4.1 Increasing Free Fatty Acid (FFA) Delivery
Subsequent human research in the past 50 years generally supported the notion of a reciprocal relationship between carbohydrate and fat oxidation in skeletal muscle, but not exclusively by means of the mechanisms defined in the original work by Randle and colleagues [
5,
6]. Most human studies attempted to increase or decrease the plasma FFA availability, without affecting many other processes. While many models have been used with varying success, including high-fat meals and diets, short- and long-term aerobic training, caffeine administration, nicotinic acid ingestion, fasting, and prolonged dynamic exercise, the acute infusion of a lipid solution coupled with heparin administration has been most commonly and effectively used. This technique has the advantage of acutely (<30 min) increasing the plasma [FFA] without changes in the availability of other substrates or alterations in metabolite and hormone levels [
8‐
10]. In contrast, dietary attempts at acutely increasing the availability of FFAs to the working muscles in humans immediately before or during exercise in an attempt to spare carbohydrate have been largely unsuccessful. This is due to the fact that fat is not digested quickly, and therefore prolonged alterations in the normal diet are required to alter the IMTG stores and spare carbohydrate. As a result, these practices are not generally in use by athletes.
If increasing fat availability decreases carbohydrate oxidation in human skeletal muscle, it would be expected that the inhibition of carbohydrate use would target the key sites regulating carbohydrate metabolism and oxidation (Fig.
1). These sites would include glucose transport (GLUT 1, 4) across the muscle membrane, glucose phosphorylation (hexokinase), glycogenolysis [glycogen phosphorylase (PHOS)], glycolysis (phosphofructokinase), and conversion of pyruvate to acetyl-CoA (PDH). These enzymes (at least PHOS, phosphofructokinase, and PDH) have been shown to be regulated by calcium, adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inorganic phosphate both through direct (allosterically) and/or indirect (phosphorylation) regulation.
During exercise at approximately 80 %
V
O
2max in moderately active individuals, the majority of energy is derived from carbohydrate use and particularly from muscle glycogen during the first 20–30 min. Exercising at this high intensity in the presence of artificially elevated FFA levels decreased net glycogen use by approximately 50 % in the initial 15 min of exercise and increased fat oxidation by approximately 15 % during 30 min of exercise [
8,
10]. The muscle contents of free ADP and AMP, activators of PHOS, were significantly reduced (increased less) in the high FFA condition during exercise, and appeared to explain the decreased PHOS activity and glycogen use. It was suggested that the mitochondrial reduced form of nicotinamide adenine dinucleotide was more abundant with high fat provision during the onset of exercise, increasing the aerobic production of adenosine triphosphate (ATP) and reducing the mismatch between ATP demand and supply, and accounting for the reduced accumulation of ADP, AMP, and inorganic phosphate [
8,
9,
11]. There were no effects on muscle citrate, acetyl-CoA, and glucose-6-phosphate contents or the proportion of PDH in the active form (PDHa) [
8,
9], and whole body glucose disappearance (glucose uptake) was also unaffected by elevated FFAs [
10]. Therefore, at this intense aerobic power output in human skeletal muscle, the fat-induced downregulation of carbohydrate oxidation was controlled at the level of PHOS. The original work on the G–FA cycle by Randle and colleagues [
4‐
6] did not include PHOS, because the diaphragm and cardiac muscles relied almost exclusively on exogenous substrates and instead focused on enzyme regulation downstream from PHOS, namely hexokinase, phosphofructokinase, and PDH.
When these experiments were repeated at lower exercise power outputs (~40 % and 65 %
V
O
2max), as well as during dynamic knee extension, high fat provision again downregulated carbohydrate oxidation, suggesting this fuel reciprocity was not dependent on exercise intensity [
11]. While the mechanism(s) of action responsible for the shift in fuel utilization again involved PHOS activity, small increases in citrate and lower PDHa levels suggested that downregulation was also present at phosphofructokinase and PDH at lower power outputs [
11]. The increase in citrate supported one of Randle’s original hypotheses, but subsequent in vitro studies that examined the inhibitory effects of citrate on phosphofructokinase activity suggested that the small increase in citrate in the high-fat trials would have minimal inhibitory effects on phosphofructokinase in contracting human skeletal muscle, and probably is not a mechanism to shift fuel preference [
12].
