Exercise training has s heparin-like effect on lipoprotein lipase activity in muscle
ws
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, DAVIDa. ESSIG LAWENCEB. ~ S C A I ,RICHARD ~ H K AAND Exercise Research Division, Department of Phj~sicakEducation, University of Illinois ab Chicago, B x 4348, Chicago, 6L 60680,U.S.A. Received November 13, 1991
OSCAI,L. B., BIKA, W. W., and Essre, DeA. 1992. Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle. Can. J. Physiol. Pharmacol. 70: 985 -909. Lipoprotein lipase (LPL) is anchored with high affinity to heparan sulphate proteoglycans on the luminal surface of the capillary endotheliurn. The levels of pre-heparin perhsate LPL activity increased from 16 f 1 to 145 f 6 Ujhindlirnb (ninefold increase) in hindlimb muscle of exercise-trained rats measured immediately after the last bout of work. At the same time, post-heparin perksate LPL activity decreased from 63 f 2 to 13 f 1 U/hindlimb ( p < 0.001). These results provide evidence that exercise-training b s a heparin-like effect on capillary-bound LPL. The total amount s f LPL (i.e., pre-heparin perfusate plus post-heparin perfusate) was twofold greater in the hindlimb s f the trained animals versus the controls. The effect of exercise on muscle LPL activity appears to last for as long as 5 days after cessation of exercise. Semm triglycerides were reduced 38% and plasma free fatty acids increased fourfold. These results provide evidence that training increases the capacity to remove triglycerides from circulation. Key words: lipoprotein lipase, skeletal muscle, exercise, serum triglycerides, heparin. OSCAH, L. B., BIKA, W. W., et ESSHG, D. AA.1992. Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle. Can. J. Physiol. Pbrmacol. 70 : 985 -909. La lipsprstdine Iipase (LPL) est like avec une haute affinitC aux protCsglycannes B hkparane-sulfate B la surface luminale de l'endsthClium capillaire. Les taux dqactivitCde LPL prBh6parine ont augmend de 16 4: 1 B 145 f 6 Ulmembre postkrieur (augmentation d'un facteur neuf) dans le muscle du membre posttrieur de rats entraiinQ 2 l'exercice, immkdiatement apr&s la dem2re pkriode d'activitt intense. De plus, l'activitk LPL post-hbparine a diminuC de 63 f 2 B 13 f 1 U/membre posttrieur ( p < 0,001). Ces rCsultats ddmontrent que l'entrainernent 2 19exercicea un effet de type hCparine sur la LPL like capillaire. La quantitC totale de LPL (6.9-d. prC-hCparine plus post-htparine) a kt6 plus ClevCe d'un facteur deux dans le membre posttrieur des a n h a u x entrainks versus les animaux tCmins. Lkffet de l'exercice sur I'activitC de LPL musculaire semble durer au rnoins 5 jours aprks I'arrCt des exercices. Les triglyctrides driques ont CtC rkduits de 38% et les acides gras libres plasmatiques ont augment6 d'un facteur quatre. Ces rCsultats dkrnontrent que l'entrainernent augmente la capacitk d'Climiner les triglycCrides de Ba circulation. Wg,e ck&s : lipprotkine lipase, muscle squelettique, exercice, triglyckrides seriques, hCparine. [Traduit par la rCdaction]
Introduction Lipoprotein lipase (LPE) is synthesized in and secreted from a variety of tissues including skeletal muscle (Borensztajn 1987). The enzyme is then anchored with high affinity to heparan sulphate proteoglycans OW the luminal surface of capillary endothelial cells. Because of this binding process, hydrolysis s f chylomicrons and very low-density lipsproteins occurs on, or close to, the luminal surface s f the vascular endothelium and not in the plasma (Blanchette-Mackie et al. 1989; Pedersen et al. 1983). It has long been recognized that exercise training markedly increases the levels s f LPL activity in skeletal muscle (NiWHB 1987). Previously (Oscai et al. 19821, it was noted that intracellular LPL activity was increased more than 2.5-fold in the soleus and fast-red fibers of the quadriceps in exercise-trained rats. Using 2m indirect method for cdculating endothelium-bound LPL activity, it was found that little enzyme was anchored to capillary walls in skeletal muscle s f trained rats. These results came as a surprise to us, since sparse mounts of EPL activity anchored t s endothelid cells would minimize the importance s f EPL in providing fatty acids for contractile activity. Therefore, the purpose of this paper was to reevaluate the effects s f exercise training on LPL activity in muscle.
unrestricted access to a diet of h r i n a laboratory chow and water. The room was maintained at a temperature between 21 and 23°C and lighted between 07:30 and 19:30. The animals were divided into two groups matched for weight. An exercising group was trained to run on a Quinton Instruments 42-15 rodent treadmill and exercised 5 dayslweek (Holloszy 1967). The speed and duration of the run were progressively increased over 12 weeks until the animals were mnning continuously for 120 min at 3 1 mimin up an 8" incline, with 12 intervals of mnning at 42 mimin, each lasting 30 s, spaced 10 min apart through the exercise sessions. They were maintained at this work level until they were sacrificed or made sedentary to determine the time course of return to control levels of LPL activity; this period varied fom 1 to 21 days. A freely eating sedentary control group was not subjected to treadmill mnning.
