KINE 451 - Protein and Amino Acids

Hello, and welcome back to our series of online sport nutrition lectures. Today's topic is going to be protein and amino acids and their role in exercise and sport metabolism.

So, as we covered in our discussion on nutrients, protein is synthesized from amino acids. Amino acids make up the building blocks of proteins, and proteins usually have up to 20 different amino acids. That we find 11 of these amino acids are considered essential, and we discussed what this means. But to review, an essential amino acid is one that is not synthesized naturally in the human body. The other nine are synthesized in the human body, mostly from bacteria in the gut and viscera.

When we think about high-quality or complete proteins—so think about these as proteins that have a protein quality score of closer to one—those proteins usually have all of the essential amino acids. So, per serving, they provide 100 percent of the essential amino acids required.

Now, when we look at the basic chemical structure of an amino acid, we'll see that regardless of which of the 20 amino acids we are talking about, they all have a central carbon atom. So, if we're taking a look over here at this chemical structure and we looked at all these different amino acids, we would find that they would all have this central carbon atom, and that it's bound to four different chemical groups. The first is a hydrogen atom; so a single hydrogen atom is bound to this central, uh, alpha carbon atom. The second is the amino group, and this is what needs to be removed for a protein to be used in metabolism, and we'll discuss where that occurs later in the presentation. The tail end of the amino acid is actually what makes it an acid; this would be the carboxylic acid group. And then what makes each amino acid unique—all of the 20 amino acids have a different, um, R group or side chain. So it's these differences in the side chain of the amino acids that actually make them unique and allow them to be used for different metabolic functions.

Now, human beings and animals are constantly engaging in a process known as protein turnover, and that turnover is the product of protein synthesis, or making new proteins, as well as protein breakdown, or breaking down stored proteins into their individual amino acids. And the degree to which protein turnover favors either synthesis or breakdown is directly related to the pool of circulating free amino acids, or amino acids that are available in the blood that are going to be able to be used for a couple of different potential purposes. So this pool of free amino acids, we get those from either eating protein and absorbing them into the bloodstream once they've been broken down into their individual amino acids, or we can—again, we can break protein down; we can break down our sources of stored protein—to then contribute to the pool of free amino acids. And then we know that this pool of free amino acids will either be used to synthesize new proteins, or it can—they can be oxidized via several metabolic processes—to carbon dioxide.

So if we think about what needs to happen in order for protein gains to occur, or for us to accrue new proteins, we need to induce a state in which protein synthesis is significantly greater than protein breakdown, and that would result in a net positive protein balance. If these two things are in equilibrium to each other, well then that would mean we would have a net protein balance; so there wouldn't be any overall loss of protein, but there also wouldn't be any accrual or gain of protein. Now, conversely, if protein breakdown—the rate of protein breakdown—is exceeding the rate of protein synthesis, then that would result in a net negative protein balance and a possible loss of proteins.

So if that's the case, and we need to maintain a net balance to maintain our stored protein sources, or we want to gain protein or create a net positive balance, why do we break down proteins at all? And there's a few different reasons why this might occur. The first, which is one of the main issues during exercise, is that we break down—remember that when we contract muscle, we create micro tears in the myofibers themselves and damage that tissue—and in order for that tissue to grow back healthier and stronger than it was before and create a positive exercise adaptation, well we ultimately need to break down and remove that damaged tissue and replace it with healthier and stronger tissue. So that's definitely something that happens as a result of all types of exercise training.

