The Physiological Regulation of Skeletal Muscle Fatty Acid Supply and Oxidation During Moderate-Intensity Exercise

The regulation of the active muscle fatty acid uptake and subsequent oxidation is complex and may be differently regulated at low, moderate, and high exercise intensities and durations of exercise. Relatively few studies have directly determined human muscle NEFA handling during exercise, with most of the outstanding studies performed in the 1960–1970s [12‐15], and even fewer studies have determined IMTAG involvement [6]. The limited, but remarkably consistent, available quantitative and kinetic information originates from prolonged moderate-intensity exercise. The regulation of the active muscle NEFA uptake can be defined by a four-step process, as depicted in Fig. 1, consisting of (1) an increased energy demand by the contracting muscle; (2) delivery of NEFA to the muscle; (3) transport of NEFA into the muscle by fatty acids transporters; and (4) activation of the fatty acids and either oxidation or re-esterified into intracellular lipids and stored into the IMTAG droplets located next to the mitochondria. A similar concept is generally acknowledged for glucose [7, 16] but differences seem to exist with respect to glycogen versus IMTAG as the intramuscular carbohydrate and fat energy stores, respectively.

The increased metabolic rate/demand of the active muscle is usually not considered to be a regulatory step. However, it is the main driving force for all physiological regulatory processes. It elicits functional hyperemia, increasing muscle blood flow, the number of perfused capillaries (recruitment), and hormone levels that affect adipose tissue NEFA release, and hence NEFA delivery to the active muscle. Moreover, resting skeletal muscle has very low energy expenditure and thus the demand for energy/adenosine triphosphate is small, implying that an increase in energy demand with exercise causes a surge for substrates that possibly creates a concentration gradient for NEFA between plasma, the interstitial space, cytosol, and entry in the mitochondria (Figs. 1, 2).

The resting muscle blood flow is low and 45–55 % of all NEFA delivered to the muscle is taken up [10, 12, 15, 17] and is clearly dependent on the NEFA concentration [6]. This suggests that the facilitated muscle NEFA uptake is limited/saturable or that the lack of substrate demand and/or re-esterification into intracellular lipids is low, reducing the NEFA gradient for uptake. During moderate-intensity exercise the blood flow to the active muscles increases linearly with the workload [18] and is easily 10- to 15-fold higher during moderate-intensity exercise [17] than at rest, and with the increase in blood flow the blood transit time through the active muscle is substantially reduced [19, 20]. The NEFA fractional extraction decreases to only ~20 % [6, 10, 12‐15, 17] as compared to the 45–55 % at rest, despite the massive increase in blood flow and reduced transit time. In addition, the NEFA factional extraction is the same over a wide range of plasma flows [12], i.e., exercise intensities, and is independent of the NEFA concentration over at least a threefold increase in NEFA concentration [6, 15]. Accordingly, the active muscle NEFA uptake is very closely and linearly related to the NEFA delivery, which is the NEFA concentration multiplied by the plasma flow to the active muscle [6, 10, 12‐15, 17]. The same relationship is found between NEFA delivery and NEFA oxidation, since 85–100 % of the NEFA taken up by the active muscle is directly oxidized [6, 10, 12‐14, 17]. Thus, during exercise the increase in NEFA uptake with delivery is dependent on the functional exercise hyperemia increase in blood flow. However, the linear increase of NEFA delivery and oxidation with the duration of prolonged moderate-intensity exercise is caused by the increase in NEFA concentration since the plasma flow is largely unchanged during continuous exercise at the same intensity, emphasizing the important role of an increased adipose tissue NEFA release to enhance the plasma NEFA concentration driving the active muscle NEFA oxidation (Fig. 1 and Sect. 3) [8]. Consistent with these findings, an increase in the NEFA concentration via intralipid/heparin infusion increases the active muscle net NEFA uptake and fat oxidation [21], and even total fat oxidation at the relative high workload of 85 % of VO_2max [22]. Conversely, if NEFA concentrations are lowered via nicotinic acid infusion, the plasma NEFA oxidation is reduced [23]. The capacity for muscle to achieve such a rapid and manyfold increase in NEFA uptake upon contraction is remarkable, particularly in view of the manyfold increase in NEFA delivery caused by the massive increase in blood flow and the substantially reduced time available for interaction with the NEFA transporters. Of course, the 10- to 15-fold increase in blood flow with moderate-intensity exercise is accompanied by an increased capillary recruitment [19, 20], also referred to as nutritive flow [24], increasing the accessibility to NEFA transporters and diffusion surface. In addition, contraction-induced translocation of NEFA transporters from the intracellular storage pool to the cell membrane [11, 25] plays an important role in enhancing muscle NEFA uptake capacity during exercise. Another contributing factor is likely the high energy demand of the active muscle creating a NEFA surge and increasing the gradient for NEFA movement from the mitochondrion to the cytosol and eventual plasma NEFA uptake.