4.2 Decreasing FFA Delivery
It is possible to take the opposite approach and decrease the availability of plasma FFAs while exercising at approximately 60 %
V
O
2max by ingesting nicotinic acid. In this situation, the respiratory exchange ratio, glycogen use (trend only), and PDHa were all higher than in the normal fat availability trial [
13]. However, there was no effect on metabolic byproducts typically associated with the G–FA cycle, namely muscle citrate, acetyl-CoA, or pyruvate contents. In addition, there were no changes in free ADP and AMP to account for the higher glycogen breakdown and oxidation in the low-fat trial.
4.3 Altering Intramuscular Triacylglycerol (IMTG) Content
Another question is whether increasing IMTG (a common aerobic training adaptation) decreases carbohydrate oxidation during exercise? The most common method to alter the IMTG availability is by long-term dietary manipulation. IMTG can be increased by 50–80 % following the consumption of high-fat diets in which fat supplies 50–70 % of the total energy intake and IMTG can be decreased when dietary fat intake is reduced from 22 to 2 % of energy intake [
14,
15]. These long-term high-fat diets reduced muscle glycogen utilization and total carbohydrate oxidation rates during moderate intensity exercise, without altering glucose uptake [
16]. Conversely, when IMTG is reduced following a low-fat diet (2 % of total energy intake), whole-body carbohydrate oxidation and muscle glycogen utilization are also increased without altering whole-body glucose uptake [
14]. These data suggest that IMTG has no effect on muscle glucose uptake during exercise, but does influence muscle glycogen utilization. However, low-fat diets also contain high carbohydrate content, and therefore while IMTG contents are reduced, glycogen levels are increased. The opposite is also true as high-fat diets lead to low muscle glycogen stores. Therefore, to examine the pure interaction between the IMTG store and carbohydrate fuel metabolism, studies must use interventions that induce acute changes in IMTG content independent of alterations in the availability of glycogen (and other substrates such as plasma FFAs).
Interestingly, Burke et al. [
17] demonstrated that the effects of a high-fat diet on reducing glycogen use during dynamic exercise persisted, even after muscle glycogen stores were returned to normal levels. In this paradigm, participants consumed either a high carbohydrate diet (9.6 g/kg/day carbohydrate and 0.7 g/kg/day fat) or an isoenergetic high-fat diet (2.4 g/kg/day carbohydrate and 4.0 g/kg/day fat) for 5 days while undergoing aerobic training. On the sixth day all participants consumed a high carbohydrate diet that normalized muscle glycogen levels, before an exercise trial on day 7. Regardless of the ‘normalized’ glycogen level, a portion of the ‘carbohydrate-sparing’ effect of the high-fat diet was still present during the day 7 exercise trial. The subjects in this study were well-trained athletes who continued to exercise at a very high level during the 5-day high-fat/low carbohydrate diet intervention. Typical high-fat dietary interventions are not associated with continued and uncompromised training, and therefore the ability to maintain and/or increase fat oxidation during aerobic training while consuming a high-fat diet may be extremely important for inducing these metabolic shifts. At the present time, the mechanisms responsible for the persistent effects of the high-fat diet are not known, but it is reasonable to speculate that a redistribution of fatty acid transport proteins to the plasma and mitochondrial membranes may contribute, but this has not been examined [
15]. Interestingly, performance has also been assessed in these studies and the high-fat diets did not improve performance, even when a day of carbohydrate restoration occurred. Consequently, these dietary manipulations are not in use by elite athletes.