Oxidative phosphorylation

Oxidative or aerobic phosphorylation production of ATP via aerobic pathways occurs within the inner membrane of mitochondria in a series of single oxidation reactions known as the electron transport or respiratory chain. Oxidation is the donation or loss of electrons (often in the form of hydrogen) from an atom or molecule, whereas reduction is the acceptance of electrons (hydrogen) by an atom or molecule. When electrons are donated, considerable chemical energy is liberated, and a portion of this energy is captured for the rephosphorylation of ADP to ATP, with the remainder being lost as heat energy (Guyton, 1986). Nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) act as hydrogen carriers (acceptors) during glycolysis, beta (β)–oxidation, and the tricarboxylic acid (TCA) cycle and, therefore, are reduced to NADH and FADH₂. These coenzymes are essential for aerobic and anaerobic phosphorylation, but their concentrations within the muscle are low. Therefore, NADH and FADH₂ must be reoxidized to NAD⁺ and FAD via the electron transport chain. Specific mitochondrial enzymes incorporated in the electron transport chain catalyze the oxidation through a process of dehydrogenation. The donated hydrogen atoms provide the electrons that are transported from one enzyme complex to another by electron carriers, for example, cytochrome c. The importance of oxygen (O₂) in this whole process is that it acts as the final hydrogen acceptor to form water. The energy released by the step-by-step transfer of electrons from NADH or FADH₂ to O₂ via the electron carriers is used to pump protons from the inner matrix of the mitochondrion into the outer chamber between the inner and outer mitochondrial membranes. This creates a strong transmembrane electric potential. Energy for the phosphorylation of ADP to ATP is obtained as the protons flow back through an inner membrane enzyme complex called ATP synthetase (Guyton, 1986; Stryer, 1988).

The availability of O₂ to the exercising muscle is the rate-limiting step for oxidative phosphorylation. Oxygen immediately available to the muscle at the onset of exercise, from myoglobin within the muscle (MbO₂), hemoglobin within the circulatory system (HbO₂) or O₂ dissolved in the body fluids, is in sufficient quantities for only a few seconds of exercise. Therefore, the delivery of O₂ to the exercising muscles via the cardiorespiratory system is crucial for the capacity to continuously produce energy via aerobic means.

The two major electron donor substrates for aerobic phosphorylation are carbohydrates (CHO) and fatty acids. Glucose is the main CHO, and if it is not used for immediate energy production, it is stored as glycogen, mostly in skeletal muscle and to a lesser extent in the liver (Hodgson et al., 1983, 1984; Lindholm, 1979). Adipose tissue constitutes the largest store of fatty acids (FA) (Robb et al., 1972; Stryer, 1988). Adipocytes store fat within their cytoplasm as triglycerides. Triglyceride storage also occurs to a much lesser extent in muscle (Lindholm, 1979).

Aerobic glycolysis

The importance of CHO as a substrate for energy production increases as exercise intensity increases (Hodgson et al., 1985; Lawrence, 1990). Glucose diffuses into the muscle cell cytoplasm from the circulation and is phosphorylated to glucose-6-phosphate (G-6-P) in a reaction catalyzed by the enzyme hexokinase (HK) and requiring one ATP molecule. G-6-P is then transferred into the glycolytic pathway for immediate energy production or reversibly converted to glucose-1-phosphate (G-1-P) and then glycogen for storage. Glycogen stores provide most of the glucose required for energy production during exercise. In the glycolytic pathway, G-6-P is phosphorylated to fructose-6-phosphate (F-6-P), which is then phosphorylated to fructose-1,6-bisphosphate (F-1,6-BP) in a reaction catalyzed by phosphofructokinase (PFK) and at the expense of one ATP molecule. F-1,6-BP is subsequently split into the triose phosphate isomers, glyceraldehyde-3-phosphate (Gl-3-P) and dihydroxyacetone-phosphate (DiH-P). DiH-P is readily converted into Gl-3-P by triose phosphate isomerase (TPI). Therefore, one molecule of glucose or glycogen gives two molecules of Gl-3-P. Gl-3-P then proceeds through a series of reactions, the end result being the production of pyruvate, ATP, NADH, water, and hydrogen ions. The net reaction for the glycolytic pathway utilizing glucose is:

