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International Journal of Obesity (1999) 23, Suppl 3, S7±S10 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo Skeletal muscle substrate metabolism H Hoppeler1* 1 Institute of Anatomy, University of Berne, Berne, Switzerland Endurance power of muscles is determined largely by the capacities to oxidize substrates in mitochondria in the process of making ATP by oxidative phosphorylation. This review explores physiological and morphological factors that may cause limitation of carbohydrate and fat utilization by muscle cells. The pathways for oxygen and substrates converge in muscle mitochondria. In mammals, a structural limitation of carbohydrate and lipid transfer from the microvascular system to muscle cells is reached at a moderate work intensity (that is, at 40 ± 50% of VO2max). At higher work rates intracellular substrate stores must be used for oxidation. Because of the importance of these intracellular stores for aerobic work we ®nd larger intramyocellular substrate stores in endurance trained athletes. The transfer limitations for carbohydrates and lipids on the level of the sarcolemma implies that the design of the respiratory cascade from lungs to muscle mitochondria re¯ects primarily oxygen demand. Keywords: mitochondria; lipid; carbohydrate; muscle What governs substrate metabolism in muscle cells? Muscle mitochondria set the demand for oxygen and substrates In order to understand substrate selection of working muscle cells we must consider how the pathways for oxygen and substrates converge at the mitochondria and how substrate ¯uxes intervene with setting oxygen consumption. The O2 pathway from the environment to muscle mitochondria is simple: it follows a single path without branches and there are only very limited O2 stores in myoglobin. The substrate pathways are more complex. Both carbohydrate and lipid pathways branch to form four parallel pathways that converge on the mitochondria (Figure 1). Proteins can be ignored because in well-fed subjects their contribution to aerobic metabolism is minimal. During exercise, when rates of substrate oxidation are highest, blood is shunted away from the gut and rates of substrate uptake are lowest. At this point, stores supply most of the fuel for oxidation. Organismic substrate stores outside muscle are in the liver and in the adipose tissue. Inside the muscle cells carbohydrates are stored as glycogen granules, and fatty acids as lipid droplets (Figure 2). We will have to delineate the importance of the parallel substrate pathways as a function of exercise intensity and duration. We would also like to know whether limiting factors are primarily structural or functional. When humans exercise at their maximal rate of oxygen consumption, the mitochondria located in their skeletal muscles consume more than 90% of the oxygen and substrates.1 Even at lower work intensities the main substrate consumer is muscle tissue. We are therefore justi®ed in concentrating on the mitochondria in this particular tissue. The mitochondrion is bounded by a continuous outer membrane and contains a second inner membrane that forms multiple infoldings, the cristae (Figure 2). Between the membranes we ®nd the `inter-membrane space'; the innermost compartment is the mitochondrial matrix. In the matrix, substrates are broken down in the Krebs cycle generating reducing equivalents. The beta-oxidation of fatty acids is also located in the matrix. Thus, the matrix volume is the appropriate structural parameter to characterize substrate catabolism, assuming a constant density of enzymes in the matrix space. In most mammals, matrix volume appears to be proportional to mitochondrial volume and the ratio between mitochondrial volume and matrix volume is also invariant under conditions of endurance exercise training.2 It follows from these constant relationships that the volume of mitochondria in skeletal muscle is a good measure of matrix enzymes, as well as the quantity of respiratory chain present. The supply of substrates from cellular stores to mitochondria *Correspondence: Dr. H. Hoppeler, Institute of Anatomy, University of Berne, BuÈhlstrasse 26, CH-3000 Berne 9, Switzerland When exercising at VO2max, substrates must be transported into the mitochondria at a rate suf®cient Substrate metabolism in muscle H Hoppeler S8 Figure 1 Model for structure function relationship of oxygen and intracellular substrate supply to the mitochondria of skeletal muscle cells. Dots indicate oxygen, open circles indicate