Acta Physiol Scand 1979, 107: 257-261
Changes in lipoprotein-lipase activity and lipid stores in human skeletal muscle with prolonged heavy exercise HANS L I T H E L L , JAN ORLANDER, RICKARD S C H E L E , BERTIL SJODIN and JAN KARLSSON Department of Geriatrics, University of Uppsala, the Department of Animal Nutrition. Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance (FOA 57). Stockholm, Sweden
LITHELL, H., ORLANDER, J., SCHELE, R., SJODIN, B. & KARLSSON, J.: Changes in lipoprotein-lipase activity and lipid stores in human skeletal muscle with prolonged heavy exercise. Acta Physiol Scand 1979, 107:257-261. Received 19 April 1979. ISSN 0001-6772.Department of Geriatrics, University of Uppsala, the Department of Animal Nutrition, Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance (FOA 57).Stockholm, Sweden. Lipoprotein-lipase (LPL) activity and intracellularly stored triglycerides were determined in muscle biopsies taken before and after an 85 km skiing race from 7 volunteers. The triglyceride stores were larger in slow twitch than in fast twitch fibres (proportions 5 : 1 before the race). The LPL activity increased and the triglyceride stores in slow twitch fibres decreased during the race. The best trained subjects had the largest TG stores before the race and their TG stores also decreased rriost during the race. These subjects also had very small increases of LPL activity. The least trained subject on the other hand showed a 6-fold increase of LPL activity. The high post-race LPL activity in less trained subjects indicates a higher capacity for uptake of fatty acids from serum TG as compared to the more trained subjects. Key words: Intermyofibrillar fat; lipoprotein-lipase activity; muscle fibre types; prolonged exercise.
During normal dietary conditions the relative exercise intensity (i.e. actual $‘,,/maximal V o 2 ) determines to what extent energy is supplied from carbohydrates o r fat (Christensen & Hansen 19390). Christensen & Hansen (19396) found that increasingly more energy was derived from fat combustion during prolonged exercise at a relative intensity of about 6 5 % , but no fat adaptation was seen a t intensities above 75% (Hermansen e t al. 1967). Fat combustion during exercise was higher after a fat-rich diet than after a carbohydrate-rich diet (Christensen & Hansen 1 9 3 9 ~o)r a normal diet (Bergstrom et al. 1967). During exercise the uptake of free fatty acids (FFA) from plasma into muscles is related to its concentration in the plasma (for ref. see Gollnick 1977). The increase in fat combustion during prolonged exercise, however, can only partly be explained by an increased uptake of plasma free fatty acids (for ref. see Froberg 1972). Fatty acids might also be supplied from plasma triglycerides (TG) which are hydrolysed by the en-
zyme lipoprotein-lipase (LPL) localized at the endothelium in the muscle capillaries (Robinson 1970). A work-induced increase of the enzyme activity should mean that an increased amount of fatty acids were made available to the muscles (Robinson 1970). In addition an increased utilization of intramuscular TG stores, that are hydrolyzed by the intracellularly located, so called hormone-sensitive lipase (Bjorntorp & Furman 1962), has been demonstrated both in animals (Baldwin et al. 1973, Reitman et al. 1973) and humans (Howald 1975, Essen et al. 1977). The objective of the present investigation has been to evaluate the changes in both LPL activity and intracellular lipid stores in muscle tissue during extremely prolonged, heavy exercise.
SUBJECTS AND METHODS Seven male physical education students who participated in an 85 km cross-country skiing race (the Vasa race 1977)
258
H. Lithell et crl.
Table 1. Clrcirtirteristics o j t h e 7 subjects Mean
S.D.
Range
26.7 175.7 76.4 4.76
3.2 4.3 6.8 0.37
23.1-32.2 169.7-180.8 66.6-83.1 4.16-5.22
62.7
6.6
46.4
14.6
2144
(Robinson 1970). Specificity tests have excluded that any of the determined enzyme activity is due to hormonesensitive lipase. Maximal oxygen uptake (Vo2max) was determined during treadmill exercise one week before the competition according to the Douglas bag procedure (Saltin & Astrand 1967). Statistical methods: Student’s t-distribution was used when testing the means of individual changes (before-after) and differences (ST-FT) and when testing correlation coefficients.