Under aerobic conditions, the hydrogen atoms are transferred to the electron transport chain, and pyruvate is transported into the mitochondrial matrix as a substrate for acetyl coenzyme A (acetyl CoA). Acetyl CoA then enters the TCA cycle by combining with oxaloacetate to form citrate. The normal function of the TCA cycle requires three NAD⁺ and one FAD to accept hydrogen atoms during the oxidative conversion of citrate back to oxaloacetate. When O₂ is available, the NADH and FADH₂ are then reoxidized back to NAD⁺ and FAD in the electron transport chain, thus replenishing the adenine dinucleotide stores and producing ATP (see Figure 3-2). The complete aerobic utilization of one mole of glucose generates 36 to 38 ATP. When O₂ is not available, pyruvate is converted to lactate as described later.

Fatty acid utilization

Following lipolysis, nonesterified fatty acids (NEFAs) are released into the circulation and are subsequently available as hydrogen donors for energy production in skeletal muscle. NEFAs likely diffuse into muscle cells down a concentration gradient as well as being actively transported across the cell membrane. At the cytoplasmic surface of the outer mitochondrial membrane, the NEFAs are esterified (activated) enzymatically forming long-chain acyl CoA molecules. The acyl CoA molecules are then linked to carnitine and shuttled across to the matrix side of the inner mitochondrial membrane. In the mitochondria, the acyl CoA molecules undergo a series of four reactions known as β-oxidation. With each cycle of β-oxidation two-carbon (C₂) units are sequentially removed from the acyl CoA molecule and acetyl CoA, NADH, and FADH₂ are produced (see Figure 3-2). NADH and FADH₂ subsequently donate their electrons in the electron transport chain, generating ATP and being reoxidized to NAD⁺ and FAD in the process. Acetyl CoA is utilized in the TCA cycle as previously described. The splitting of C₂ units from the parent acyl CoA molecule is repeated until the whole chain has been cleaved into the acetyl CoA molecules. The number of carbon atoms in the parent FA chain will determine the net energy yield from β-oxidation. Complete oxidation of one palmitic acid molecule produces 129 molecules of ATP.

Anaerobic phosphorylation

The pathways of anaerobic phosphorylation occur solely in the muscle cell cytoplasm, with no reactions in the mitochondria as there are for aerobic phosphorylation. With the initiation of exercise, there is a lag period before oxidative energy production becomes an important source of ATP. During this time, rapid supplies of ATP must still be available if muscular contraction is to continue. Stores of ATP in skeletal muscle are limited (4–6 millimoles per kilogram [mmol/kg] wet muscle) and contribute little to the total energy supply (Lindholm and Piehl, 1974; McMiken, 1983). Until aerobic phosphorylation makes a substantial contribution to energy supply, rapid regeneration of ATP must occur in the absence of O₂. The anaerobic phosphorylation of ADP is achieved by three pathways: (1) the phosphocreatine reaction, (2) the myokinase reaction, and (3) anaerobic glycolysis. The former two pathways may be described as anaerobic alactic reactions because no lactate is produced as it is in the latter process (Clayton, 1991).

Regulation of aerobic and anaerobic pathways

At all exercise levels, both systems of energy supply are active; however, one will predominate, depending, in particular, on the intensity and duration of the activity. A complex method of metabolic regulation controls the input of each pathway. Substrate and enzyme availability, end product concentrations, and various feedback mechanisms contribute to pathway dynamics.