fatty acids, squares indicate glucose; row of squares indicates glycogen and triangles indicate acetyl CoA. The horizontal arrows indicate the pathways of intracellular substrate breakdown from the intracellular stores to the terminal oxidase in the mitochondrial membrane (black square). The vertical arrows indicate the supply routes of oxygen and substrates from the capillaries, with dotted arrows for the supply route to intracellular stores (temporally split from the phase of oxidation; modi®ed from 6). CHOic intracellular glucose stores, glycogen Gls glycolysis KC= Krebscycle b OX beta oxidation FATic interacellular lipid stores. Figure 2 Electron micrograph of longitudinal section of skeletal muscle tissue. In the center, on the level of the z-line, we ®nd inter®brillar mitochondria with a lipid droplet immediately adjacent. li lipid droplet mc central mitochondria mf myo®laments marker indicates 0.5 mm. to fuel oxidative phosphorylation. The oxidation of one mole of glucose consumes 6 moles of oxygen, and one mole of fat consumes 23 moles of O2. It has been clearly established in dogs and goats that circulatory carbohydrates and lipids can only cover a fraction of the total substrate demand of mitochondria in working muscles.3,4 Both carbohydrate and lipid transport from capillaries into muscle cells appear to be maximal at quite low exercise intensities, corresponding to approximately 40% of VO2max and cannot be upregulated at higher work intensities. Instead, the mitochondria depend on obtaining their fuels from intracellular substrate stores whereby at VO2max over 80% of the fuel is supplied from glycogen granules. These results were obtained in a comparative setting of adaptive variation, but they are very similar to the situation observed in exercising humans where a heavy reliance on intracellular substrate stores at higher work intensities has also been demonstrated.5 From this it follows that substrates for mitochondrial oxidation at work intensities of around 80% of VO2max must primarily be supplied from glycogen granules and lipid droplets inside the muscle cells, with no more than 20 ± 30% of the fuel coming from the capillaries. There is evidence that this is due to a limitation of fuel supply from the capillary with the sarcolemma as the main barrier.6 What are the structures that determine ¯ow through the intracellular substrate pathways? On careful observation, one ®nds that all of the lipid droplets are in direct contact with the outer mitochondrial membrane.6 They form a tight contact which is often marked by a dent in the mitochondria (Figure 2). It seems likely that the fatty acids are transported across the mitochondrial membrane preferentially in these areas of contact, allowing them to circumvent transport problems associated with their low solubility in the cytosol. The maximal ¯ow through the cytosolic pathway is reached at low exercise intensities, and it can supply at best about 20% of the fuel required at VO2max . From this it follows that inter®brillar mitochondria likely depend substantially on lipid droplets for fatty acid supply. The importance of intracellular lipid stores for aerobic performance has long been suspected. Already in 1973 it was noted that highly endurance trained subjects had considerably larger substrate stores in the form of lipid droplets in their muscles.7 Later it was found that six weeks of endurance exercise training was suf®cient to increase intracellular lipid concentration by a factor of two from 0.5% to 1% of the muscle ®ber volume. Highly trained cyclists or endurance runners may have even larger lipid stores in their trained muscles.8 Moreover, it was demonstrated in 1976 that the lipid (and glycogen) stores disappeared almost completely after a 100 km run9,10 indicating that the intracellular lipid droplets represent the metabolically accessible form of substrate reserves in muscle tissue. In situations in which the reliance on fatty acid metabolism is reduced, such as in high altitude residents, we ®nd that intracellular substrate stores are massively reduced also.11 An additional observation which is likely relevant to the understanding of lipid oxidation in skeletal muscle tissue relates to the distribution of mitochondria Substrate metabolism in muscle H Hoppeler within the muscle cell. Whereas a large fraction of mitochondria is found interspersed between myo®brils, there is a smaller population of mitochondria found massed under the sarcolemma close to capillaries (subsarcolemmal mitochondria). The location of these