The Proposed Effects of Nicotinamide Adenine Dinucleotide (NAD) Supplementation on Energy Metabolism

Contents

1. Introduction

2. Discussion

3. Conclusion

References

1. Introduction

Energy metabolism is a process that is essential in the maintenance of life and has obvious roles with regards to sporting/exercise performance. The body can produce energy both aerobically and anaerobically and the regulatory mechanisms underlying these pathways of energy modulation are complex (40). Under aerobic conditions the Krebs cycle is crucial for energy production, the hydrogen’s removed during the cycle are transferred to the electron transport chain and the energy released during electron transport is utilised in the formation of ATP (1). Oxygen’s role in aerobic respiration is to act as the final hydrogen/electron accepter to form water. If this is not present the whole aerobic pathway cannot occur and so the body will rely on energy produced anaerobically. The question instantly raised is to whether oxygen is ever in short supply, does it become a limiting factor for energy metabolism? Or are other factors limiting? Can increasing or maintaining NAD+ concentrations sustain the action of the Krebs cycle and bring about the continuation of oxidative phosphorylation and therefore reducing build up of lactate as a consequence? If this hypothesis were to be true then this could have advantageous implications in sporting performance (Fig. 1).

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Figure 1. Flow diagram representing the stages in energy metabolism and how NAD affects each of these stages. NAD is required largely during the Krebs cycle but also is required during glycolysis and for the conversion of pyruvate at the end of glycolysis for its entry into the Krebs cycle. Author ’ s own figure.

Arterial oxygen content does not decrease at exercise intensities <75% of VO2max (49).

VO2 max is a measure of the ability of working muscles to oxidise metabolic substrates, with eventually a plateau in oxygen uptake occurring despite increases in work rate therefore achieving maximal oxygen uptake. This capacity is exceeded before circulating delivery of oxygen is limiting (28, 55). This is a significant, as it suggests that oxygen delivery is only limiting at VO2max where beyond this point oxygen uptake and delivery will become limiting. With regards to the majority of sporting events, exercise is carried out sub maximally for the athlete and so oxygen supply will not be limiting. An early experiment concluded that it seems unwarranted at present to ascribe alterations in body lactate to oxygen deficiency (30). This paper states that oxygen saturations exceeded 96% at every intensity set for the experiment (mild or severe). The phenomenon of the O2 debt formation is a manifestation of the need for oxygen by the body tissues during exercise which is not met at the time. This occurs despite the rate of delivery of oxygen to the tissues being greater per minute than normal (30). This is more supportive evidence that oxygen is not a limiting factor and is in fact transported efficiently to meet the demand, at least at exercise intensities up to 85-90% VO2max. Hence, why other metabolic factors appear to be limiting to such a degree that the cessation of aerobic respiration occurs. Blood flow redistribution is important to help compensate for the limits on O2 delivery and uptake set by maximal cardiac output and O2 extraction (55). The oxyhaemoglobin dissociation curve demonstrates the extreme efficiency of haemoglobin at combing with O2 in the lungs and unloading at tissues, this can be up to 90% of the O2 carried by haemoglobin during intense exercise (49). The myoglobin in the muscles functions as an oxygen store and transporter (35, 43). With regards to the respiratory system it has been identified that it only becomes limiting in untrained individuals with the endurance of respiratory muscles markedly improving in trained individuals (11).

When the oxidative potential of a cell has diminished pyruvate can be converted to lactic acid by lactate dehydrogenase. This is important as energy can still be produced through the continuation of glycolysis. The rates of the oxidation for energy metabolism are not affected until NAD+ is affected (29). If oxidative energy metabolism is so greatly impacted by NAD+, knowing that even during intense exercise oxygen is not in short supply nor are the delivery mechanisms efficiency, can increasing NAD+ concentration allow aerobic metabolism to continue? [Lactate] = [pyruvate] x k[DPNH2]/[DPN], the equation suggests, on a theoretical basis, that all instances of lactate production by tissues are influenced by the ratio of NAD+ and NADH (DPN or Diphosphopyridine nucleotide is another name for NAD) thus leading one to assume that lactate production can be manipulated by altering the ratio.