Animal care and exercise program Male rats of a Wistar strain weighing about 90 g were purchasd from Charles River (Wilmington, Mass.). The rats were provided
Pre- and post-baeparin EPL activity measure pre-heparin LPL activiv, one hindlimb was perfklsed (5 mL/min) with Krebs-Ringer bicarbonate buffer ( p '9.4) ~ containing 5 % rat serum. After perfusion with 200 mL of medium, the per&sates were free from LPL activity. Next, the hindlimb was perfused
B A ~ t h ofor r correspondence. Printed in Canada I HrnprirnC ;aaa Canada
Tissue prepamtion Experiments were started at 08:15 and concluded by 1O:15. Experiments were performed on rats after an overnight fast. The rats were anesthetized with sodium pentobarbital (5 - 18 mgi1W g body weight). Samples s f red vastus muscle were obtained from the Qeepest portion, closest to the femur, of the whole quadriceps muscle (Holloszy and Booth 1976). Surgical preparation of the hindlimb was performed as described by Ruderrnan et al. (19'71) with minor modifications (Qlscai 1979). During surgery and subsequent perfusion, rats were kept warm with a heating pad and an overhead heat lamp,
CAN. J. BBYSIOL. PHARMACOL. VOL. 70, 1992
TABLE1. The effect of exercise training on pre-heparin and post-heparin perfusate lipoprotein lipase (LPL) activity from rat hindlimb
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Pre-heparin perfusate LPL activity (U/hindlirnb) Control Exercise trained taken imediately after exercise Exercise trained taken 24 h after exercise
Post-heparin perfusate LPL activity (U/hindlirnb)
Total LPL activity (pre-heparin plus post-heparin perfusate) (Ulhindlimb)
B6f l 145f 6*
89f I*
NOTE:Values are expressed as means f SEM. 'Phere were eight animals per group. *Significantly different from controls, p
< 0.001.
TABLE2, The effect of exercise training on free fatty acids (FFA) and triglycerides (TG)in serum
Serum TG Semm %;FA (mg/lW mL) (ymol F F A l d serum) -
Control Exercise trained taken imediately after exercise Exercise trained taken 24 h after exercise
5 5 f 3 (5)
0.21 40.01 (10)
34 9 2 (5)"
0.87 f0.02 (10)" 0.57f 0.01 (l5)*
NOTE:Values are expressed as means f %EM.The number d animals per group is given in parentheses. "Significantly different from controls, p < 0.001.
FIG. 1. $re-heparin perfusate (e) and post-heparin perfusate (ma) lipoprotein lipase activity from hindlimb muscle of exercise-trained rats sacrificed imediately after the last bout of work. Pre-heparin perhsate (m) and post-heparin perfusate (0)lipoprotein lipase activity from hindlimb muscle of sedentary control animals.
with 200 mL of buffer containing heparin (5 U/mL) to release LPL bound to the capillary walls. The medium was drawn from a reservoir, then perfaased through the hindlimb by a Gilson Minipuls 2 peristaltic pump in a nonrecirculatory system. Amay methods LPL activity was measured by the method described by Borensztajn et aall. (1972). The tissue and perfusates were homogenized in 0.025 M NH,/HCl buffer (pH 8.1) with a Duall ground-glass grinder (Kontes Glass. Evanston, Ill). The fresh tissue concentration of the homogenates was 50 mg/mL. The composition of the assay medium and the procedures for incubation, extraction, and measurement of unesterified fatty acids released into the assay medium have been described previously (Borensztajn et al. 1972). Skeletal muscle lipolytic activities had the characteristics of LPL; e.g., the reaction was inhibited in the absence of serum and in the presence of 1 M NaCl. LPL activities are expressed as units f SEM, 1 U representing 1 pmol of unesterified fatty acids released into the assay medium per hour of incubation per millilitre of perfusate or per gram wet weight tissue. Plasma free fatty acids (FFA) were measured using the titrim metric assay of Trout et al. (1960). Plasma triglycerides were meas u r d by the method of Fletcher (1968). Adikcytes were isolated according to the method of Rodbell (1964). The diameter of fat cells was measured dcroscopicdly.
Statistics Results are expressed as means f SEM. Student's t-test was used to evaluate the significance of difference between the means.
Results Table 1 shows that exercise training resulted in a nine-fold increase in pre-heparin perfusate LPE activity in rats sacrificed immediately after the last bout of work. Pre-heparin perfusate activity was still elevated (5.6-fold) 24 h after exercise in the hindlimb muscle of the trained animals. Table 1 also shows that post-heparin perfusate EPL activity was significantly reduced both immediately and 24 h after exercise in the treadmill runners. When totd amounts of enzyme activity (i.e., pre-heparin perfusate plus post-heparin perhsate LPE activity) were calculated, the trained rats sacrificed immediately after work had twice as much LPL in circulation than did the controls. The increase in totd amount of enzyme activity was still evident 24 h after exercise in the trained animals. The possibility exists that the rates of enzyme synthesis and release could have been altered by the prolonged perhsion period. l[n csntrast wi& &is notion, Fig. 1 shows that qproxirnately two-thirds of the pre-heparin perfusate LPL activity from the hindlimb of the treadmill mnners was released in the first 80 mL of perfusion. Very little pre-heparin perfusate LPL activio was detected in the find 40 mL of perfusion' The same line. d- reasoning could be to the anirnaisTables 1 and 2 reveal that at a time when tot& EPL activiegr was markedly elevated in the trained animals, triglycerides
TABLE 3. Lipoprotein lipase activity from whole homogenates sf red vastus muscle after cessation sf training
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Days after cessation of training
Lipoprotein lipase activity (Uig tissue)
Control
Exercise trained
Difference*
p
-
NOTE:Values are expressed as means f SEM. There were five or six rats
per group. *Control minus exercise trained.
were decreased by 38% and FFA were increased by fourfold measured immediately after the last bout of work. Semm-free fatty acids were still elevated 2.7-fold measured 24 h after exercise. Table 3 shows that training markedly elevated LPL activity in whole homogenates of red vastus muscle in rats sacrificed immediately after the last bout of treadmill mnning. Three days later, enzyme activity was twice as high, and 5 days later nearly 50% higher in muscle of trained versus control rats. Afer 7 days, LPL activity in muscle of the mnners was back to control levels. Fat cell size was used as a marker of the trained state. We felt justified in using adipocyte diameter as an index of training, since the adaptation reflects a decrease in size and since the diameter remains significantly reduced for at least 9 days after exercise has stopped (Craig et al. 1983). The finding that adipocyte diameter was significantly smaller in the mnners (74 f 2 pm) compared with that in the control rats (106 f 4 pm) provides evidence that the mnners were highly trained.