Over recent decades there has been considerable debate regarding whether NEFA transport across the plasma membrane occurs via passive diffusion or via facilitated transport by means of membrane-associated proteins. The physical properties of NEFA with a non-polar carbon chain and the polar head group would make passive diffusion possible, and this is seemingly supported by the above-described connection between NEFA concentration and active muscle NEFA uptake. However, over the past decade it has been shown that the majority of NEFA is transported across the cell membrane via protein-mediated mechanisms. Moreover, some of these fatty acid transporters demonstrate reversible translocation, from the intracellular storage pool to the cell membrane [11, 25] under the influence of contraction and insulin, analogous to the well-described glucose transporter type-4 (GLUT-4) translocation for muscle glucose uptake [7, 16]. Furthermore, studies with FAT/CD36 knockout or overexpressing mice [26, 27] have lent support to the conclusion that these fatty acid transporters critically regulate skeletal muscle fuel selection, performance, and training-induced adaptation of NEFA oxidation [26, 27]. However, while these studies undoubtedly demonstrate the importance of NEFA transporters in skeletal muscle NEFA transport from the plasma into the active muscle, they do not provide information on whether these transporters are rate limiting for NEFA oxidation. The well-described human in vivo increase in NEFA oxidation with exercise duration and the close linear relationship between NEFA delivery and uptake into the active muscle suggests that NEFA uptake is not solely controlled by the rate of sarcolemmal transport. This does not exclude an important regulatory role for fatty acid transporters and translocation of resting muscle NEFA uptake and its role in NEFA clearance from the circulation, particularly after food ingestion and during exercise in untrained individuals or patients with, for example, type 2 diabetes mellitus.

Once NEFA is taken up by the active muscle cell it can be oxidized or incorporated into intracellular lipid-like membranes and/or re-esterified and stored into IMTAG. The rate of resting muscle NEFA re-esterification into IMTAG is high [6, 28, 29], accounting for ~50 to 60 % of the total NEFA uptake [6, 30]. During exercise the majority (85–100 %) of NEFA taken up is directly oxidized [6, 10, 12‐14, 17] and the rate of re-esterification into IMTAG is reduced, accounting for ~10 % of the total NEFA uptake [6]. A net IMTAG utilization with prolonged moderate-intensity exercise is observed for mixed muscle as a result of a reduced rate of IMTAG synthesis [6] and potentially an increased IMTAG lipolysis by adipose tissue TAG lipase responsible for the contraction-induced IMTAG lipolysis [31, 32]. However, in contrast to glycogen, the mixed muscle IMTAG store does not seem to be utilized to a large extent (20–50 % of the pre-exercise total IMTAG [5, 6, 33‐35]), and this was even the case after 12 h of exercise [36]. The mixed muscle net IMTAG breakdown during exercise may underestimate IMTAG utilization by the different fiber types since only type I fibers have been suggested to utilize IMTAG during 2 h of 60 and 75 % of VO_2max bicycling exercise [37, 38] as well as after 2 h of high-intensity knee-extensor exercise [39]. Moreover, type II fibers have a much lower IMTAG content, but this does not decrease during exercise [37‐39]. The high IMTAG synthesis rate that is reduced but still continues during muscle contraction together with a seemingly sub-maximal utilization rate suggests that the role of IMTAG is not solely as a rapidly responsive intracellular energy store such as glycogen, particularly since the higher circulatory NEFA oxidation shows that the NEFA oxidation capacity of mitochondria is not limiting for IMTAG utilization.