The second is that protein can provide energy. Some of the individual amino acids—and we'll investigate the roles of amino acids in metabolism here in a moment—but some of these amino acids can actually be converted into the essential materials needed for oxidative metabolism, specifically those that are related to the Krebs cycle and the generation of electron carriers, which produces large yields of ATP once those electrons are carried to the electron transport chain. We also know that individual amino acids can be used to synthesize other protein-based compounds, other than things like muscle tissue or collagen or some of our structural proteins that we potentially damage during exercise. For example, we know that our neurotransmitters, such as serotonin for example, are highly dependent upon protein for their formation. We know that hormones such as insulin and growth hormone—or polypeptide hormones—are chemically based in amino acids; these are protein-based compounds and mixtures of multiple different peptides that are bonded together that make these hormones, which function in a very specific way to alter metabolism and perform a host of other physiological functions. And then lastly, we know that amino acids can actually be used to form other energy-yielding compounds, specifically glucose and ketones. And so this is going to be an important concept to visit here as we discuss the different ways in which proteins—or amino acids, rather—can enter the metabolic pathways and provide a fuel source for energy expenditure.

So before any amino acids can be metabolized or enter our metabolic pathways after those proteins are digested and broken down into their individual amino acids in the gastrointestinal system, the amino group ultimately needs to be removed, and this happens in the liver. And when we think about the role of amino acids in metabolism, all of these things are occurring in the mitochondria of the liver itself. And we can see here depicted the different places, relative, um, to oxidative metabolism in the mitochondria where different amino acids can enter and contribute to energy metabolism. And in order for that to happen, once that amino group is removed, it's actually the remaining carbon skeleton that is going to enter the metabolic pathways, which can happen in one of two different ways. The first is that some amino acids are considered glucogenic. So if we take a look at our metabolic pathways here, any of the amino acids that are printed in red print are considered glucogenic amino acids, and the reason that these are considered glucogenic is because ultimately they can enter, um, in one of three different places: the first is that they can be used to form pyruvate; they can also be converted into oxaloacetate; or they can be converted into one of the intermediates of the Krebs cycle itself. And the reason that this is important and the reason that these amino acids are considered glucogenic is because ultimately what they can do is they can be converted—or into oxaloacetate—either via conversion from pyruvate, direct conversion to oxaloacetate, or used to form oxaloacetate by providing one of the other intermediate steps of the Krebs cycle that precede the formation of oxaloacetate. And this is important because oxaloacetate can then be converted to phosphoenolpyruvate in the liver, which can ultimately be converted into glucose. So the liver can use these ultimately to produce new glucose or undergo the process of gluconeogenesis. So all of the glucogenic amino acids can enter at one of three places that ultimately can result in the formation of new glucose in the liver.

The other metabolic fate of certain amino acids is that they can become ketogenic, and we've talked about ketone formation in our discussion of ketogenic diets, and we know that one of the three ketone bodies that can be formed is acetoacetate. And so there are five different amino acids that can be converted into this ketone body, acetoacetate. Now we also can convert certain amino acids—specifically one amino acid, isoleucine—can be directly converted into acetyl-CoA, which can then also be used to form the ketone body acetoacetate. So the ketogenic amino acids ultimately enter, um, the metabolic pathway either by being converted directly to acetyl-CoA or into the ketone body acetoacetate.

Now you might be asking yourself, well if these are converted into acetyl-CoA, well then ultimately once they enter the Krebs cycle aren't they going to form oxaloacetate, which can then be used to make new glucose? And the answer to that has to do with exactly what we were just discussing in terms of where they're entering the actual metabolic pathways. If we remember from our knowledge of the Krebs cycle, the carbon atoms from acetyl-CoA are actually oxidized to carbon dioxide before they actually ever make it, um, to the other side of the Krebs cycle and form oxaloacetate. So that's why these ketogenic amino acids ultimately aren't thought of as amino acids that are able to be used to make new glucose.

