~ g l e were ground to a fine paste in liquid nitrogen with a mortar and pestle, and then allowed to thaw. The resulting suspension was centrifuged for 30 min at I500 g and the supernatant fluid used for the assay of glycogen branching enzyme by the method of Rarsselll and 'Taylor (1974). l'issue to be later analyzed for- glycogen using the method of Lo cb a / . (1970) was immediately frozen in Dry Ice and ethanol. The data were treated by an analysis of variance technique, the significance of the difference between me:zns being determined by Trskey9s "w9" test (Steel and Torm-ie 1960). OK)

Results Significant increases were observed in MV,,,, h4Vo2,/kg, and work time to fatigue after the training proeram. demomstrating thc effectiveness of the exercise regimen to increase the oxidative c a ~ a c i t vsf the subiects (Table I ). No goupdifferellces were noted between ""'"r~ and active P""'" ""i'g @Y"gen values. The training program, however, C

1

U

C I

I

J

J

-

- -

- -

39 (2 1-75)

-

-

-

-

-

resarlted in a significant increase ( P < 0.01) irn the glycogen content of the vastus lateralis musclc. At fatigue glycogen concentratioras were decreased for all threc groups after subrasaxirasal exercise (apprc~xinaately50% for the sedentary and pretrai~lii~ggroups and 75% for the trained group), although no differences between groups were noted at exhaustion (TabBc 2 ) . Significant training increases wcrc observed in resting glycogen branching enzyme activities ( P < 0.81) as measured by nanomolcs of glucose incorporated per minute per grain of bet tissue. he active prctraining-group demonstrated greater resting values than the scdentary group ( P < 0.05)- although the decreases at fratiguc: in each group were sin-ailar (405 0 % ) (Table 3 ) . Immediately after exhaustive inaximal exercise of 3-5 min duration, the skeletal TBPIJSC~C samples of all grcsups exhibited a small incrcase in glycogen branching enzyme activity (Table 3). Because caf the short term of this maximal exercise test it is doubtful that tlae notcd TABLE2. Skeletal muscle glycogen levels in sedentary, active, and trained subjects with submaximal exercise -

- - --- ---- -- --- -- ---.-- -- --- -

Glycogen Group

--

Sede~~tary pretraining Active, trained

4ctive7

Rest 1.10

0.09 * Oeil 2.52 + 0.23b

Fatigue 0.54 2 0.81" 0.57 k 0.09" 0.59 4 8.08"

-. - - - - - - - - - - - - QQ~Wrences between resting and fat~guevalues significant (P < 0.01). bDigferences between warned and nontrained subjects significant < 0 01) (pNclT;. ,lucs arc mean$ i S.E.M. in of ylyLogccnper (00 g wet niluscle for eight subjects per group.

-


7'.4YLOK E'T' AI-.: Gl,YCOGEN BRANCHING l.,NLYME .ACTIVITIES

TABLE3.

Glycogen brarmching enzyme activities in skeletal rnuscle from sedentary, active, and trained human subjects with subrnaximal and rnaximal exercise Activities Submaxirnal

Group

Rest

-

--

Fatigue

-

--

Sedentary Active, pretraining Active, trained -

72.4 2

118.4 132.8

+ +

-

Maximal

41.7 k 3.8" 67.3 11 .6"" 77.3 -t 8.1'"

6.0

+

14.2' 12.bd --

-

-

-- -- -

Rest

76.4 & 8.3 111.4 10.2" 139.3 16.3d

+ +

- -- --

---

Fatigue

-

84.3 _t 6.9" 136.5 2 1 6 . P 154.6 _+ 16.1'" -

- -. - -

aD~fferencesbetween fatigue and restlng values significant ( P < 0.01). bDitierences between fdt~gueand rertlng values signnficant ( P < 0.05). 'Difference between actlve pretrdlned and sedentary groups significant ( P /- 0.05). *DiEeaence betweeti trarned and non-tra~ned%ilbje&t§significant ( P .: 0.01). NOTE: Actlv~t~es are expressed as nanomoles of glucose ~ncorporatedpel ~ninutcper glam of tlssue. kaluer are mean3 for elyht subjects per group.

+

- .

S.L.M.