Prolonged moderate-intensity exercise in the post-absorptive state causes a continuous increase in the NEFA concentration and oxidation with exercise duration [10, 17, 34, 40]. Moreover, the continuous increase in the NEFA concentration with duration of exercise implies that the rate of NEFA entering the circulation is higher than the uptake rate of the active muscles. Most of the circulatory NEFAs originate from TAG lipolysis in adipose tissue. Direct measurements across abdominal subcutaneous adipose tissue show a substantial increase in NEFA release at low exercise intensities [41]. Moreover, no or only a modest increase in adipose tissue NEFA release is found with higher exercise intensity [9, 41]. Therefore, low-intensity exercise provides an adequate stimulus for abdominal adipose tissue NEFA mobilization. The release of NEFA from subcutaneous adipose tissues depends on the relative contribution of three processes: (1) adipose tissue TAG lipolysis; (2) the rate of adipose tissue NEFA re-esterification into TAG; and (3) adipose tissue blood flow. The adipose tissue NEFA uptake is undetectable at rest [9, 42] and during exercise [9]; hence, NEFA re-esterification does not play an important role and the increase in adipose tissue NEFA release can be attributed to an increase in TAG lipolysis. However, the exercise-induced subcutaneous adipose tissue blood flow [9, 41, 43] and likely capillary recruitment [44, 45] plays a crucial role in the increase in NEFA release during exercise. The difference between the arterial and adipose tissue venous NEFA concentration is very similar at rest and during exercise. Therefore, the 2- to 3-fold increase in adipose tissue blood flow during exercise is responsible for the increase in the circulatory rate of NEFA [9]. The importance of adipose tissue blood flow for NEFA release is substantiated by observations in patients with type 2 diabetes who exhibit an exercise-stimulated adipose tissue lipolysis but in whom a high fraction of the liberated NEFA caused by TAG lipolysis could not be released into the circulation since the exercise-induced increase in adipose tissue blood flow was much smaller than in healthy subjects [46]. The exercise-induced increase in adipose tissue lipolysis and blood flow has mainly been attributed to the elevated catecholamine concentration with exercise [47, 48]. Moreover, circulating epinephrine is more important than sympathetic nervous activity for the stimulation of adipose tissue lipolysis during exercise [49, 50], albeit differences in various adipose tissue depots may exist [51]. Whereas epinephrine may be primarily responsible for the exercise-induced increase in lipolysis, selective β-adrenergic blockage does not completely prevent the increase in lipolysis [47, 48, 52], suggesting that other mediators play a role in the stimulation of lipolysis during exercise. Atrial natriuretic peptide is a potential candidate since there is evidence that it stimulates adipose tissue lipolysis and is produced during exercise in an intensity-dependent manner [53‐55]. Growth hormone and cortisol increase adipose tissue lipolysis [56‐58] and are potentially involved in the regulation of adipose tissue lipolysis during exercise. Growth hormone usually increases with exercise but its effect on lipolysis is not manifested until after 1–2 h of exercise [59] and thus is likely to become more important during prolonged exercise or be involved in the enhanced lipolysis during recovery from exercise [59]. Interestingly, 4 weeks of high-dose growth hormone supplementation raised the basal lipolytic rate substantially but did not change the exercise-induced increase in lipolysis during 30 min of exercise at 65 % of VO_2max [60], suggesting that growth hormone does not play an important role in the exercise-induced increase in adipose tissue lipolysis. Cortisol may increase to some extent with prolonged exercise [61] but this does not coincide with the rapid increase of adipose tissue TAG lipolysis upon the start of exercise. It has been suggested that infused cortisol and growth hormone stimulate systemic and adipose tissue lipolysis in an additive manner; however, it has to be mentioned that the cortisol levels were substantially higher than seen for any form of exercise [62]. Interleukin-6 (IL-6) is produced by skeletal muscle during exercise in increasing amounts with increased duration [63], and when recombinant human IL-6 (rhIL-6) is infused it increases systemic lipolysis [64]. Thus, IL-6 could be a signal from muscle to adipose tissue to coordinate the NEFA supply from adipose tissue with demand from the exercising muscle. However, concomitant IL-6 infusion and exercise fails to raise fatty acid delivery or oxidation [65], and at rest rhIL-6 infusion does not increase adipose NEFA release but does increase NEFA release from skeletal muscle [66]. Therefore, IL-6 is not a regulator for the increased adipose tissue NEFA release during exercise, but may be involved in skeletal muscle TAG utilization during exercise.