If we take a look then at our legend here, we can see that most amino acids—about 14 of them—are considered glucogenic. So there's a heavy reliance on amino acids, or I guess ability of amino acids to be used to make new glucose, and it expands pretty widely across the different amino acids. So about 14 of them are able to be used to produce new glucose, either by entering as oxaloacetate, as pyruvate which is then converted to oxaloacetate and used to make new glucose, or by entering the, uh, Krebs cycle as an intermediate which fuels the, um, which fuels the formation of oxaloacetate and production of new glucose. So most of our amino acids are able to perform this function. When we look at those amino acids that are exclusively ketogenic, those are going to be our two amino acids here depicted in green, leucine and lysine, and together these are kind of known as the L-shaped amino acids, and these amino acids can only enter, um, the metabolic pathways by being converted to the ketone body acetoacetate. So it's only these two amino acids that are exclusively ketogenic and cannot be used to form new glucose. And then we have four amino acids, and those amino acids are phenylalanine, tyrosine, tryptophan, and isoleucine, which can be used both glucogenically and ketogenically. So, for example, we can see here if we take isoleucine for example, isoleucine can be ketogenic in that it can produce acetyl coenzyme A and be converted into the ketone body acetoacetate, or it can also be glucogenic in that it can be converted into the Krebs cycle intermediate succinyl coenzyme A and ultimately used to fuel the formation of oxaloacetate and production of new glucose. So these four amino acids—tyrosine, phenylalanine, and isoleucine and tryptophan—are able to enter the metabolic pathways both from a glucogenic standpoint and a ketogenic standpoint.

So how do these amino acids contribute to, um, fuel or energy expenditure, uh, during rest and exercise? And we kind of referenced that during exercise we don't think of these as a hugely important fuel source, but at rest, um, protein turnover or breakdown—or the oxidation of proteins, rather—amino acid oxidation contributes about 15 percent of energy expenditure in resting conditions. Now if we compare that to what happens during exercise, the relative contribution of protein drops pretty drastically to about 5 percent, and this is due to all of the things that we've discussed previously about the large increases in the reliance on carbohydrates and fats as a relative percentage of energy expenditure that occurs during exercise. And so this is what happens under—under normal conditions—but as you may, uh, be thinking right now, there are some situations, um, specifically situations where carbohydrate availability becomes limited, and we've discussed a couple of these factors: one being exercise, prolonged exercise that results in glycogen depletion in the liver and in the muscle, which is obviously going to reduce the availability of carbohydrate. So we're going to ultimately need to use amino acids in these scenarios to produce more glucose in the liver and release that glucose into circulation so that it can be used to supply blood glucose. And we know that this could become even more pronounced, um, if exercise is being performed, um, in a fasted state where energy availability and carbohydrate availability may be even further challenged, or in something like a ketogenic diet where, due to low intake of carbohydrates, that availability of carbohydrate during exercise may become even more significantly challenged. So during exercise in these situations, it is very possible that amino acid oxidation and the contribution of amino acids to energy expenditure will need to increase. And if we look at studies that have, um, that have investigated the contribution of these fuel sources—or amino acids and protein as a fuel source—during exercise in these situations, protein can contribute a maximum of about 10 percent to energy expenditure. So not as much as a relative percentage as what is observed at rest; however, if we think about the fact that energy expenditure in these situations is much higher than it is during rest, the absolute amount of protein that would need to be broken down and oxidized would actually be larger. So there is an increased need for amino acid oxidation, specifically in situations where carbohydrate availability is limited or depleted.

So where do we get these stored proteins, or where do we store the majority of our protein? And not surprisingly, as we've already discussed when we looked at fuel sources for exercise metabolism, the largest source of protein storage in the human body is in skeletal muscle. And skeletal muscle, as a percentage—on average, obviously this depends a bit on body composition and there are some individual differences—but on average skeletal muscle contains about 40 percent of the stored protein and amino acids found in the human body.

Now when we take a look at what types of proteins primarily comprise skeletal muscle, well not surprisingly, um, 80 to 90 percent of that protein are the contractile proteins, um, actin and myosin. So our thin and thick filaments, um, that are involved in producing the sliding of filaments and force generation and muscle contractions. So the majority of the protein in skeletal muscle is this contractile element; the remaining would be more structural proteins such as troponin, tropomyosin, and titan that are there to either serve regulatory functions or provide physical structure to the muscle itself.