( 1968) has stated that thc rclative activities of the two cnr.yrnes control thc structure of the glycogcn molecule, i.e. high synthctase activity rcsults in a relatively low degree sf Discussion branching and a low synthetase activity allows Endurance exercise has becn shown to I-esult branching enzyrale to introduce a greater numin two- to fourfold incrcases in the activities ber of ( 1+6) bonds. The results of the preof the enzymes of the mitochondrial respiratory vious study (Taylor r t nl. 1972tr), which indichain (Holloszy and Qscai 19691, citric acid cated a twofold increase in thc synthetase cycle related enzymes (Holloszy et al. 1970), activity and the corresponding twofold increasc and respiratory enzymes linking the oxidation in glycogen branching enzyme activity found of NADH and of succiisate to oxygen (Holloszy in this study with exercise training, suggest st ul. 1971 ) as mcasured by the incorporation that thc ratio of the two enzymes remains inof substrate per gram of wct tissue per minute tact and the length of the N- ( I +4) -glucosidic and also by the increased mitochondrial protein chain remains relatively constant. It is now generally accepted that insulin content. The rcsults of the present study suggest that regular cndurancc or aerobic exercise activates glycogen synthetase. causing a rapid is a sufficient stimulus to increase thc activities deposition of liver glycogen (Stciner ct 01. of the glycogen cycle enzymes as well as thosc 1961; 1,arner et a/. 19681, and Manners of thc oxidative pathways. This finding seems (1968) has indicated that the activity of acceptable since muscies exercising submaxi- branching enzyme is correspondingly increased. mally utilizc oxidative and anaerobic substrates The decrease in plasma insulin noted by Pruett sim~altancously(Tssekutz and Paul 1966). It is ( 1970) after prolonged subrnaxinmal work of generally acccptcd that skeletal mliscle glyco- approximately 70% M V , ~ ,could possibly acgen is a multiply branched molecule consisting count for part of the decrement in the branchof chains of a- ( 1+4) -linked D-glucose resiclucs ing enzyme activity. Hermanscn ct al. ( I 970) joined together by a-(1+4)-glucosidic inter- noted an increase in plasma insulin immediately chain linkages to form a trec-likc structure after inaximal work, which similarly correlates (klanners 1957 ) . Tlle wcll known increases with the increase in branching enzyme activity glycogen store$ after excrcise after maximal work found in this study. in skeletal n~~ascle training (Hultman 1967h; Taylor et rrl. 1971) The results of the present and prcvlious rcsult in increased work time of the subjects. studies indicate that cxercise training can proThis finding, however, has not indicated duce a twofold increase in thc activities of the whether the increase was due to a grcater num- enzymes of the glycogen cycle. These increases ber ol' glycogcn macromolecules or to increased in enzyme activity undoubtedly play a major numbers of ( 1 4 6 ) -glucosidic linkages. Smith rolc in thc rapid resynthesis of glycogen found

enzyme activity increase was due to altered tissue hydration although water content was not mcasured in the present study.


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C A N . J . PHYSIOI,. PHAKMAC:OL. V O L . 5 2 , 1974