Biscnssisn Bre-heparin perhsate LBL activity increased ninefold measured immediately after the last bout of work in the hindlimb of the exercise-trained rats. At the same time, post-heparin perfusate LPL activity was markedly decreased in the trained animals. These results provide evidence that exercise-training has a heparin-like effect on capillary bound LPL activity. The total amount of LPL activity (i.e., pre-heparin perhsate plus post-heparin perfusate) was twofold greater in the treadmill mnners than in the controls seen immediately after exercise. These results provide evidence that exercise increases the capacity to clear triglycerides from circulation. The data in the present study were collected on trained rats. There is evidence that muscle LPL activity is also increased in response to acute exercise. For example, Paulin et al. (199 1) reported that LPC activity was elevated in post-heparin plasma measured 24 h after a I-h mn on a treadmill in previously untrained rats. In another study in the heart, Goldberg et d . (1984) reported that both heparin-releasable and tissue-bound LPL activity were elevated after a single bout of treadmill mnning in previously untrained rats. It has long been recognized that heparin causes the release of LPL from capillary beds in a variety of tissues including skeletal muscle and that the release occurs rapidly (Borensztajn 1987; Robinson and Harris 1959). It has been hypothesized h a t the release mechanism probably involves the formation of a heparin-enzyme complex; the enzyme could be detached from the cell surface heparan sulfate either
through competition between the soluble heparin and the membrane-bound heparan sulfate for the same binding site or as a result of a conformationd change in the protein molecule induced by heparin (8livecrona et d. 197'7). The mechanism(~)by which exercise causes the release of LPL activity from capillary walls similar to that seen with heparin is not clear. However, at least two possibilities can be advanced. First, the release of capillary-bound LBL activity could have been mediated by an exercise-induced increase in plasma free fatty acids (FFA). It has long been recognized that exercise increases the concentration of plasma FFA (Friedberg et al. 1963; Have1 et al. 1964; Oscai 1979). In turn, FFA have recently been shown to remove LPL activity from endothelial cells [Peterson et al. 1990; Saxena et d.1989). A second possibility is that the increased force caused by the increased rate of blood flow is responsible. A third possibility is that an exercise-induced conformational change could have taken place brought about by a blood-borne factor h a t is yet to be identified. The vascular endothelium releases a variety of substances that interact directly with other tissues. For example, it has been demonstrated that endothelin- l interacts with intestinal and uterine smooth muscle, impacts on the central nervous system, and has cardiac as well as mitogenic actions (Yanagisawa and Masaki 1989). It has long been recognized that exercise exerts pleiotropic effects in a variety of tissues. Therefore, it is conceivable that the exercise-induced release s f LPE from capillary walls might be mediated by blood-borne substances contributed by the neurocrine or endocrine system and (or) brought about by the autcscrine or paracrine system. It could be argued that LLPL activity measured in the perhsates originated from both the extracehlar compartment and the intracellular pool. Moreover, the endothelium could have internalized enzyme that could have been blocked by the presence of FFA. In contrast with this notion, there is evidence that only extracellular LPL activity was measured in the medium. In a previous paper (Oscai et al. 19821, intracellular LPL activity was determined in heparin-perfused hindimb of rats under conditions similar to those used in this study. As a check on the technique of perfusing the hindlimb with heparin to fractionate LPL activity in skeletal muscle in the earlier study, rats were treated with Triton WR-1339. Triton selectively inhibits LPL activity that is directly involved in the hydrolysis of circulating triglycerides in capillary beds. The finding that LLP activity in the intracellular fraction of the soleus md fast-twitch red fibers of the quadriceps was essentidly the same for both techniques provides suggestive evidence that enzyme was not being pulled from the intracellular fraction during perfusion. Further support for the notion that o d y extracellular enzyme was being measured comes from the finding in the present study that LPL activity was not detectable at the end of the perfusion period. There is evidence that substantial amounts of TG can be hydrolyzed when LPL activity is released into circulation by action of either heparin or exercise. With respect to heparin, HAn (1943) found that when dogs with marked lipemia were injected with heparin, the lipemia disappeared completely in blood samples taken 3 -5 min later. This early finding led to the discovery of LPL. In another study, Remie et al. (1976) fed rats 5 mL of corn oil by stomach tube. Three hours later, when their plasma was visibly lipemic, the animals were given 200 units of sodium heparin subcutaneously. Administration of heparin to the corn oil fed animals resulted in an increase in plasma fatty acid concentration to more than twice the control
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CAN. J. PHYSIOL. PMARMACOL. VOL. 70, 1992
value after 18 rnin and to more than four times the control value after 40 rnin in sedentary animals. With respect to exercise, Thompson et d.(1988) injected exercise-trained humans with 10% TravamuHsion (1 mE/kg body weight). The rate of clearance sf the fat emulsion increased nearly 50% above pretraining values after 32-48 weeks of exercise. Exercise decreases muscle TG stores (Carlson et al. 1971; Havel et al. 1967; Holloszy and Booth 1976; Weitman et al. 1973). The question is whether plasma LPL plays a role in replenishing the decreased lipid stores in muscle. There are two lines of evidence suggesting that plasma TG are used to replenish the decreased fat stores in muscle. First, LPEdeficient mice (cld) develop severe hyperchylornicronernia because of an inability to clear TG from circulation (Blanchette-Mac~eet al. 1986; Paterniti et al. 1983). These affected mice have no lipid droplets in myedcytes of heart and diaphragm, whereas unaffected mice have numerous lipid droplets in their muscle cells. These results suggest an important sole for LPL in the replenishment s f muscle lipid stores. Second, TG uptake from circulation is highly related to muscle LPL activity ((Linder et al. 1976; Tan et al. 1977; Terjung et al. 1983). Muscle LPL activity is markedly increased with exercise training (Nikkila 1987; Oscai et al. 1982). The results of the present study do not provide evidence that would distinguish between the use of LPE-released fatty acids as a direct source s f energy for contractile activity or as precursors for the replenishment of triglycekde stores. The results do, however, show that because of the marked increase in muscle enzyme activity, the total amount of LPL activity in circulation (i.e., pre-heparin perksate plus post-heparin perfusate) was much greater in trained rats compared with comparable sedentary controLs measured both immediately and 24 h after exercise. Semm TGs were decreased and plasma FFA increased at a time when extracellular LPE was markedly elevated. These results suggest that training increases the capacity to clear TG from circulation. Further, this effect of exercise training on muscle EPE activity appears to last for as long as 5 days after cessation of work. Finally, it should be noted that the mechanism(s) responsible for activating LPL in muscle in response to exercise remains unknown.