So which amino acids comprise the majority of this protein that is found in skeletal muscle? Well, the—the largest, um, contribution or the largest percentage of amino acids in skeletal muscle, uh, are actually a class of amino acids known as the branched-chain amino acids, and there are three, um, that have this branched structure. So they're all very similar; their R chains are a little bit different, but they possess this similar branched R chain structure, and those amino acids are the amino acids leucine, valine, and isoleucine. So combined, these make up about 20 percent of the total amino acids in skeletal muscle, and we'll see, um, in a few moments how the contribution—or the oxidation, specifically—of these BCAAs increases during—during endurance exercise specifically. And if we take a look then at how much the turnover of skeletal muscle represents in terms of the total daily amount of protein that is turned over, about a third—a quarter to a third—25 to 35 percent of protein turnover is accounted for by the turnover of the proteins found in skeletal muscle. So a relatively large amount of this turnover occurs in our skeletal muscle, given that it contains nearly 40 percent of the protein found in the body. So this is something that we're going to need to account for and may even be more of a concern when we are exercising—muscle breaking down proteins and needing to synthesize new proteins in order to repair damaged muscle or provide amino acids as a fuel source for oxidation.

So let's take a look at what the overall dietary protein requirements are for the general population, and then investigate whether there are elevated protein requirements that are for athletes and exercisers who may be breaking down and turning over more protein in absolute terms compared to sedentary individuals. And so this, um, looks at this particular figure. If we take a look at this, this is a—again, a reflection of protein balance. So this dotted red line would indicate a state in which protein breakdown and protein synthesis are in equilibrium of each other. So this would be a net protein balance; so no overall loss of protein would occur, but also no overall gain in protein would occur, um, so anything above this line would be a positive protein balance. So in these situations, um, this would be a protein synthesis—the—would outweigh protein breakdown, and we would have a net positive balance. And then the opposite would be true if protein breakdown, um, exceeded protein synthesis and we had a net loss of protein or a net negative protein balance, um, so that's what we're looking at here on our y-axis, and this is looking at across studies in sedentary individuals in which they were looking at the ingestion of protein in grams of protein per kilogram of body weight per day and looking at the minimum amount that would be needed to maintain a, um, an even protein balance in individuals who don't engage in extra moderate vigorous physical activity. And this is how the recommended dietary allowance, or the RDA for protein, was set. And if we look at the findings from these studies, it looks like most individuals who are sedentary—remember these are not individuals who would be considered athletes or exercisers—will reach this kind of even protein balance at a rate of ingestion of about 0.6 grams of protein per kilogram of body weight per day. However, the RDA is set at 0.8 grams of protein per kilogram of body weight per day, and the reason for that is because these recommended dietary allowances, um, they are kind of built with a little bit of a safety net in that they're set so that they would meet the needs of at least 97 percent of the population. So other than cases of extreme outliers, um, these protein intakes at 0.8 grams per day—while most people can achieve balance with 0.6 grams per day without any extra requirements—this 0.8 grams per day RDA is set to meet the needs of nearly all of the population in sedentary conditions. But of course this begs the question, um, do athletes and exercisers need to consume higher amounts of protein, and why? Or what would the basis for those extra protein requirements be?