Oxygen supply to muscles and the ratio of ATP to ADP are the most significant regulators of the energy producing pathways. Although there is adequate O₂ available, aerobic phosphorylation persists, providing a driving force for substrate to enter the TCA cycle and produce high concentrations of citrate. Citrate retards the activity of PFK, the enzyme responsible for the irreversible conversion of F-6-P to F-1,6-BP in the glycolytic pathway. This has the effect of inhibiting the metabolism of glucose and glycogen. Accumulation of glycolytic intermediates before the PFK step, including G-6-P, inhibits the activities of HK and phosphorylase, thus also dampening the glycolytic pathway. High cellular concentrations of ATP (i.e., a high ATP:ADP ratio) also inhibits PFK activity. Pyruvate kinase, the enzyme which converts phosphoenolpyruvate to pyruvate, is the third control site in glycolysis. Pyruvate kinase is inhibited by ATP and activated by F-1,6-BP. When the relatively slow production of energy by oxidative phosphorylation is unable to meet the demands for ATP, the ATP:ADP ratio swings in favor of ADP, and this, in turn, stimulates PFK and enhances glycolysis and the utilization of glucose and glycogen stores. The pyruvate produced is metabolized to lactate, and this allows the reoxidization of NADH to NAD⁺ for continued electron acceptance in the glycolytic pathway.

Control of the TCA cycle starts with regulation of the irreversible oxidative decarboxylation of pyruvate to acetyl CoA by pyruvate dehydrogenase (PDH). Acetyl CoA, NADH, and ATP inhibit PDH activity. Once acetyl CoA is formed, its combination with oxaloacetate to produce citrate is inhibited by high concentrations of ATP. At least two other sites in the TCA cycle have regulatory mechanisms that respond to the cellular concentrations of ATP. Overall, the rate of the TCA cycle is reduced when abundant ATP is present, but it should also be remembered that the TCA cycle operates only under aerobic conditions because of the constant need for NAD⁺ and FAD, regenerated or reoxidized from NADH and FADH₂ by the transfer of their electrons to O₂ during oxidative phosphorylation.

The rate of oxidative phosphorylation is regulated by the cellular ADP concentration. This is known as respiratory control. For electrons to flow through the electron transport chain to O₂, the simultaneous phosphorylation of ADP to ATP is usually required. When ATP is utilized, ADP concentrations increase; therefore the rate of oxidative phosphorylation increases, that is, if an adequate O₂ supply is available. So, oxidative phosphorylation is coupled to ATP utilization via the relative ADP concentration.

Enzymes in the β-oxidation pathway are inhibited by NADH and acetyl CoA; however, the rate of FA oxidation is determined greatly by substrate availability.

Energy pathway contributions in the exercising horse

The duration and intensity of exercise determine the metabolic requirements of muscle. At any given time, the most effective combination of the various energy-producing pathways will occur; again, it should be emphasized that no form of exercise by the horse is entirely aerobic or anaerobic in nature.

At the initiation of exercise, the immediate source of energy is locally available ATP. As previously noted, this supply of ATP is limited and rapidly depleted; therefore, for exercise to continue, ATP must be replenished by other processes. Creatine phosphate and the phosphocreatine pathway provide the next rapidly available ATP source; however, this energy supply is also of limited capacity (McMiken, 1983). The myokinase reaction provides a further means of regenerating ATP, but it is restricted to certain muscle fiber types and is used in anaerobic exercise only when these fibers are recruited (Meyer et al., 1980). The myokinase reaction is believed to have a minor role in energy production overall.

The glycolytic pathway with the production of pyruvate and lactate provides the main ongoing ATP source and reaches peak metabolism within about 30 seconds of the onset of exercise. The delay in maximal glycolytic output is possibly caused by the multiple and complex reactions required (McMiken, 1983). The large stores of glycogen in equine muscle (Hodgson et al., 1984; Lindholm, 1979; Lindholm and Piehl, 1974; Lindholm et al., 1974; Nimmo and Snow, 1983) allow this pathway to provide an early consistent source of energy, but still there is a finite limit to this substrate.

Aerobic mechanisms for ATP replenishment represent the most efficient means of substrate production. However, it is the slowest pathway to respond to exercise demands, owing to the cardiovascular lag in supplying O₂ to the cells and the intricacy of the reactions. Oxidative processes are in full production within 1 minute after the onset of exercise, and then muscular energy is more likely to be dependent on the rate of O₂ transport to the cells rather than substrate availability (McMiken, 1983). In fit racehorses, the time to peak energy production via oxidative processes can be as short as 20 seconds, especially following warm-up (Tyler et al., 1996).