It has been stated that conclusions about tissue oxygen supply should not be drawn from

determining lactate alone, suggesting the interaction between the anaerobic and aerobic energy systems are intricate. For example epinephrine has been found to increase lactate in muscle which is not due to diminished blood flow or blood arteriovenous oxygen difference (16). Lactate produced during high intensity endurance activities appear to be occurring when the maximum rate of fat oxidation is inadequate to meet the demands of muscle contracting. This causes intracellular signalling events to occur which ultimately lead to the rate of pyruvate delivery to the mitochondria progressively exceeding the ability of the mitochondria to convert and transfer it into the Krebs cycle causing accelerated generation of lactic acid (34). It has been argued that lactate formation will occur when NADH and pyruvate are available to lactate dehydrogenase regardless of how much O2 is present (26). Lactate dehydrogenase can convert lactate back to pyruvate for further utilisation in the Krebs cycle, the reaction does make use of NAD+ (68). The problem becomes one of fuel availability when exercise extends beyond approximately two hours but events lasting 15-30minutes (e. g. 5km and 10km running) the anaerobic contribution can be 10-20% of total ATP turnover. Total ATP turnover during endurance performance reflects the interplay of aerobic and anaerobic metabolism with lactate generation functioning to maintain the NAD+ needed for continuation of glycolysis. If more NAD+ could be supplied or synthesised could lactate be converted back to pyruvate for use in the Krebs cycle and could aerobic metabolism be sustained for longer with reduced lactate build up? Lactate is produced regardless of how much O2 is present as long as pyruvate is available but with increased NAD+ pyruvate would be converted to Acetyl CoA for its entry into the Krebs cycle. The exact mechanisms by which lactate plays a role in fatigue has remained elusive but it’s clear it has some debilitating role as endurance trained individuals produce less (26). Although only assumptions presently, the implications that NAD+ could have on prolonging aerobic metabolism on exercise performance could be incredible, especially when considering the small margins between winning and losing in many sporting environments.

The human body will adapt in a variety of manners to physical training, these can effect both major systems/organs and more microscopic changes cellularly. These adaptations occur as a result of prolonged exposure to particular situations in an attempt to become a more efficient system. There is evidence that rats see an increase in mitochondria along with certain enzyme activities per gram/muscle (NADH dehydrogenase and NADH cytochrome c reductase), increasing approximately two fold in response to training. This results in a increased capacity of the electron transport chain which was associated with a concomitant rise in the capacity to generate ATP via oxidative phosphorylation (26). A similar study conducted on rabbits using electrical stimulation of the muscle draws the same conclusion with an increased volume of mitochondria (50). The exercise induced adaptation of increased mitochondria content appear to be essential for trained muscle to exhibit an increased O2 flux capacity, illustrating the significance of mitochondrial adaptations (53). Trained endurance runners saw at least a 2. 5 times higher activity value in succinate dehydrogenase than untrained individuals, implying that enzyme activity of the Krebs cycle increases and adapts (26). It is known that beta oxidation of fatty acids involves FAD and NAD, so it would seem feasible to suggest that increasing NAD concentration/synthesis could help increase or maintain utilisation of fat in doing so sparing glucose.

The Krebs cycle itself is an elaborate chain of intermediate compounds, enzymes and reactions. The cycle is responsible for approximately 67% of all generated reducing equivalents per molecule of glucose, highlighting the importance of Krebs cycle flux for oxidative phosphorylation. An increase in the total concentration of the Krebs cycle intermediates is also necessary to augment and maintain Krebs cycle flux during exercise (12). NAD+ plays a central role throughout the cycling of reactions and so with the suggestion that Krebs cycle intermediates increase during exercise training, increasing NAD+ biosynthesis and therefore concentration/pool size could have beneficial effects on exercise performance. Research has been carried out on maximal one leg exercise and the results show that as maximal oxygen uptake increased to the muscle the maximal enzyme activity of citrate synthase, α-ketoglutarate dehydrogenase and succinate dehydrogenase increased to match demand (9). α-ketoglutarate dehydrogenase average maximal activity is almost the same as the average flux through the Krebs cycle. This indicates that the enzyme activity is fully activated during maximal exercise (one leg exercise) and is one factor limiting the flux through the Krebs cycle. Enzyme activity within the Krebs cycle appears to increase in adaptation to exercise training but more research is needed to confirm if activity/concentration of all enzymes within the Krebs cycle adapt to training. It is interesting to note that α-ketoglutarate dehydrogenase is highlighted as one limiting factor in Krebs cycle rate/flux because this point coincides with an increased demand for NAD+ to oxidise isocitrate then α-ketoglutarate. Could NAD+ increased demand at this specific point potentially explain why the enzyme α-ketoglutarate dehydrogenase has been described as limiting? Would α-ketoglutarate dehydrogenase not be limiting if there was a sufficient input of NAD+? The evidence does possibly strengthen the theorised importance of NAD (Fig. 2).