It should be noted that most research is performed in the post-absorptive state and little information exists on adipose tissue NEFA release during exercise in the post-prandial state. Ingestion of a mixed meal 1 h before exercise causes much lower plasma NEFA concentrations but does not influence adipose tissue lipolysis [67]. However, the adipose tissue net NEFA release is substantially lower during exercise in the post-prandial than in the fasted state as a result of the high adipose tissue NEFA uptake and re-esterification into TAG. In addition, the exercise-induced increase in adipose tissue blood flow is absent post-prandially [67]. Thus, the decrease in the muscle fat oxidation during exercise after a mixed meal may in part be caused by reduced NEFA delivery to the active muscle as a consequence of the decreased fatty acid mobilization from adipose tissue.

Lipolysis of circulating TAG-rich lipoproteins in the capillaries of skeletal muscle is a source of NEFA for uptake and either oxidation or re-esterification in the active muscles. The majority of TAG-rich lipoproteins are the very-low-density lipoproteins (VLDL-TAG) produced in the liver in the fed and fasted state and contains one molecule of apolipoprotein B₁₀₀ and 5–25,000 TAG molecules in its core [68]. In the fed state chylomicron TAG (CM-TAG) is secreted by the intestines and begins to appear in the blood approximately 1 h after the oral fat intake. The large CM-TAG contain apolipoprotein B₄₈ and are very rich in TAG with a half-life estimated at 5 min [69]. Both VLDL-TAG and CM-TAG undergo lipolysis by lipoprotein lipase (LPL) anchored to heparin sulphate on the endothelial surfaces in muscle. The affinity of LPL for CM-TAG is reported to be 50-fold higher than for VLDL-TAG; however, this is balanced by the usually much higher concentration of VLDL-TAG. Moreover, complex differences exist between tissues in LPL expression, activity, and specific nutritional regulation [70, 71]. Circulating CM-TAGs are suggested to provide a more readily available source of NEFA for muscle oxidation than VLDL-TAG [72]. However, this obviously requires oral fat intake before or during the exercise, something that is not common practice among athletes. A key problem in the quantification of muscle CM-TAG and VLDL-TAG lipolysis is the technical difficulty of measuring TAG extraction across a muscle, a problem that is exaggerated during exercise when blood flow to the active muscle increases manyfold [73]. In general, no significant extraction of TAG, CM-TAG, and VLDL-TAG by the active or inactive muscle can be determined in the fasted state [10, 73, 74]. However, in spite of this inability to measure small quantities of muscle TAG extraction, the potential amount of NEFA for the muscle can contribute substantially to the total fat oxidation. Enevoldsen et al. [75] did not observe leg TAG uptake 1 h after meal ingestion, but during the following hour a significant TAG uptake of ~45 μmol/min was observed in only the exercising leg during 1 hour of cycle ergometer exercise at 50 % of VO_2max. The NEFA uptake in the exercising leg was of similar magnitude; in other words, the NEFA uptake was only one-third of the TAG uptake in NEFA equivalents. Moreover, these results suggest that exercise per se increases muscle LPL activity, which has previously been shown not to occur via more direct muscle LPL activity measurements [76, 77]. However, Enevoldsen et al. [75] argued that not all capillaries are recruited in the resting muscle, but that the functional hyperemia of the active muscle increases the number of perfused capillaries [24, 78] and, hence, the accessible amount of LPL as depicted in Fig. 2. Evidence against a considerable contribution of circulating TAG to active muscle fat oxidation include findings that in the fasted state the muscle NEFA uptake and oxidation account for most of the fat oxidation [6, 10, 12, 14, 15, 17] and also for the majority of whole-body fat oxidation [37, 79], which suggests that relatively little of the oxidation of NEFAs originates from CM-TAG and VLDL-TAG. More research is needed to quantify the contribution of VLDL-TAG and CM-TAG to muscle fat oxidation and whether nutritional interventions before or during exercise can enhance its utilization without compromising performance.