So let's set up a little bit of the physiological rationale for why athletes and exercisers may have higher protein requirements than the 0.8 grams per kilogram of body weight per day that is set for the recommended dietary allowance. And the first thing that we need to take a look at is how these proteins are utilized during exercise. So we'll take a look at endurance exercise first, and specifically the main issue during bouts of endurance exercise is that acute endurance exercise actually increases the oxidation specifically of the branched-chain amino acids, and this occurs as a function of exercise intensity. So if we take a look at light-intensity aerobic exercise all the way up to 100 percent, uh, of VO2 max, we see that BCAA oxidation—so this is, um, this is leucine oxidation; the same would be true for isoleucine and valine if we looked at this—the oxidation of these branched-chain amino acids increases somewhat linearly from light intensity all the way up to maximal intensity aerobic exercise. So why do specifically the oxidation of branched-chain amino acids increase during these bouts of endurance exercise? And the reason for this is because the branched-chain aminotransferase enzymes—so these are enzymes that are able to, uh, transfer the amino group from one amino acid to another compound and form, uh, and enter the oxidative pathways—these enzymes, the rate of activity and the ways in which they're able to do this allows for them to really easily enter those oxidative pathways. And as we've suggested previously, while the oxidation of these branched-chain amino acids increases during exercise as a proportion of relative energy expenditure—or the rate of these—and of these increases—we know that it's going to be even more pronounced when glycogen is depleted. So when carbohydrate availability becomes challenged—say in the later stages of a prolonged bout of exercise—if glycogen becomes depleted, or the more that glycogen is depleted, the, um, the greater the overall contribution of these branched-chain amino acids to oxidation, um, will occur. And this is important for several reasons, and the first being that all three of the branched-chain amino acids are actually essential. So we don't make the branched-chain amino acids naturally in the human body; they're not synthesized in the gut from gut bacteria, and we need to consume them in the diet in order to maintain appropriate levels of these branched-chain amino acids, um, and then if we reference that back to where our largest stores of branched-chain amino acids are, well we know that branched-chain amino acids make up about 20 percent of the proteins found—or the amino acids found—in skeletal muscle proteins. So the supposition here is that if the

Oxidation of BCAAs increases during this type of exercise, and it increases even more if glycogen is depleted for any of the reasons that we've discussed. Well, then it stands to reason that the oxidation of these branched-chain amino acids is most likely largely coming from our stored protein sources, such as skeletal muscle, where there is a large concentration of these branched-chained amino acids.

Now, relative to this demand on branched-chain amino acid oxidation, not surprisingly, resistance exercise doesn't usually increase the oxidation of these branched-chain amino acids. It's really due to the fact that we're not as reliant on our oxidative metabolic pathways during this type of exercise; we're much less likely to deplete glycogen, but also we just don't have as much of a need to supply our oxidative energy pathways or supplement our oxidative energy pathways with the breakdown of these branched-chain amino acids during this type of exercise. However, we know that resistance exercise is going to cause a lot of damage to muscle tissue, and so elevated protein needs for athletes engaging in resistance training is likely more due to the role of amino acids and proteins in providing enough amino acids in the free amino acid pool so that we can synthesize and make new proteins that will be able to replace some of those damaged proteins.

Now, in respect to the effects of exercise and eating on building proteins, as we just discussed, which is something that needs to happen in response to both resistance and endurance exercise, we'll talk about which types of proteins are favored in terms of protein synthesis that results from exercise and feeding in response to both of these different types of exercise. We know, again, that amino acids are going to serve as the precursors for building proteins. If we look at the effects of exercise and feeding again on protein balance, so we've got protein gains or a net positive protein balance above this center line, and protein loss or protein degradation, a net negative protein balance, look at looking over here below the line, well, we can look at studies, and this has been shown both in response to resistance and endurance exercise, that have looked at the effects on protein balance in a rested and fasted state: so no exercise and no feeding versus no exercise with a protein feeding, and then we can also look at exercise without feeding as well as exercise with feeding.

What we will notice, first of all, is that at rest, we again, because we know that about 15% of the total energy expenditure at rest can come from the breakdown and oxidation of amino acids, we see that when not fed and not exercising, there can be a significant uptick in protein breakdown and not much of a spike in protein synthesis, which is going to ultimately result in a net negative protein balance and protein loss. Now, we can shift that in the favor of protein gains or net positive protein balances at rest by simply feeding protein. So, by eating, that eating protein, we can then digest and absorb it into its amino acids and use those amino acids to add to the free amino acid pool, and more of them can then be used for protein synthesis. And if enough of a feeding is is provided, then that overall net gain in protein can happen at rest in the fed state.