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Figure 2. Diagram depicting the Krebs cycle in more depth. The red area highlights the limiting enzyme α- ketoglutarate and the increased demand for NAD at this point. Author ’ s own figure.

Enzymes involved in aerobic metabolism become limiting only when the energy need of the cell requires a rate of substrate catabolism that exceeds the maximum catalytic ability of the rate limiting enzymes. However adaptations such as increased enzyme activity and increase in mitochondria in trained individuals results in these enzymes not being rate limiting (26).

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Table 1. Table representing an experiment on untrained, medium trained and well trained individuals and their ability to generate ATP aerobically and anaerobically.

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Table. 1 represents the results of an investigative experiment carried out on untrained, medium trained and well trained humans. The values of the maximum rate of O2 uptake (VO2max) were measured and the results show that values were 21% higher in medium trained and 49% higher in well trained compared to untrained individuals. α-ketoglutarate dehydrogenase activity was also analysed and the activities of the enzyme were 39% higher in medium trained and 90% higher in well trained compared with untrained individuals (8). The results demonstrate that training increases the capacity to generate ATP aerobically and that α-ketoglutarate dehydrogenase activity also increases with training. Hexokinase (enzyme involved in phosphorylating glucose-6-phosphate) has been reported to change in parallel with the Krebs cycle enzymes as a result of exercise training and electrical stimulation of skeletal muscle (9). The enzymes citrate synthase and NAD+ linked isocitrate dehydrogenase, both involved in the Krebs cycle, are inhibited by ATP and low concentrations of calcium respectively and activated by ADP. Although these inhibitory and activating effects are removed when concentrations of isocitrate are high (1, 61). This evidence implies that as long as the Krebs cycle can continue the enzymes citrate synthase and NAD+ linked isocitrate dehydrogenase are not limiting factors in aerobic metabolism. In the same study it was found that in insect flight muscles the activities of both citrate synthase and NAD+ linked isocitrate dehydrogenase are high, indicating that the muscles involved in flight for insects depend on aerobic metabolism for energy production. The study concerns insect flight muscles but the premise is the same, in order for aerobic metabolism to continue at a high rate these enzymes and substrates involved (i. e. NAD) need to be high. It has been demonstrated that if the concentrations of NAD and isocitrate are sufficient the activities of the enzyme NAD+ linked isocitrate dehydrogenase can remain maximal, emphasising the key role NAD+ has in the continuation of the Krebs cycle and thus aerobic metabolism (1). The slowest step of the Krebs cycle has been found to be between oxaloacetate and citrate and its at this point that pyruvate is converted to acetyl CoA with NAD+ being utilised in this reaction (67). Citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are major know sites for regulation and represent important branch points for the overall flux through the Krebs cycle (67, 68). All of these enzymes are effected by NAD+ concentrations. Citrate synthase and α-ketoglutarate dehydrogenase both have favourable conditions when NADH concentrations are low and NAD+ concentrations are high. Isocitrate dehydrogenase is stimulated by NAD+. So all three of the most important enzymes involved in the Krebs cycle need NAD+ concentrations to remain high.

The oxidative capacity of skeletal muscle is highly plastic in humans with adaptations occurring to the cardiovascular and respiratory systems. Changes are also seen in mitochondrial concentrations and in the activities of the various enzymes involved throughout the process of aerobic metabolism. All of these adaptation are beneficial from an exercise performance standpoint and increase the efficiency of the complex metabolic process. There are still however questions surrounding the idea that NAD+ plays a key role in this process and whether increasing the synthesis/concentration would be advantageous for the trained individual.