At rest and during low-intensity exercise (walking and trotting), aerobic pathways provide most energy requirements after the initial lag period. At this exercise intensity, the ratio of ATP to ADP will be high; PFK will, therefore, be inhibited and β-oxidation of fatty acids will provide the main method for ATP regeneration (Hodgson et al., 1985; Lawrence, 1990). Such is the case during endurance rides, in which case, it is well recorded that blood concentrations of nonesterified fatty acids (NEFA) increase and the glycogen utilization rate is low (Hodgson et al., 1983; Hodgson et al., 1985; Lucke and Hall, 1980; Snow et al., 1982) (Table 3-1 and Table 3-2).

As exercise intensity increases, ADP accumulates, and this stimulates anaerobic glycolytic energy production and a dramatic increase in the use of CHO substrates (see Table 3-1). Galloping and bursts of intense exercise such as during polo games and jumping rely heavily on anaerobic energy supply. The self-limiting nature of anaerobic power output (substrate exhaustion) means the horse can only maintain maximal speed for about 600 to 800 m. After this distance, energy supply falls back to the slower aerobic pathways, necessitating a reduction in speed of exercise (Hodgson et al., 1985; McMiken, 1983).

Energy substrates

Scientific data on the relationship between nutrition and equine performance has occupied the attention of many researchers in recent years; however, it is difficult to design controlled experiments that only isolate nutritional influences on performance. Subtle, yet important effects of nutritional alterations may go undetected, partly because the power of statistical studies is limited by the small numbers of horses often used in experiments (Hintz, 1994). The relationship between energy and exercise is complex and inseparable. The amount of energy required depends on the type and duration of activity and the horse’s body weight. Maintenance digestible energy (DE) requirements are linearly related to body weight (Pagan and Hintz, 1986a). During submaximal exercise energy expenditure is exponentially related to speed and proportional to the body weight of the riderless horse or the combined weight of the horse plus rider (Pagan and Hintz, 1986b). The method used by Pagan and Hintz (1986b) for calculating energy requirements was based only on the amount of work performed and may not account for any follow-on demands for energy in recovery that the work bout stimulates (Lawrence, 1990).

The stores of major fuels in the horse for muscular contraction are outlined in Table 3-3 as calculated by McMiken (1983). It is clear that “fast” energy stores (i.e., ATP, creatine phosphate, and glycogen) are limited despite the high capacity for glycogen storage in equine muscle. The primary dietary sources of energy stores for the horse are soluble and fiber derived CHOs and fats. Protein is considered to play a minor role as an energy source.

Effects of dietary alterations on energy substrate utilization

Many published reports have described the effect that altering components of the normal diet has on substrate utilization and performance in the horse. The consumption of large amounts of digestible CHO within a few hours of strenuous activity may depress the performance of that exercise (Åstrand and Rodahl, 1986). This is possible because insulin-stimulated uptake of blood glucose results in hypoglycemia, a greater dependence on muscle glycogen, and, therefore, earlier onset of fatigue. Free FA mobilization is also inhibited by insulin (Frape, 1988). The effects of consumption of CHO or fat before or during exercise on metabolism are summarized in Table 3-4.