Now, if we look at what happens during exercise, let's compare exercise in the fasted condition versus rest in the fasted condition. We'll see that there is a small uptick in protein synthesis that occurs; however, we also have an elevated rate of protein breakdown, and so when we add those two things together, we end up with a net negative protein balance that isn't quite as pronounced as it is in the rested state. So, exercise by itself causes an increase in protein synthesis, but it's not enough for it to overcome the rate of protein breakdown and result in a positive protein gain unless we feed protein. So this net negative result or this net negative protein balance can can last for many hours following a bout of exercise if we do not provide protein feeding and provide amino acids to the amino acid pool via digestion and absorption. However, when we add the effects of exercise—so again, exercise by itself will cause an increase in protein synthesis—but when we add that with the increase in protein synthesis that comes from feeding, we have a significantly higher net positive protein balance that occurs when we combine the effects of exercise and feeding protein. So this is going to be an optimal strategy, not surprisingly, for accruing more proteins, and this happens in response to both feeding and exercise in models of endurance exercise and resistance exercise.

However, the key difference is where or what types of protein synthesis are being favored when this net positive protein balance is achieved. And not surprisingly, when we look at where the majority of that protein synthesis is directed during endurance exercise, most of it is going to be an increase in mitochondrial protein synthesis. So the proteins that make up the mitochondria, the enzymes that are needed to break down fuels oxidatively in the mitochondria, are all increased. So these protein synthesis that ultimately will enhance oxidative metabolism by either increasing the capacity or the overall amount of mitochondria that we have available are favored following a bout of endurance exercise. And then, of course, because the bigger issue during resistance exercise is the damage that it causes—so it it causes greater amounts of tears and muscle damage than endurance exercise does—the majority of this protein synthesis is going to go towards myofibrillar protein synthesis, and ultimately, over time, those smaller upticks on a daily basis from resistance training and feeding will ultimately accrue enough protein to where the size of the muscle can increase or the muscle can hypertrophy. So again, these effects of exercise and feeding are going to produce much larger net positive protein balances than than feeding without exercise will, and where that protein synthesis is directed is dependent upon the exercise stimulus that is provided or induced.

So with that in mind, we can take a look at the daily protein recommendations that have been established for athletes. And if we look at the recommendations for strength athletes, for example, the recommended amount is about 1.6 to 1.7 grams per kilogram of body weight per day, and I think that this is pretty well accepted, and most people understand this in the context of protein and its role in repairing damaged muscle and why individuals who engage in high levels of strength training need more than twice the RDA for protein in order to facilitate the repair and recovery of those damaged muscles. But I don't think that people think about this as much for endurance athletes, even though studies in endurance athletes have shown that similarly, at least 1.2 grams of protein per kilogram of body weight per day are required to maintain protein sufficiency and improve mitochondrial protein synthesis, and that that can get up to close to or actually the same amount that is recommended for strength athletes during periods of high-volume resistance or endurance training where the overall energy requirements and expenditure and possible depletion of muscle glycogen become even more of an issue, and protein may be even more heavily relied upon as an energy source.

Now, if we then look at sports like soccer or rugby or basketball, and we've kind of discussed these, we discussed these previously, these sports involve a mixture of both high-intensity or strength or power bouts as well as endurance bouts. So training for these sports and performing these sports involves the performance of different types of exercise that would fall into the category of strength and endurance, and and not surprisingly, we see that the recommendations for for protein intake for these athletes are also significantly higher than they are for the general population. So studies have shown that about 1.2 to 1.4 grams of per kilogram of body weight per day are sufficient to to recover from these types of sports and training. Now, again, we know that in some situations, extreme situations, and this could be any of these types of athletes could could fall into this category, we've highlighted how protein requirements can be elevated during times of fasting, during performance of ketogenic diets, and also in scenarios where glycogen may be getting depleted either multiple times in a day or are in close succession to exercise bouts or exercise bouts in close succession to each other in the course of an intense training period. These needs for athletes may increase to up to two and a half grams per kilogram of body weight per day.