429 8.07
291 1.39
l o g 1 000 5.32-9.62
RESULTS
~
Age (yrs) Height (cm) Weight (kg) V o , max (I x min-I) V,,xmax (ml x kg-’ x min-I) Muscle composition (%ST) Amount of ski training (km) Performance time (h)
57.3-78.0
volunteered for the study. The subjects were of normal body weight and height but varied considerably in capacity (within the upper part of the normal range) and training level as indicated in Table 1 . Mean performance time was 8.07 h (range 5.32-9.62). Biopsies were taken from the vastus lateralis the day before the race and immediately after the race with a needle technique (Bergstrom 1962). The biopsies were divided into three portions. One was embedded in a plastic medium for histochemical analysis by staining sections for myosin ATPase and identification of slow twitch (ST) and fast twitch (FT) fibres (Gomori 1941, Padykula & Herman 1955). The second portion was used for electron microscopy: Small pieces of muscle tissue were fixed in 4 % phosphate-buffered glutaraldehyde, pH 7.4 (at least 2 h at O T ) , washed, post-fixed in 1 % veronal-buffered osmium tetroxide, washed in Tyrode’s solution, and dehydrated in ethanol. After treatment with propylene oxide, the muscle pieces were embedded in Epon. Approximately 60-70 nm thick longitudinal sections were cut with a LKB Ultrotome I , stained with uranyl acetate and lead citrate, and examined in a Siemens Elmiskop 101. About 25 micrographs (final magnification about 25 000) were taken at random from two tissue blocks per biopsy. Volume fractions of myofibrils, mitochondria, lipid droplets and sarcoplasm (including sarcoplasmic reticulum and transverse tubules) were estimated stereologically according to Weibel (1969). The study was restricted to the fibrillar space of the muscle fibres. The fibres were classified as ST or FT according to Z-line width (Payne et al. 1975, Wroblewski & Jansson 1975, Eisenberg & Kuda 1976, Tomanek 1976, Bylund et al. 1977) measured to the nearest 0.1 mm with a measuring magnifier. Only clearly demarcated Z-lines were chosen for measurement, excluding the fuzzy matrix present at the Z-line border. Muscle LPL activity was determined in the third portion of the biopsy as described by Lithell & Boberg (1978). The biopsies were incubated in a reaction medium containing :%H-trioleatelabelled Intralipid (Vitrum, Stockholm, Sweden). The release of %H-oleicacid was used as a measure of LPL activity. The activity was expressed as nmol fatty acids (FA) released per minute and per gram wet weight of muscle tissue (nmol FA X g-l X min-I). This method determines the activity of the heparin-releasable LPL, located at the capillary endothelium
Skeletal muscle LPL activity averaged 18.4 nmol FA x g-’ x min-’ (range 11.9-31.4) before the race and the average increase during the race was 238 % (range 8 4 3 7 % ) (Table 2 ) . The increase in LPL activity was negatively related to V o 2 max (r=-O.85, P
Changes in lipoprotein-lipase activity and lipid stores in human skeletal muscle with prolonged heavy exercise HANS L I T H E L L , JAN ORLANDER, RICKARD S C H E L E , BERTIL SJODIN and JAN KARLSSON Department of Geriatrics, University of Uppsala, the Department of Animal Nutrition. Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance (FOA 57). Stockholm, Sweden
LITHELL, H., ORLANDER, J., SCHELE, R., SJODIN, B. & KARLSSON, J.: Changes in lipoprotein-lipase activity and lipid stores in human skeletal muscle with prolonged heavy exercise. Acta Physiol Scand 1979, 107:257-261. Received 19 April 1979. ISSN 0001-6772.Department of Geriatrics, University of Uppsala, the Department of Animal Nutrition, Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance (FOA 57).Stockholm, Sweden. Lipoprotein-lipase (LPL) activity and intracellularly stored triglycerides were determined in muscle biopsies taken before and after an 85 km skiing race from 7 volunteers. The triglyceride stores were larger in slow twitch than in fast twitch fibres (proportions 5 : 1 before the race). The LPL activity increased and the triglyceride stores in slow twitch fibres decreased during the race. The best trained subjects had the largest TG stores before the race and their TG stores also decreased rriost during the race. These subjects also had very small increases of LPL activity. The least trained subject on the other hand showed a 6-fold increase of LPL activity. The high post-race LPL activity in less trained subjects indicates a higher capacity for uptake of fatty acids from serum TG as compared to the more trained subjects. Key words: Intermyofibrillar fat; lipoprotein-lipase activity; muscle fibre types; prolonged exercise.