However, a lack of available CHO during submaximal exercise can also limit performance; there is strong evidence supporting the use of high CHO diets in humans for endurance exercise. In humans, muscle glycogen loading was achieved by performing intense exercise and then consuming a CHO-rich diet (Lindholm, 1979). Current practice is to combine a program of decreased activity with increased CHO consumption a few days before competition to achieve a glycogen load. Glycogen loading in horses has been accomplished, but no obvious improvement in work performance has been demonstrated (Frape, 1988; Lawrence, 1990; Snow, 1994; Topliff et al., 1983; 1985). Intravenous, but not oral, glucose supplementation has increased glycogen repletion rates after exercise (Davie et al., 1994; 1995). Although glycogen loading is not recommended in the horse, adequate CHO intake must still be ensured (Hintz, 1994). A low CHO diet and regular exercise lead to glycogen depletion and decreased performance in horses (Topliff et al., 1983; 1985). In a study in which fit Standardbreds were exercised strenuously for 3 consecutive days to achieve a 55% depletion of the muscle glycogen store, anaerobic, but not aerobic, capacity was seen to be impaired (Lacombe et al., 1999). However, the association between glycogen depletion and impaired anaerobic metabolism is not conclusive as confounding effects of other exercise-induced changes on performance could not be eliminated (Lacombe et al., 1999). When muscle glycogen was depleted by 22%, there was no significant effect on the performance of Thoroughbreds exercising at high intensities (Davie et al., 1996). A CHO supplement taken an hour or two before exercise does not seem to benefit endurance performance, but intake of glucose during exercise may supplement waning plasma concentrations and delay the onset of fatigue (Lawrence, 1990).

The beneficial effects of feeding high-fat diets to horses on substrate utilization and performance remain controversial, although benefits for horses with problems such as recurrent tying up and gastric ulceration are unequivocal. Differences in the condition of the horses, type of exercise, the length of the adaptation period to the diets, the type of fat used as the supplement, and the level of fat supplemented, particularly in relation to CHO, make comparing the published results difficult. Many variations in study designs influence the results. Feeding an increased level of fat is suggested to cause metabolic adaptations that permit horses to preferentially utilize fat and spare glycogen during exercise, but the evidence to support such a proposal is inconclusive (Hintz, 1994; Lawrence, 1990; Snow, 1994).

Interest in amino acid requirements of the performance horse is growing, and there is a suggestion that improvements in oxidative capacity can be brought about by certain amino acid supplements (Hintz, 1994; Lawrence, 1990). Higher-than-necessary protein diets are often fed to performance horses, but studies to demonstrate that this practice enhances exercise capabilities are lacking (Lawrence, 1990). In the case of CHO-rich and fat-rich diets, plasma concentrations of glucose, ammonia, lactate, alanine, and the muscle concentrations of G-6-P and lactate were higher at the end of exercise, compared with normal diets (Essén-Gustavsson et al., 1991). Higher pre-exercise muscle glycogen concentrations and FFA concentrations were present when horses were fed a CHO-rich diet compared with the fat-rich and normal-diet fed periods. No significant difference in performance during trotting at submaximal intensity on a horizontal treadmill was detected between the three diets (Essén-Gustavsson et al., 1991) with the average time to fatigue being 51 to 56 minutes. Whether or not the diets would alter performance in shorter or longer exercise periods remains unanswered. The effects on protein metabolism need to be further investigated as both the CHO-rich and the fat-rich diets, compared with normal diet, were associated with significant increases in branched-chain amino acids in plasma during and at the end of exercise (Essén-Gustavsson et al., 1991). The resting plasma concentration of the branched-chain amino acids was increased by 26% with the fat-rich diet but only by 8% with the CHO-rich diet. A 9% (control) or 18.5% (high) crude-protein diet had no effect on hepatic or muscular glycogen utilization and did not affect exercise performance in Quarterhorses exercising at submaximal intensities (Miller-Graber et al., 1991). Performance of Arabian endurance horses was not augmented by excessive protein in the diet (Hintz, 1983). In contrast, Standardbreds fed a high-protein diet (20%) or high-fat diet (15% soybean oil) showed greater muscle and liver glycogen utilization during prolonged exercise than when fed a control diet of 12% crude protein (Pagan et al., 1987). During higher-intensity, shorter-duration exercise, glycogen utilization was less when horses were fed high-protein or high-fat diets.

The timing of feeding and what to feed before exercise have considerable influence on the metabolic and physiologic responses to exercise (Harris and Graham-Thiers, 1999; Lawrence et al., 1995). In one study, it was concluded that feeding only hay shortly before exercise would not adversely affect performance but feeding grain would and that grain therefore should be withheld (Pagan and Harris, 1999).

Of course, many other nutritional components not discussed in this chapter may play roles in equine performance. These include water, electrolytes, acid–base balance, minerals, and vitamins. For more information see Chapter 4.