mitochondria puts them in close vicinity to the vascular supply of both oxygen and substrates. It has been demonstrated by mathematical modelling that mitochondrial distribution within muscle cells is not likely to play a major role for oxygen diffusion because diffusivity of oxygen is high in muscle ®bers partly related to the myoglobin content of muscle.12 As a consequence of their location under the sarcolemma, problems of cytosolic transport of substrates is minimal for subsarcolemmal mitochondria. It can be argued that this puts these mitochondria in a favourable position with regard to oxidizing lipids supplied by capillaries (for a review of lipid transfer to mitochondria see reference 13). The notion of a preferential oxidation of vascular lipids by subsarcolemmal mitochondria is further supported by the circumstantial evidence indicating a greater relative increase of subsarcolemmal versus inter®brillar mitochondria with endurance exercise training.14 This ®nding can be interpreted as indicating a general shift towards a larger reliance on fat metabolism which has two structural components a) an increase of substrate stores favouring lipid metabolism of inter®brillar mitochondria and b) a large increase in subsarcolemmal mitochondria favouring oxidation of lipids taken from the vasculature. It is currently unclear what throttles triacylglycerol oxidation in exercising muscle. Is it the transfer of fatty acids to the mitochondrial matrix or is it boxidation?13 Likewise, the control of the balance between lipid and carbohydrate (CHO) metabolism in muscle cells is poorly understood. In resting muscle there is some indication that the glucose ± fatty acid cycle (Randle cycle) is operative, however under conditions of exercise the mechanism of regulation is currently debated.15 A possible regulation could also occur through malonyl-CoA. The increase in the use of lipids as a substrate with long-term exercise of low intensity is probably related to the increase in plasma free fatty acids,5,15. However, again we do not know how the muscle cell selects between the two substrates and what governs use of intracellular versus extracellular substrates. The rate at which glycogen stores can supply pyruvate to the Krebs cycle or to further degradation to lactate is not really limited. Glycogen stores supply pyruvate up to the limit of oxidation by the mitochondria, and then can achieve a further several-fold increase in break down in order to fuel anaerobic glycolysis. Clearly, therefore, oxidation in the mitochondria is not limited by the supply of fuel but rather by the supply of oxygen from the capillaries Ð or by the amount of mitochondria that can perform oxidative phosphorylation. Microcirculatory supply of oxygen and substrates Oxygen diffuses from the capillaries to the mitochondria. In general terms, the maximal conductance must be related to the total volume of capillaries in skeletal muscles, a structural parameter that has been measured. Capillary volume is directly proportional to the capillary surface area available for diffusion of oxygen out of the blood, since the diameter of capillaries does not change with both size or adaptation. It has been shown that there is a reasonably good match between structure and the oxygen supply function at the level of the capillaries. It is generally found that the higher rates of oxygen consumption are matched by corresponding differences in the amount of capillary structure.16 What about the transport of substrates out of the capillaries? The maximum rate appears to be reached at very low exercise intensities, as noted above. Even at 40% of VO2max, only about 20 ± 30% of the substrates are supplied from the circulating blood, and the absolute rate does not increase with increasing intensity. Very similar results have been obtained in exercising humans as well as in exercising dogs and goats.5,17 At work intensities above 40% VO2max intracellular substrate stores, in particular glycogen, are used to fuel aerobic work, as noted above. The transport of glucose from capillaries to myocytes has been studied by comparing `athletic' dogs to `sedentary' goats.6 The transfer of glucose from the plasma to the interstitial ¯uid is assumed to occur through the small pore system at the endothelial intercellular junction. Calculations indicate that neither the pore system nor the interstitial space between the capillary and the myocyte introduce a major resistance to glucose ¯ux across the sarcolemma. Based on morphologic evidence it is concluded that