So regardless of what types of athletes we're talking about, there is an increased reliance on protein as an energy source or as a need for providing protein to repair damages to tissue and regenerate different things like enzymes and proteins that are involved in mitochondrial protein synthesis and mitochondrial function. That athletes do have higher protein requirements than the general population, but then the question becomes, is this an issue for most athletes? And if we look at studies that have looked at reported protein intake in athletes, we'll see that most athletic populations across all of these different types of performance, the intake of protein is either well within these ranges or well above these recommended ranges. And this is because protein insufficiency really mainly becomes an issue when energy intake is low, and that's because most foods that are eaten in the Western diet contain at least some protein. So when we match energy intake to expenditure and we look at the effects of increasing energy intake on protein intake, we see that in most cases, more than ample protein is usually consumed by athletes, so it's not necessarily much of a concern in terms of their ability to achieve these elevated rates of protein intake. Most athletes do a pretty good job of maintaining a sufficient overall daily intake.

Now, of course, this becomes more of an issue when you have athletes with specific dietary restrictions. So one example—now, this is not to say that there are not a multitude of strategies available for vegan and vegetarian athletes to meet these protein requirements, and we'll talk about that later in the quarter when we look at our last research article that is specifically geared towards recommendations for vegan athletes and exercisers—but this can sometimes become an issue in terms of overall protein intake because the sources of protein available in foods are a little more limited to specific types of vegetarian or vegan foods for these athletes. And then, of course, we know that individuals who are either practicing calorie restriction or energy restriction or carbohydrate restriction might need to make some specific provisions to meet these protein requirements specifically related to the fact that energy intake is usually lower in these individuals who are engaging in these practices in order to to lose weight.

So more specifically, if we've established that the overall protein requirements for athletes are higher than they are for the general population, and the first step in making sure that positive protein balances and exercise-induced adaptations can occur is going to be just getting enough protein or enough amount of daily amount of protein intake, there are other factors that are related to these positive exercise and feeding adaptations, and the first is the timing of the protein intake relative to exercise. So if we take a look at studies that have been done where they look at ingesting protein close to the time of exercise—so in the black bars, this is going to be individuals who ingested protein immediately before and after a bout of exercise versus individuals who took in their protein in the morning and evening several hours before and after the exercise session—we'll see that if we look at over time the increases in lean body mass that occur as a result of something like resistance training as well as the overall changes in body fat percentage—so remember, if we increase lean mass and don't have any change in fat mass, which neither of these groups did—well, then body fat percentage of body composition would improve as lean body mass increases. We see that protein consumed close to exercise enhances the maintenance and building of skeletal muscle compared to protein intake that occurs farther away from the exercise session itself.

So then the question from these studies that came up was twofold: Is there an optimal window of time that an individual should consume protein relative to a bout of exercise, and does it matter whether the protein is taken in before the exercise or after the exercise? And the majority of studies have shown, first, that similar increases in muscle protein synthesis are actually observed when protein is consumed either immediately before or immediately after exercise. So it doesn't seem to make too much of a difference in terms of the overall increases in protein synthesis that occur when you exercise and combine that exercise with a protein feeding, but what does seem to matter is that the window of time occurs either at least one hour before the exercise session or within one hour after the exercise session, and this is due to the effects, the pronounced effects or anabolic effects of the exercise itself on protein synthesis. So whether you feed the protein before or after, you're going to get an increase in protein synthesis that is related to just the increase in amino acid availability that occurs from absorbing the amino acids that are broken down from the protein consumed, but in order to combine that effectively with the increase in protein synthesis that occurs as a result of the bout of exercise, you want that ingestion to occur within one hour before or after the exercise session itself.