by Multman ( 1967b) after severe exercise. This resynthesis has recently been observed to start within 5 min after exhaustive maximal interval exercise (Piehl 1974) and is found only in skeletal muscle previously depleted of glycogen stores (Saltin, B., ailid Piehl, K.: personal communication). Supported in part by grant D N H W 55-04022 from the Department of National Health and Welfare, Ottawa, Canada. We thank Miss Diane Tougas and h4iss Nancy Smith for technical assistance. This work was completed while A. W. Taylor was under the tenure of a research associateship from the Department of National Health and Welfare, Ottawa, Canada. HERMANSEN, I,., P H Z U E ~E.' ~ D. ' , R., OSNES, J. B,, and G ~ E R EF., A. 1978. Blood glucose and plasma insulin in response to maximal exercise and glucose infusion. 9 . Appl. Physiol. 29, 13-16. HOI.LOSZY, J. ( I . , and O s c ~ rL. , 13. 1969. Effect of exercise ow (Y-glycerophosphate dehyda-ogenase activity in skeletal muscle. Arch. Rioshem. Biophys. 13Q. 653-656. HOI-LOSLY, J . O., OSCAI.1,. B., DON,1. J.. and MOLL,P. A. 1970. Mitochondrial citric acid cycle and related enzymes: adaptive response to exercise. Biochem. Wiophys. Kes. Cornmula. 40, 1368-1 373. HC)L~LOSZY, J. 0..OSCAI,I.. B., MOIJ?,P. A. and DON,I. J. 1971. Biochemical adaptations to endurance exercise in skeletal muscle. In Muscle nletabolism during exercise. Plenum Press, Corp. London. pp. 51-61. I-1111.I M A N , E. 19670. Muscle glycogen in man determined by needle biopsy specimens. Scand. J. Ciin. Lab. Invest. 19, 209-217. 1947h. Studies on muscle metabolism of glycogen and active phosphate irm man with special reference to exercise and diet. Scand. J. Clin. Lab. Invest. 94, 1-43. T s s ~ ~ u . rSz.,, JR., and PAUL,P. 1966. 'The role of extramuscular energy sources in the metabolism of the exercising dog. Fed. Proc. 25, 934. I,ARNEK.J., VII.I.AR-PALASI, C., C;OL.DWERG. N. D., Brsfnol,, J. S., HUIJING, F., WENGER, %. I.. SASKO, M., and BROWN, N . S . 1968. Hormonal and non-hormonal control of glycogen synthesis-control of transferax phosphatase and transferase I kinase. Ita Control of

glycogen metabolism. L4cadernicPress Inc., 1,ondon. pp. 1-18. L t LOIR, I .. F. 1964. Role of ~ ~ r i d i diphosphate ne glucose in the synthesis of glycogen. I,, Control of n,etabolism. J. and A . Churchif],Idondon. pp. 68-81. I'LLOIR,I A .F., and CRDINI, C. E. 1957. Biosynthesis of glycogen from uridine diphosphate glucose. J. Am. Chem. Soc. 79, 6340-6341. I,o, S., RUSSELL, J. C., and TAYLOR, A. W. 1970. Deterrnination of glycogen in small tissue samples. J. Appl. Physiol. 28, 234-236. MANNI:RS,D. J . 1957. The molecular structure of glycogens. Adv. Carbohydr. Chem. 12, 261-298. 1968. Branching enzymes. 112 Control of glycogen metabolism. ,4cademic Press Inc., 1,ondon. pp. 83-100. P I I H LK , . 1974. 'rime course for refilling of glycogen \toms in human muscle fibers following exercise-induced glycogen depletion. Acta Physicpl. Scand. In press. PKIJETT,E. D. R. 1970. Glucose and insulin during proBonged work stress in men living on different diets. J . Appl. B'hysiol. 28, 199-208. RUSSELL, J. G., TAYI.C~R. A. W. 1974. .4n assay for glycogen branching enzyme in tissue. Clin. Chem. In press. SMITH,E. E. 1966. Action pattern of glycogen phosphorylase. Biochem. J. 100. 22 P. 1968. Enzymic control of glycogen structure. 111 Control of glycogen metabolism. Academic Press Inc., London. pp. 203-213. Sr-I-EI., R. G. D., and TORRIE, J . H. 1960. Principles and procedures of statistics. McGraw-Hill Book Ccp., New York, N.Y. STEENER, D. F.. KAUDA, V., and WILLIAMS, K. H. 1961. Effects of insulin, glucagon and glucocorticoids upon hepatic glycogen synthesis from uridine ciiphosphate glucose. J. Biol. Chem. 236, 299-304. TAYLOR. A. W., BOOTH, M. A., and RAO, S. 1972rr. Human skeletal nmuscle phosphoryiase activities with exercise and training. Can. J. Physiol. Pharnlacol. SO, 1038-1032. TAYLOR, A. W.. LAPPAGE K., and KAO,S. 1971. Skeletal nmalscle glycogen stores after submaximal and nlaximal work. Med. Sci. Sports; 3, 75-78. TAYLOR, A. W.. THAYCR, R., and KAO, S. 1972h. Human skeletal muscle glycogen synthetase activities with exercise and training. Can. J. Physi~9l.Pharmacol. 50, 41 1-415. VERHUII, W., and HERS.H. G. 1966. A study of the reaction catalysed by the liver branching enzyme. Biochem. J. 99, 222-227.