2. Discussion

The nicotinamide coenzymes are biological carriers of reducing equivalents. The most common function of NAD+ is to accept two electrons and a proton (H+ equivalent) from a substrate undergoing metabolic oxidation to produce NADH, the reduced form of the coenzyme. The chemistry of the NAD+ molecule allows it to readily accept electrons to transfer to the electron transport chain where donation of the electrons results in the concomitant generation of ATP, a molecule universally required for most energy consuming cellular processes (54, 68). Energy metabolism is largely mediated by the electron transport chain found within the mitochondrion and NADH plays a vital role in furnishing reducing equivalents to fuel oxidative phosphorylation (56). The molecule NAD+ is formed from simple compound precursors such as nicotinic acid, nicotinamide, nicotinamide riboside and tryptophan. All of which can be taken up in the diet. Cells can also take up extracellular NAD+ from the surroundings. There are several pathways for NAD+ formation: 1. In the liver and other animal tissues, tryptophan degradation forms, among other products, quinolinic acid which is converted to nicotinate mononucleotide (or deamidonicotinamide mononucleotide/deamido-NMN) by quinolinate phosphoribosyltransferase, 2. In the cytosol of cells in many mammalian tissues there is the enzyme nicotinate phosphoribosyltransferase that forms deamido-NMN from nicotinate, 3. A very similar phosphoribosyltransferase present in the cytosol of all animal tissues acts on nicotinamide (Fig. 3).

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Figure 3. Diagram showing the different pathways of NAD formation. Author ’ s own figure.

These transferases are responsible for the utilisation of nicotinate and nicotinamide in the diet with the role of ATP in the reaction unclear. Flavin adenine dinucleotide (FAD) also plays a similar role in energy metabolism as NAD+ but it does not appear as significant. FAD is formed from riboflavin which is an essential dietary constituent for mammals. The requirement for multiple synthesis/recycling pathways is a question with which a satisfactory explanation is needed. The most obvious rationalisation would be that the continued generation and preservation of NAD+ levels is so essential for metabolic processes that evolutionary selective processes resulted in the development of several pathways (21). Therefore emphasising the critical role NAD+ has to play in the metabolic energy systems, again strengthening the claim that increasing NAD+ levels would bring about beneficial consequences.

Before elaborating on the biosynthesis of NAD the malate-aspartate shuttle and the glycerol-3- phosphate shuttle will first be looked at. It is known that one rate limiting step for aerobic metabolism is the shuttling of both NADH and pyruvate from the cytoplasm into the mitochondria. In this regard it is interesting to note that well conditioned and trained athletes actually have a higher number of mitochondria in their muscle cells, a possible adaptation to overcome the rate limiting factor of shuttling NADH and pyruvate into the mitochondria (14).

The malate aspartate shuttle is a beautifully complex array of reactions that occur at the inner membrane of the mitochondria. NADH that is generated in the Krebs cycle takes place in the mitochondrial matrix and so has direct access to the electron transport chain. NADH generated during glycolysis on the other hand cannot reach the electron transport chain directly as there is no direct mechanism for the transfer of NADH across the mitochondrial membrane. The malate aspartate shuttle has evolved so that the energy of the reduced NADH can move across the membrane in the form of other reduced molecules. In the intermembrane space NADH donates its hydrogen and electrons to oxaloacetate to form malate, which can then move into the matrix of the mitochondria through the malate α-ketoglutarate transporter. The electrons and hydrogen present in malate are removed by NAD+ to generate NADH and oxaloacetate, with malate dehydrogenase catalysing the reaction. Therefore the electrons from glycolysis have entered the matrix and now inside can enter the electron transport chain (Fig. 4). Approximately 2. 5 molecules of ATP are formed from each NADH that is oxidised in the mitochondria, the shuttling system does not produce as much ATP as can be obtained from mitochondrial NADH (68). This is the same with the glycerol-3-phosphate shuttle.

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Figure 4. Diagram of the malate aspartate shuttle. Author ’ s own figure.

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