Acknowledgments The assistance of Vinod Singh is gratefully acknowledged. This research was supported by National Institutes sf Health research grants AM- 17357 and AR-39872. BBanchette-Macfie, E. J., Wetzel, M.G . , m d Chernick, S. S., et al. 1986. Effect of the combined lipase deficiency mutation (cld/cld) on ultrastmcture of tissues in mice diaphragm, hear%,brown adipose tissue, lung, and liver. Lab. Invest. 55: 347 -362. Blanchette-MacBaie, E. J., Masuno, W., and Bvvyer, N. K . , et al. 1989. Lipoprotein lipase in myscytes and capillary endothelium of heart: immunocytochernical study. Am. J . Phy siol . 256: E8 1 8 E828. Borensztajn, J. 1987. Heart and skeletal muscle lipoprotein lipase. In Lipoprotein liipase. Edited by J. Borensztajn. Evener Publishers Ine., Chicago, gap. 133- 148. Borensztajn, J., SamoBs, B. W., and Rubenstein, A. H. 1972. Effects of insulin on lipoprotein lipase activity in the rat heart and adipose tissue. Am. I. Physiol. 223: 1271 - 1275. Carlson, E. A., Ekelund, E.-G., and Frbberg, S. 0.1971. Concentration of triglycerides, phsspholipids and glycogen in skeletal muscle and of free fatty acids and @-hydroxybutyricacid in blood in man in response to exercise. Eur. J. Clin. Invest. 1: 248 -254.
Craig, B. W., Thompson, K., and Holloszy, J. 8.1983. Effects of stopping training on size and response to insulin of fat cells in female rats. J. Appl. Physiol. 54: 571-575. Fletcher, M. J. 1968. A colorimetric method for estimating serum triglycerides. Clin. Chirn. Acta, 22: 393-397. Friedberg, S. J., Sher, P. B., Bogdonoff, M. B., and Estes, E. H., Jr. 1943. The dynamics of plasma free fatty acid metabolism during exercise. J. Lipid Res. 4: 34-38. Goldberg, D. I., Rumsey, W. L., and Kendrick, %. $1'. 1984. Exercise activation of myocardial lipoprotein lipase in male and estrogen-treated female rats. Metabolism, 33: 964 -969. Hahn, P. F. 1943. Abolishment of alimentary lipemia following injection of heparin. Science (Washington. D.C .), 98: 19 -28. Havel, R. J., Carlson, L. A., Ekelund, La-G., and Holmgren, A. 1964. Turnover rate and oxidation of different free fatty acids in man during exercise. 3. Appl. Physiol, 19: 613 -618. Havel, R. J., Pernow, B., and Jones, N. L. 1967. Uptake and release of free fatty acids and other metabolites in the legs of exercising men. J. Appl. Physiol. 23: 90-96. Holloszy, 5. 8.1967. Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J. Biol. Chem. 242: 2278 -2282. Holloszy, B. O., and Booth, F. W. 1976. Biochemical adaptations to endurance exercise in muscle. Annu. Rev. Physiol. 38: 273 -291. Linder, C., Chernick, S. S., Fleck, T. R., and Scow, R. 8. 1974. Lipoprotein lipase and uptake of chylomicron triglyceride by skeletal muscle of rats. Am. J. Physiol. 231: 860-864. Niwla, E. A. 1987. Role sf lipoprotein lipase in metabolic adaptation to exercise and training. Hn Lipoprotein Bipase. Edited by I. Borensztajn. Evener Publishers, Inc., Chicago. pp. 187 - 299. Olivecrona, T., Bengtsson, G., a d Maridrand, S.-E., et al. 1977. Heparin - lipoprotein lipase interactions. Fed. Proc. Fed. Am. Soc. Exp. Biol. 36: 60-65. Oscai, L. B. 1979. Effect of acute exercise on tissue free fatty acids in untrained rats. Can. J. Physiol. Phamacol. 57: 485 -489. Oscai, E. B., Camso, R. A., an Wergeles, A. C . 1982. Lipoprotein lipase hydrolyzes endogenous triacylglycerols in muscle of exercised rats. J. Appl. Physiol. 52: 2059 - 1063. Paterniti, J, W., Brown, W. V., and Ginsberg, H. N. 1983. Combined lipase deficiency (cld): a lethal mutation on chromosome 17 of the mouse. Science (Washington, DOC.), 221: 164 - 169. Paulin, A., Lalonde. J.. and Deshaies. Y . 1991. B-Adrenergic blockade and iipoprotein lipase activity in rat tissues'after acute-exercise. Pam, J. Physiol. 261: R891 -R897. Pedersen, M. E., Cohen, M., and Schotz, M. C. 1983. Imunocytochemical localization of the functional fraction of lipoprotein lipase in the perfused heart. J. Lipid Res. 24: 5 12 - 521. Peterson, J., Bihain, B. E., and Bengtsson-Olivecrona, G., et al. 1990. Fatty acid control of lipoprotein lipase: a link between energy metabolism and lipid transport. Proc. Natl. Acad. Sci. U.S.A. 87: 9639-913. Reitman, J., Baldwin, R.M . , and Holloszy, J. 0. 1973. Intramuscular triglyceride utilization by red, white, and intermediate skeletal muscle and heart during exhausting exercise. Proc. Soc . Exp . Biol . Med. 142: 428-631. Rennie, M. J., Winder, W. W., and Holloszy, 5. 0. 1974. A sparing effect of increased plasma fatty acids on muscle and liver glycogen content in the exercising rat. Biochem. J. 156: 647 -655. Robinson, D. S., and Harris, P. M. 1959. The production of liplytic activity in the circulation of the hind limb in response to heparin. Q . J. Exp. Physiol. 44: 80-90. Rodbell, M. 1964. Metabolism of isolated fat cells. I. Effects s f hsrmones on glucose metabolism and lipolysis. J. Biol. Chem. 239: 375 -380. Rudermn, N. B . , Houghton, C. R. S., and Hems, R. 1971. Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism. Bisehem. J. 124: 639 -65 1. Saxena, U.,Witte, L. D., and Goldberg, I. S. 1989. Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids. J. Biol. @hem. 264: 4349-4355, ,
