Endurance exercise
9.2 Effects of exercise intensity
9.2.2 Lipid metabolism
It is well documented that the contribution of lipid sources to ATP production is inversely related to exercise intensity and that it declines beyond intensities greater than 65% VO2max. There are a number of potential mechanisms underpinning this intensity-induced shift from lipid to CHO oxidation, and we will now discuss the potential sites involved in regulating these processes.
Adipose tissue lipolysis and FFA availability/delivery
The first stage of failure in lipid oxidation rates with increased exercise intensity is often cited as
a reduction in adipose tissue lipolysis. However, there is strong evidence to suggest that it is not a failure in lipolysisper se, which is the limitation, but rather an inability to deliver FFA to the exer- cising muscles primarily as a result of inadequate adipose tissue blood flow.
Indeed, Romijn et al. (1993) assessed plasma FFA (remember, FFA is non-soluble and requires albumin for transport) and glycerol (which is water soluble) during exercise at 25, 65 and 85%
VO2max (see Figure 9.6). These authors observed that plasma FFA declined during exercise at 85%
VO2max, although these data should be not be interpreted as a reduction in lipolysis, given that plasma glycerol increased during exercise.
The use of plasma glycerol per se as an indi- cator of lipolysis is, of course, not without limita- tions, as glycerol may also be taken up by other tissues, namely the liver. Nevertheless, stable iso- tope methodology demonstrated that the rate of appearance of glycerol (considered an accurate indication of the rate of lipolysis) was not reduced at 85% VO2max, compared to 65% VO2max. It was therefore suggested that the reduced plasma FFA availability was not to due to reduced lipolysis, but rather a reduction in adipose tissue blood flow (given that more blood is now being directed to the active muscles) and, as such, there was less albu- min available to transport FFA. In such cases, FFA may become re-esterified within the adipocyte due to this inadequate perfusion (Frayn, 2010).
Evidence supporting this theory of blood flow limitation is also provided in the recovery period after exercise, where it can be seen that as soon as exercise terminates (and blood flow is thus available for adipose tissue again), plasma FFA suddenly increases, whereas plasma glycerol (and, indeed, rate of appearance) actually declines, therefore indicating reduced lipolysis post-exercise (see Figure 9.6).
In a subsequent study, Romijn et al. (1995) tested the hypothesis that reduced plasma FFA availability is limiting to lipid oxidation rates during high-intensity exercise by intravenous infusion of lipid and heparin during exercise at 85% VO2max. In this way, plasma FFA was artificially maintained above 1 mmol.L−1, which
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Figure 9.6 Plasma glycerol (red symbols) and FFA (green symbols) during and in recovery from exercise at (a) 25, (b) 65 and (c) 85% VO2max(adapted from Romijn et al., 1993)
was in marked contrast to control exercise con- ditions (0.2–0.3 mmol.L−1). However, although lipid oxidation rates were increased with lipid and heparin infusion (34µmol.kg−1. min−1)compared with control (26µmol.kg−1. min−1), they were still not restored to levels observed during exercise at 65% VO2max (43µmol.kg−1. min−1).
Based on these data, it can be assumed that approximately only half of the reduced lipid oxidation rates observed with increased exercise intensity can be explained by reduced FFA avail- ability. Furthermore, the actual rate of appearance of plasma FFA exceeded lipid oxidation rates (61 versus 34µmol.kg−1. min−1, respectively) thus suggesting there may be a failure in FFA transport across the plasma and/or mitochondrial membranes.
FFA transport into the cytosol
In order for FFAs to be oxidized by skeletal mus- cle, they must firstly pass through the endothelium, the interstitial space, the sarcolemma and, finally, the mitochondrial membranes. In principle, there could therefore be a failure of FFA to actually get into the muscle itself even before they pass through the mitochondrial membrane. The process of transport into the cytosol is regulated by a number of transport proteins, including fatty acid translocase (FAT/CD36), membrane fatty acid binding protein (FABPpm) and fatty acid transport protein (FATP).
However, while these proteins may be limit- ing in other situations (as discussed later in this chapter), it is unlikely that they are a contributing factor to the reduction in lipid oxidation which occurs with increased exercise intensity. Indeed, Kiens et al. (1999) observed intra-muscular con- tent of long chain fatty acids (LCFA) to actu- ally increase as intensity progressed from 65 to 90% VO2max, despite reductions in lipid oxida- tion rates. Such data therefore point to a failure of the mitochondria to increase lipid utilization, either through a failure in transport across mitochondrial membranes and/or metabolic oxidation.
FFA transport across mitochondrial membranes
In recapping what we discussed in Chapter 6, you should remember that, once in the cytosol, fatty acids combine with CoA to form fatty acyl-CoA through the action of acyl-CoA synthase. Theacti- vated fatty acid can now pass through the outer mitochondrial membrane by combining withcar- nitine to form acylcarnitine in a reaction catal- ysed by CPTI. Acylcarnitine is, in turn, trans- ported through the inner mitochondrial membrane via the acylcarnitine/carnitine translocase system.