During normal dietary conditions the relative exercise intensity (i.e. actual $‘,,/maximal V o 2 ) determines to what extent energy is supplied from carbohydrates o r fat (Christensen & Hansen 19390). Christensen & Hansen (19396) found that increasingly more energy was derived from fat combustion during prolonged exercise at a relative intensity of about 6 5 % , but no fat adaptation was seen a t intensities above 75% (Hermansen e t al. 1967). Fat combustion during exercise was higher after a fat-rich diet than after a carbohydrate-rich diet (Christensen & Hansen 1 9 3 9 ~o)r a normal diet (Bergstrom et al. 1967). During exercise the uptake of free fatty acids (FFA) from plasma into muscles is related to its concentration in the plasma (for ref. see Gollnick 1977). The increase in fat combustion during prolonged exercise, however, can only partly be explained by an increased uptake of plasma free fatty acids (for ref. see Froberg 1972). Fatty acids might also be supplied from plasma triglycerides (TG) which are hydrolysed by the en-
zyme lipoprotein-lipase (LPL) localized at the endothelium in the muscle capillaries (Robinson 1970). A work-induced increase of the enzyme activity should mean that an increased amount of fatty acids were made available to the muscles (Robinson 1970). In addition an increased utilization of intramuscular TG stores, that are hydrolyzed by the intracellularly located, so called hormone-sensitive lipase (Bjorntorp & Furman 1962), has been demonstrated both in animals (Baldwin et al. 1973, Reitman et al. 1973) and humans (Howald 1975, Essen et al. 1977). The objective of the present investigation has been to evaluate the changes in both LPL activity and intracellular lipid stores in muscle tissue during extremely prolonged, heavy exercise.
SUBJECTS AND METHODS Seven male physical education students who participated in an 85 km cross-country skiing race (the Vasa race 1977)
258
H. Lithell et crl.
Table 1. Clrcirtirteristics o j t h e 7 subjects Mean
S.D.
Range
26.7 175.7 76.4 4.76
3.2 4.3 6.8 0.37
23.1-32.2 169.7-180.8 66.6-83.1 4.16-5.22
62.7
6.6
46.4
14.6
2144
(Robinson 1970). Specificity tests have excluded that any of the determined enzyme activity is due to hormonesensitive lipase. Maximal oxygen uptake (Vo2max) was determined during treadmill exercise one week before the competition according to the Douglas bag procedure (Saltin & Astrand 1967). Statistical methods: Student’s t-distribution was used when testing the means of individual changes (before-after) and differences (ST-FT) and when testing correlation coefficients.
429 8.07
291 1.39
l o g 1 000 5.32-9.62
RESULTS
~
Age (yrs) Height (cm) Weight (kg) V o , max (I x min-I) V,,xmax (ml x kg-’ x min-I) Muscle composition (%ST) Amount of ski training (km) Performance time (h)
57.3-78.0
volunteered for the study. The subjects were of normal body weight and height but varied considerably in capacity (within the upper part of the normal range) and training level as indicated in Table 1 . Mean performance time was 8.07 h (range 5.32-9.62). Biopsies were taken from the vastus lateralis the day before the race and immediately after the race with a needle technique (Bergstrom 1962). The biopsies were divided into three portions. One was embedded in a plastic medium for histochemical analysis by staining sections for myosin ATPase and identification of slow twitch (ST) and fast twitch (FT) fibres (Gomori 1941, Padykula & Herman 1955). The second portion was used for electron microscopy: Small pieces of muscle tissue were fixed in 4 % phosphate-buffered glutaraldehyde, pH 7.4 (at least 2 h at O T ) , washed, post-fixed in 1 % veronal-buffered osmium tetroxide, washed in Tyrode’s solution, and dehydrated in ethanol. After treatment with propylene oxide, the muscle pieces were embedded in Epon. Approximately 60-70 nm thick longitudinal sections were cut with a LKB Ultrotome I , stained with uranyl acetate and lead citrate, and examined in a Siemens Elmiskop 101. About 25 micrographs (final magnification about 25 000) were taken at random from two tissue blocks per biopsy. Volume fractions of myofibrils, mitochondria, lipid droplets and sarcoplasm (including sarcoplasmic reticulum and transverse tubules) were estimated stereologically according to Weibel (1969). The study was restricted to the fibrillar space of the muscle fibres. The fibres were classified as ST or FT according to Z-line width (Payne et al. 1975, Wroblewski & Jansson 1975, Eisenberg & Kuda 1976, Tomanek 1976, Bylund et al. 1977) measured to the nearest 0.1 mm with a measuring magnifier. Only clearly demarcated Z-lines were chosen for measurement, excluding the fuzzy matrix present at the Z-line border. Muscle LPL activity was determined in the third portion of the biopsy as described by Lithell & Boberg (1978). The biopsies were incubated in a reaction medium containing :%H-trioleatelabelled Intralipid (Vitrum, Stockholm, Sweden). The release of %H-oleicacid was used as a measure of LPL activity. The activity was expressed as nmol fatty acids (FA) released per minute and per gram wet weight of muscle tissue (nmol FA X g-l X min-I). This method determines the activity of the heparin-releasable LPL, located at the capillary endothelium
Skeletal muscle LPL activity averaged 18.4 nmol FA x g-’ x min-’ (range 11.9-31.4) before the race and the average increase during the race was 238 % (range 8 4 3 7 % ) (Table 2 ) . The increase in LPL activity was negatively related to V o 2 max (r=-O.85, P