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Tan, M. B., Sab, T., and Havel, R. J. 1977. The significance of lipoprotein lipase in rat skeletal muscles. 9. Lipid Res. 18: 363 370. Terjung, R. L., Mackie, B. G . , Dudley, G. A., and Kaciuba-Uscilko, H. 1983. Influence of exercise on chylornicron triacylglycerol metabolism: plasma turnover and muscle uptake. Med. Sci. Sports Exercise, 15: 340 -347. Thompson, B. D.,Cullinane, E. M., and Sady, S. B., et al. 1988. Modest changes in high-density lipoprotein concentration and
metabolism with prolonged exercise training. Circulation, 78: 25 -34. Trout, D.L., Estes, E. H.,and Friedberg, S . J. 1960. Titration of free fatty acids of plasma: a study of current methods and a new modification. 9. Lipid Res. 1: 199 -202. Yaraagisawa, M . , and Masaki, T. 1989. Molecular biology and biochemistry of the endsthelins. Trends Phamacsl. Sci. 10: 374 - 378.
ws
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, DAVIDa. ESSIG LAWENCEB. ~ S C A I ,RICHARD ~ H K AAND Exercise Research Division, Department of Phj~sicakEducation, University of Illinois ab Chicago, B x 4348, Chicago, 6L 60680,U.S.A. Received November 13, 1991
OSCAI,L. B., BIKA, W. W., and Essre, DeA. 1992. Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle. Can. J. Physiol. Pharmacol. 70: 985 -909. Lipoprotein lipase (LPL) is anchored with high affinity to heparan sulphate proteoglycans on the luminal surface of the capillary endotheliurn. The levels of pre-heparin perhsate LPL activity increased from 16 f 1 to 145 f 6 Ujhindlirnb (ninefold increase) in hindlimb muscle of exercise-trained rats measured immediately after the last bout of work. At the same time, post-heparin perksate LPL activity decreased from 63 f 2 to 13 f 1 U/hindlimb ( p < 0.001). These results provide evidence that exercise-training b s a heparin-like effect on capillary-bound LPL. The total amount s f LPL (i.e., pre-heparin perfusate plus post-heparin perfusate) was twofold greater in the hindlimb s f the trained animals versus the controls. The effect of exercise on muscle LPL activity appears to last for as long as 5 days after cessation of exercise. Semm triglycerides were reduced 38% and plasma free fatty acids increased fourfold. These results provide evidence that training increases the capacity to remove triglycerides from circulation. Key words: lipoprotein lipase, skeletal muscle, exercise, serum triglycerides, heparin. OSCAH, L. B., BIKA, W. W., et ESSHG, D. AA.1992. Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle. Can. J. Physiol. Pbrmacol. 70 : 985 -909. La lipsprstdine Iipase (LPL) est like avec une haute affinitC aux protCsglycannes B hkparane-sulfate B la surface luminale de l'endsthClium capillaire. Les taux dqactivitCde LPL prBh6parine ont augmend de 16 4: 1 B 145 f 6 Ulmembre postkrieur (augmentation d'un facteur neuf) dans le muscle du membre posttrieur de rats entraiinQ 2 l'exercice, immkdiatement apr&s la dem2re pkriode d'activitt intense. De plus, l'activitk LPL post-hbparine a diminuC de 63 f 2 B 13 f 1 U/membre posttrieur ( p < 0,001). Ces rCsultats ddmontrent que l'entrainernent 2 19exercicea un effet de type hCparine sur la LPL like capillaire. La quantitC totale de LPL (6.9-d. prC-hCparine plus post-htparine) a kt6 plus ClevCe d'un facteur deux dans le membre posttrieur des a n h a u x entrainks versus les animaux tCmins. Lkffet de l'exercice sur I'activitC de LPL musculaire semble durer au rnoins 5 jours aprks I'arrCt des exercices. Les triglyctrides driques ont CtC rkduits de 38% et les acides gras libres plasmatiques ont augment6 d'un facteur quatre. Ces rCsultats dkrnontrent que l'entrainernent augmente la capacitk d'Climiner les triglycCrides de Ba circulation. Wg,e ck&s : lipprotkine lipase, muscle squelettique, exercice, triglyckrides seriques, hCparine. [Traduit par la rCdaction]
Introduction Lipoprotein lipase (LPE) is synthesized in and secreted from a variety of tissues including skeletal muscle (Borensztajn 1987). The enzyme is then anchored with high affinity to heparan sulphate proteoglycans OW the luminal surface of capillary endothelial cells. Because of this binding process, hydrolysis s f chylomicrons and very low-density lipsproteins occurs on, or close to, the luminal surface s f the vascular endothelium and not in the plasma (Blanchette-Mackie et al. 1989; Pedersen et al. 1983). It has long been recognized that exercise training markedly increases the levels s f LPL activity in skeletal muscle (NiWHB 1987). Previously (Oscai et al. 19821, it was noted that intracellular LPL activity was increased more than 2.5-fold in the soleus and fast-red fibers of the quadriceps in exercise-trained rats. Using 2m indirect method for cdculating endothelium-bound LPL activity, it was found that little enzyme was anchored to capillary walls in skeletal muscle s f trained rats. These results came as a surprise to us, since sparse mounts of EPL activity anchored t s endothelid cells would minimize the importance s f EPL in providing fatty acids for contractile activity. Therefore, the purpose of this paper was to reevaluate the effects s f exercise training on LPL activity in muscle.