Once on the matrix side, the fatty acyl group is transferred back to CoA, thus reforming fatty acyl- CoA in a reaction catalysed by CPTII. In this way, the free carnitine that has been cleaved from acylcarnitine can be transported back to the outer mitochondrial membrane to participate in the ini- tial reaction involving CPTI.
Of the various components in this transport shut- tle system, CPTI activity is thought to be rate limiting. It is important to note that only LCFAs (i.e. those with carbon chain lengths of 12 to18) are transported using this mechanism, whereas the medium and short chain fatty acids can freely dif- fuse into the mitochondrial matrix.
To test the hypothesis that FFA transport across mitochondrial membranes is limiting during high-intensity exercise, Sidossis et al. (1997) conducted a study using isotope methodology during which oleate (a LCFA) and octanoate (an MCFA which does not require facilitated transport) were infused during exercise at both 40 and 80% VO2max. Lipid and heparin were also infused during high-intensity exercise, in order to offset the reduction in plasma FFA that occurs at these intensities and therefore to ensure that plasma FFA was similar between trials.
Interestingly, the percentage of oleate uptake that was oxidized during exercise was signifi- cantly decreased during exercise at 80% VO2max, compared with 40% VO2max, whereas octanoate oxidation did not change with exercise intensity (see Figure 9.7). These data therefore suggest that an inhibition of LCFA entry into the mitochondria
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Figure 9.7 Percentage of oleate and octanoate tracer uptake oxidized during exercise at 40 (blue bars) and 80% VO2max (pink bars) (adapted from Sidossis et al., 1997)
is a contributing factor to the reduction in lipid oxidation rates with high-intensity exercise.
Does malonyl-CoA regulate LCFA uptake?
The mechanisms underpinning the reduction in LCFA entry to the mitochondria likely involve CPTI activity. In this regard, early attention focused on malonyl-CoA, which is a potent inhibitor of CPTI under resting conditions and light exercise. Indeed, under these conditions, muscle malonyl-CoA decreases, thus relieving the inhibition on CPTI and permitting LCFA mitochondrial uptake. In contrast, if malonyl-CoA inhibits LCFA uptake during high-intensity exercise, then muscle malonyl-CoA content should progressively increase with increments in exercise intensity.
Malonyl-CoA is formed from acetyl-CoA in a reaction catalysed by acetyl-CoA carboxylase (ACC). Given that acetyl-CoA would be increased with high-intensity exercise (due to increased glycolytic flux), it is reasonable to suggest that an increased formation of malonyl-CoA may coordinate the down-regulation of lipid and the up-regulation of CHO metabolism. However, evidence supporting this mechanism during high- intensity exercise is limited. ACC is under control through allosteric activation bycitrate and is also deactivated through phosphorylation through 5’
AMP activated protein kinase (AMPK). AMPK
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Figure 9.8 Skeletal muscle malonyl-CoA content at varying exercise intensities (adapted from Odlandet al., 1998a)
activity is greater with high-intensity exercise compared with moderate intensity exercise, and thus ACC activity is lower, favouring decreased malonyl-CoA production. Accordingly, muscle malonyl-CoA remains unchanged during exercise at 90% VO2max, compared with 65 VO2max (see Figure 9.8).
Does free carnitine availability regulate LCFA uptake?
Carnitine is required as a substrate for CPTI and is therefore required for the transport of an acti- vated LCFA across the inner mitochondrial mem- brane. If acetyl-CoA formation from the oxidation of pyruvate exceeds the rate of utilization by the Krebs cycle, PDH activity could be reduced due to product inhibition. As a result, exercise intensity would be reduced and performance impaired. In such situations, carnitine can also act as a buffer for excess acetyl-CoA production by combining with acetyl-CoA to form acetylcarnitine through the action of carnitine acetyl transferase (CAT). In this way, PDH activity and Krebs cycle flux can continue, thereby allowing exercise to continue at high-intensity.
However, the down side of this is that the use of carnitine in this way reduces the amount of free carnitine available to act as a substrate for CPTI,
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Figure 9.9 The percentage of carnitine present in free (pink bars) form or as acetylcarnitine (blue bars) in relation to exercise intensity (adapted from Stephens et al., 2007)
and hence its activity is reduced. Indeed, the amount of free carnitine is reduced and acetylcar- nitine is increased in accordance with increases in exercise intensity (see Figure 9.9). Furthermore, there is a strong positive and negative correlation with acetyl carnitine concentration and RER and fat oxidation rate, respectively, as shown in Figure 9.10. Based on these data, a proposed model for carnitine mediated regulation of lipid oxidation is outlined in Figure 9.11.
Does exercise-induced decreases in muscle pH reduce CPTI activity?
In addition to potential carnitine mediated limi- tations in LCFA uptake, it is also possible that reduced pH induced by high-intensity exercise may limit CPTI activity. Indeed, in vitro data demonstrate that a fall in pH from 7.1 to 6.8 resulted in a 30–40% decrease in CPTI activity in both sarcolemmal and inter-myofibril mito- chondria (Bezaire et al., 2004). However, further studies to test this hypothesis in exercising human skeletal muscle are warranted.