the glucose transporters of the sarcolemma are likely saturated at low to moderate exercise intensities and are responsible for the major part of the resistance in the carbohydrate pathway. The transport and utilization of fatty acids in muscle has recently been reviewed extensively.13 Based on data published in many studies these authors come to the conclusion that it is the delivery rather than the oxidative metabolism in myoytes that is the rate ± limiting factor in lipid oxidation during exercise. However, many uncertainties remain and lipid oxidation could be regulated at various steps in the route of transport as well as within the metabolic pathways. The general consensus seems to be that endurance exercise training increases the importance of fatty acids as a source of aerobic energy. Likewise, recent evidence suggests that high fat diets do have a potential for shifting metabolism towards a higher reliance on fatty acid metabolism.13 These general observations could be of importance for athletes S9 Substrate metabolism in muscle H Hoppeler S10 competing in endurance events, in particular in very long term endurance events where intensities are low and the importance of fat oxidation high. In this context it may be noted that for certain long term endurance events lasting over days or weeks, such as multi stage bicycle races, the concept of maximal sustained metabolism may be more important than that of VO2max.18 In these events racers are limited in their performance by the need of having to balance work output to substrate uptake. For humans the limit of energy uptake with nutrition seem to be approximately 7 000 kcal=d. Therefore, professional cyclists are capable of elevating basal metabolism acutely by some 20-fold (aerobic scope) while average 24 h metabolism (maximal sustained metabolism) can only be increased 4-fold. Reviewing the transfer processes for oxygen and substrates from capillaries to myocytes we can conclude that muscle microvasculature is optimally designed for the transfer of oxygen, but not for the transfer of substrates. Because of the lack of substantial oxygen stores, oxygen has to be supplied by the circulation so as to match the demand of the contracting muscle cells. By contrast, only a limited quantity of substrates is supplied from the vasculature during exercise. Animals and humans working at intensities above 30% of VO2max rely increasingly on intracellular substrate stores mainly on glycogen at high work intensities. These stores will eventually be exhausted during exercise leading to muscle fatigue. The intracellular stores can then be replenished during periods of rest at low transfer rates. Conclusions Our analysis shows that human endurance capacity is limited mainly by structural constraints. The entire respiratory system from lung to muscle mitochondria is built on the constraint of supplying oxygen rather than substrates to active muscle mitochondria. Carbohydrate and lipid supply rates are likely throttled by transport processes at the level of the sarcolemma. In order to ascertain adequate substrate supply at high work loads both lipids and carbohydrates are stored within muscle cells. These substrate stores are replenished at low ¯ux rates during periods of rest to reach a size adequate for high rates of combustion in exercise. References 1 Mitchell JH, Blomqvist G. Maximal oxygen uptake. N Engl J Med 1971; 284: 1018 ± 1022. 2 Davies KJA, Packer L, Brooks GA. Biochemical adaptation of mitochondria, muscle, and whole-animal respiration to endurance training. Arch Biochem Biophys 1981; 209: 539 ± 554. 3 Weber J-M, Brichon G, Zwingelstein G, McClelland G, Saucedo C, Weibel ER, Taylor CR. Design of the oxygen and substrate pathways. IV. Partitioning energy provision from fatty acids. J Exp Biol 1996; 199: 1667 ± 1674. 4 Weber J-M, Roberts TJ, Vock R, Weibel ER, Taylor CR. Design of the oxygen and substrate pathways. III. Partitioning energy provision from carbohydrates. J Exp Biol 1996; 199: 1659 ± 1666. 5 Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, Wolfe RR. Regulation of endogeneous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am J Physiol 1993; 265: 380 ± 391. 6 Vock R, Hoppeler H, Claassen H, Wu DXY, Billeter R, Weber JM, Taylor CR, Weibel ER. Design of the oxygen and substrate pathways. VI. Structural basis of intracellular substrate supply to mitochondria in muscle cells. J Exp Biol 1996; 199: 1689 ± 1697. 7 Hoppeler H, LuÈthi P, Claassen H, Weibel ER, Howald H. 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