unrestricted access to a diet of h r i n a laboratory chow and water. The room was maintained at a temperature between 21 and 23°C and lighted between 07:30 and 19:30. The animals were divided into two groups matched for weight. An exercising group was trained to run on a Quinton Instruments 42-15 rodent treadmill and exercised 5 dayslweek (Holloszy 1967). The speed and duration of the run were progressively increased over 12 weeks until the animals were mnning continuously for 120 min at 3 1 mimin up an 8" incline, with 12 intervals of mnning at 42 mimin, each lasting 30 s, spaced 10 min apart through the exercise sessions. They were maintained at this work level until they were sacrificed or made sedentary to determine the time course of return to control levels of LPL activity; this period varied fom 1 to 21 days. A freely eating sedentary control group was not subjected to treadmill mnning.
Animal care and exercise program Male rats of a Wistar strain weighing about 90 g were purchasd from Charles River (Wilmington, Mass.). The rats were provided
Pre- and post-baeparin EPL activity measure pre-heparin LPL activiv, one hindlimb was perfklsed (5 mL/min) with Krebs-Ringer bicarbonate buffer ( p '9.4) ~ containing 5 % rat serum. After perfusion with 200 mL of medium, the per&sates were free from LPL activity. Next, the hindlimb was perfused
B A ~ t h ofor r correspondence. Printed in Canada I HrnprirnC ;aaa Canada
Tissue prepamtion Experiments were started at 08:15 and concluded by 1O:15. Experiments were performed on rats after an overnight fast. The rats were anesthetized with sodium pentobarbital (5 - 18 mgi1W g body weight). Samples s f red vastus muscle were obtained from the Qeepest portion, closest to the femur, of the whole quadriceps muscle (Holloszy and Booth 1976). Surgical preparation of the hindlimb was performed as described by Ruderrnan et al. (19'71) with minor modifications (Qlscai 1979). During surgery and subsequent perfusion, rats were kept warm with a heating pad and an overhead heat lamp,
CAN. J. BBYSIOL. PHARMACOL. VOL. 70, 1992
TABLE1. The effect of exercise training on pre-heparin and post-heparin perfusate lipoprotein lipase (LPL) activity from rat hindlimb
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Pre-heparin perfusate LPL activity (U/hindlirnb) Control Exercise trained taken imediately after exercise Exercise trained taken 24 h after exercise
Post-heparin perfusate LPL activity (U/hindlirnb)
Total LPL activity (pre-heparin plus post-heparin perfusate) (Ulhindlimb)
B6f l 145f 6*
89f I*
NOTE:Values are expressed as means f SEM. 'Phere were eight animals per group. *Significantly different from controls, p
< 0.001.
TABLE2, The effect of exercise training on free fatty acids (FFA) and triglycerides (TG)in serum
Serum TG Semm %;FA (mg/lW mL) (ymol F F A l d serum) -
Control Exercise trained taken imediately after exercise Exercise trained taken 24 h after exercise
5 5 f 3 (5)
0.21 40.01 (10)
34 9 2 (5)"
0.87 f0.02 (10)" 0.57f 0.01 (l5)*
NOTE:Values are expressed as means f %EM.The number d animals per group is given in parentheses. "Significantly different from controls, p < 0.001.
FIG. 1. $re-heparin perfusate (e) and post-heparin perfusate (ma) lipoprotein lipase activity from hindlimb muscle of exercise-trained rats sacrificed imediately after the last bout of work. Pre-heparin perhsate (m) and post-heparin perfusate (0)lipoprotein lipase activity from hindlimb muscle of sedentary control animals.
with 200 mL of buffer containing heparin (5 U/mL) to release LPL bound to the capillary walls. The medium was drawn from a reservoir, then perfaased through the hindlimb by a Gilson Minipuls 2 peristaltic pump in a nonrecirculatory system. Amay methods LPL activity was measured by the method described by Borensztajn et aall. (1972). The tissue and perfusates were homogenized in 0.025 M NH,/HCl buffer (pH 8.1) with a Duall ground-glass grinder (Kontes Glass. Evanston, Ill). The fresh tissue concentration of the homogenates was 50 mg/mL. The composition of the assay medium and the procedures for incubation, extraction, and measurement of unesterified fatty acids released into the assay medium have been described previously (Borensztajn et al. 1972). Skeletal muscle lipolytic activities had the characteristics of LPL; e.g., the reaction was inhibited in the absence of serum and in the presence of 1 M NaCl. LPL activities are expressed as units f SEM, 1 U representing 1 pmol of unesterified fatty acids released into the assay medium per hour of incubation per millilitre of perfusate or per gram wet weight tissue. Plasma free fatty acids (FFA) were measured using the titrim metric assay of Trout et al. (1960). Plasma triglycerides were meas u r d by the method of Fletcher (1968). Adikcytes were isolated according to the method of Rodbell (1964). The diameter of fat cells was measured dcroscopicdly.
Statistics Results are expressed as means f SEM. Student's t-test was used to evaluate the significance of difference between the means.
Results Table 1 shows that exercise training resulted in a nine-fold increase in pre-heparin perfusate LPE activity in rats sacrificed immediately after the last bout of work. Pre-heparin perfusate activity was still elevated (5.6-fold) 24 h after exercise in the hindlimb muscle of the trained animals. Table 1 also shows that post-heparin perfusate EPL activity was significantly reduced both immediately and 24 h after exercise in the treadmill runners. When totd amounts of enzyme activity (i.e., pre-heparin perfusate plus post-heparin perhsate LPE activity) were calculated, the trained rats sacrificed immediately after work had twice as much LPL in circulation than did the controls. The increase in totd amount of enzyme activity was still evident 24 h after exercise in the trained animals. The possibility exists that the rates of enzyme synthesis and release could have been altered by the prolonged perhsion period. l[n csntrast wi& &is notion, Fig. 1 shows that qproxirnately two-thirds of the pre-heparin perfusate LPL activity from the hindlimb of the treadmill mnners was released in the first 80 mL of perfusion. Very little pre-heparin perfusate LPL activio was detected in the find 40 mL of perfusion' The same line. d- reasoning could be to the anirnaisTables 1 and 2 reveal that at a time when tot& EPL activiegr was markedly elevated in the trained animals, triglycerides
TABLE 3. Lipoprotein lipase activity from whole homogenates sf red vastus muscle after cessation sf training
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Days after cessation of training
Lipoprotein lipase activity (Uig tissue)
Control
Exercise trained
Difference*
p
-
NOTE:Values are expressed as means f SEM. There were five or six rats
per group. *Control minus exercise trained.
were decreased by 38% and FFA were increased by fourfold measured immediately after the last bout of work. Semm-free fatty acids were still elevated 2.7-fold measured 24 h after exercise. Table 3 shows that training markedly elevated LPL activity in whole homogenates of red vastus muscle in rats sacrificed immediately after the last bout of treadmill mnning. Three days later, enzyme activity was twice as high, and 5 days later nearly 50% higher in muscle of trained versus control rats. Afer 7 days, LPL activity in muscle of the mnners was back to control levels. Fat cell size was used as a marker of the trained state. We felt justified in using adipocyte diameter as an index of training, since the adaptation reflects a decrease in size and since the diameter remains significantly reduced for at least 9 days after exercise has stopped (Craig et al. 1983). The finding that adipocyte diameter was significantly smaller in the mnners (74 f 2 pm) compared with that in the control rats (106 f 4 pm) provides evidence that the mnners were highly trained.
Biscnssisn Bre-heparin perhsate LBL activity increased ninefold measured immediately after the last bout of work in the hindlimb of the exercise-trained rats. At the same time, post-heparin perfusate LPL activity was markedly decreased in the trained animals. These results provide evidence that exercise-training has a heparin-like effect on capillary bound LPL activity. The total amount of LPL activity (i.e., pre-heparin perhsate plus post-heparin perfusate) was twofold greater in the treadmill mnners than in the controls seen immediately after exercise. These results provide evidence that exercise increases the capacity to clear triglycerides from circulation. The data in the present study were collected on trained rats. There is evidence that muscle LPL activity is also increased in response to acute exercise. For example, Paulin et al. (199 1) reported that LPC activity was elevated in post-heparin plasma measured 24 h after a I-h mn on a treadmill in previously untrained rats. In another study in the heart, Goldberg et d . (1984) reported that both heparin-releasable and tissue-bound LPL activity were elevated after a single bout of treadmill mnning in previously untrained rats. It has long been recognized that heparin causes the release of LPL from capillary beds in a variety of tissues including skeletal muscle and that the release occurs rapidly (Borensztajn 1987; Robinson and Harris 1959). It has been hypothesized h a t the release mechanism probably involves the formation of a heparin-enzyme complex; the enzyme could be detached from the cell surface heparan sulfate either
through competition between the soluble heparin and the membrane-bound heparan sulfate for the same binding site or as a result of a conformationd change in the protein molecule induced by heparin (8livecrona et d. 197'7). The mechanism(~)by which exercise causes the release of LPL activity from capillary walls similar to that seen with heparin is not clear. However, at least two possibilities can be advanced. First, the release of capillary-bound LBL activity could have been mediated by an exercise-induced increase in plasma free fatty acids (FFA). It has long been recognized that exercise increases the concentration of plasma FFA (Friedberg et al. 1963; Have1 et al. 1964; Oscai 1979). In turn, FFA have recently been shown to remove LPL activity from endothelial cells [Peterson et al. 1990; Saxena et d.1989). A second possibility is that the increased force caused by the increased rate of blood flow is responsible. A third possibility is that an exercise-induced conformational change could have taken place brought about by a blood-borne factor h a t is yet to be identified. The vascular endothelium releases a variety of substances that interact directly with other tissues. For example, it has been demonstrated that endothelin- l interacts with intestinal and uterine smooth muscle, impacts on the central nervous system, and has cardiac as well as mitogenic actions (Yanagisawa and Masaki 1989). It has long been recognized that exercise exerts pleiotropic effects in a variety of tissues. Therefore, it is conceivable that the exercise-induced release s f LPE from capillary walls might be mediated by blood-borne substances contributed by the neurocrine or endocrine system and (or) brought about by the autcscrine or paracrine system. It could be argued that LLPL activity measured in the perhsates originated from both the extracehlar compartment and the intracellular pool. Moreover, the endothelium could have internalized enzyme that could have been blocked by the presence of FFA. In contrast with this notion, there is evidence that only extracellular LPL activity was measured in the medium. In a previous paper (Oscai et al. 19821, intracellular LPL activity was determined in heparin-perfused hindimb of rats under conditions similar to those used in this study. As a check on the technique of perfusing the hindlimb with heparin to fractionate LPL activity in skeletal muscle in the earlier study, rats were treated with Triton WR-1339. Triton selectively inhibits LPL activity that is directly involved in the hydrolysis of circulating triglycerides in capillary beds. The finding that LLP activity in the intracellular fraction of the soleus md fast-twitch red fibers of the quadriceps was essentidly the same for both techniques provides suggestive evidence that enzyme was not being pulled from the intracellular fraction during perfusion. Further support for the notion that o d y extracellular enzyme was being measured comes from the finding in the present study that LPL activity was not detectable at the end of the perfusion period. There is evidence that substantial amounts of TG can be hydrolyzed when LPL activity is released into circulation by action of either heparin or exercise. With respect to heparin, HAn (1943) found that when dogs with marked lipemia were injected with heparin, the lipemia disappeared completely in blood samples taken 3 -5 min later. This early finding led to the discovery of LPL. In another study, Remie et al. (1976) fed rats 5 mL of corn oil by stomach tube. Three hours later, when their plasma was visibly lipemic, the animals were given 200 units of sodium heparin subcutaneously. Administration of heparin to the corn oil fed animals resulted in an increase in plasma fatty acid concentration to more than twice the control
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908
CAN. J. PHYSIOL. PMARMACOL. VOL. 70, 1992
value after 18 rnin and to more than four times the control value after 40 rnin in sedentary animals. With respect to exercise, Thompson et d.(1988) injected exercise-trained humans with 10% TravamuHsion (1 mE/kg body weight). The rate of clearance sf the fat emulsion increased nearly 50% above pretraining values after 32-48 weeks of exercise. Exercise decreases muscle TG stores (Carlson et al. 1971; Havel et al. 1967; Holloszy and Booth 1976; Weitman et al. 1973). The question is whether plasma LPL plays a role in replenishing the decreased lipid stores in muscle. There are two lines of evidence suggesting that plasma TG are used to replenish the decreased fat stores in muscle. First, LPEdeficient mice (cld) develop severe hyperchylornicronernia because of an inability to clear TG from circulation (Blanchette-Mac~eet al. 1986; Paterniti et al. 1983). These affected mice have no lipid droplets in myedcytes of heart and diaphragm, whereas unaffected mice have numerous lipid droplets in their muscle cells. These results suggest an important sole for LPL in the replenishment s f muscle lipid stores. Second, TG uptake from circulation is highly related to muscle LPL activity ((Linder et al. 1976; Tan et al. 1977; Terjung et al. 1983). Muscle LPL activity is markedly increased with exercise training (Nikkila 1987; Oscai et al. 1982). The results of the present study do not provide evidence that would distinguish between the use of LPE-released fatty acids as a direct source s f energy for contractile activity or as precursors for the replenishment of triglycekde stores. The results do, however, show that because of the marked increase in muscle enzyme activity, the total amount of LPL activity in circulation (i.e., pre-heparin perksate plus post-heparin perfusate) was much greater in trained rats compared with comparable sedentary controLs measured both immediately and 24 h after exercise. Semm TGs were decreased and plasma FFA increased at a time when extracellular LPE was markedly elevated. These results suggest that training increases the capacity to clear TG from circulation. Further, this effect of exercise training on muscle EPE activity appears to last for as long as 5 days after cessation of work. Finally, it should be noted that the mechanism(s) responsible for activating LPL in muscle in response to exercise remains unknown.
Acknowledgments The assistance of Vinod Singh is gratefully acknowledged. This research was supported by National Institutes sf Health research grants AM- 17357 and AR-39872. BBanchette-Macfie, E. J., Wetzel, M.G . , m d Chernick, S. S., et al. 1986. Effect of the combined lipase deficiency mutation (cld/cld) on ultrastmcture of tissues in mice diaphragm, hear%,brown adipose tissue, lung, and liver. Lab. Invest. 55: 347 -362. Blanchette-MacBaie, E. J., Masuno, W., and Bvvyer, N. K . , et al. 1989. Lipoprotein lipase in myscytes and capillary endothelium of heart: immunocytochernical study. Am. J . Phy siol . 256: E8 1 8 E828. Borensztajn, J. 1987. Heart and skeletal muscle lipoprotein lipase. In Lipoprotein liipase. Edited by J. Borensztajn. Evener Publishers Ine., Chicago, gap. 133- 148. Borensztajn, J., SamoBs, B. W., and Rubenstein, A. H. 1972. Effects of insulin on lipoprotein lipase activity in the rat heart and adipose tissue. Am. I. Physiol. 223: 1271 - 1275. Carlson, E. A., Ekelund, E.-G., and Frbberg, S. 0.1971. Concentration of triglycerides, phsspholipids and glycogen in skeletal muscle and of free fatty acids and @-hydroxybutyricacid in blood in man in response to exercise. Eur. J. Clin. Invest. 1: 248 -254.
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