Now, of course, we've talked about the optimal overall daily amount of protein that's needed to maintain protein synthesis and positive protein balances in athletes in terms of how much in a given day is needed, but if we are now saying that some of that protein intake should occur as close to the exercise session as possible or within the hour before or after exercise, we also then would want to look at: Is there an optimal amount of protein to intake during this period of time? And this is a study that looked at individuals who were all around 70 kilograms of body weight, and they gave these individuals varying amounts of protein in response to a volume-controlled resistance exercise session. So they had them ingest different amounts of protein, so we can see five; they gave they had one condition in which they fed them no protein, five grams of protein, 10 grams of protein, 20 grams of protein, and all the way up to 40 grams of protein. And if we look at the effects of what the individual researchers saw on overall muscle fractional synthesis rate—so this would be a reflection of protein synthesis within skeletal muscle, so a higher value would indicate a higher rate of synthesis—we see that up to the optimal amount for these individuals was about 20 to 25 grams of high-quality protein, and so that's key: the protein doesn't need to be given in only a sufficient amount; it also needs to contain all of the essential amino acids. So you would want a protein that has a quality score of closer to one so that you're getting all of the essential amino acids that you need from this protein ingestion, and it looks as though this protein synthesis in these in these individuals topped out at about 20 to 25 grams of ingestion of high-quality protein, and that there didn't seem to be much of an added benefit when they increased the amount of protein intake to a higher degree. So when we look at, for example, providing 40 grams of protein to these individuals, there really wasn't much of an added benefit in terms of the increase in muscle protein synthesis rate.

Now, of course, this was, as I stated, this particular study was done in individuals who were about 70 kilograms, so if we were to look at this in larger individuals, we would see that the better recommendation would be that this post-exercise meal contains about 0.4 grams of protein per kilogram of body weight per meal in terms of the maximal effect on muscular protein synthesis rate. So this 20 to 25 grams for a 70-kilogram individual would fall into that approximate rate of ingestion of about 0.4 grams per kilogram of body weight per meal. So for a larger individual, that requirement would be would be slightly higher because they have more muscle mass, and studies have shown that this rate of ingestion—again, because if this is coming from high-quality proteins—that this should provide this rate of ingestion should provide about 8 to 10 grams of essential amino acids. So if we're thinking about the optimal amount of protein intake following a bout of exercise or in that hour before or after an exercise session or as close to the exercise session as possible, we would want that meal to have about 0.4 grams per kilogram of body weight per meal, and that it would provide 8 to 10 grams of the essential amino acids or amino acids that are not naturally synthesized by the body.

So that tells us a lot about the amount of optimal protein intake in either a pre- or post-exercise meal and its effects on muscle protein synthesis, but what it doesn't tell us is: Is there an optimal pattern? If we know that we need to, for instance, for a strength-trained athlete, provide 1.6 to 1.7 grams per kilogram of body weight per day, and that some of that is going to come from the meal that is close in relation to the exercise session itself, well, what about the pattern of intake over the course of the rest of the day? And so what this particular study looked at was, again, this would be a bout of resistance exercise, and they were looking at myofibrillar protein synthesis rate over a 12-hour period following a bout of exercise. And what these researchers did was they looked at the effects of feeding the same amount of protein during this 12-hour period in different patterns. And so the first, which would be considered an intermediate pattern of protein ingestion in the 12-hour exercise recovery period, was a pattern in which they gave these individuals four servings of 20 grams of protein. So again, this would be high-quality protein every three hours starting in the hour following the exercise session, and this would be again equivalent to about 0.4 grams of protein per kilogram of body weight per meal. So again, these individuals were closer to like 70 kilograms in weight, but again, this if we wanted to to make this relative for individuals who are larger in size, this would be a rate of ingestion of about 0.4 grams per kilogram per meal. So in this pattern over that three-hour period, they got this amount of protein every three hours at a rate of 0.4 grams per kilogram. The second condition was what the researchers termed a larger bolus of protein ingestion. So they got the same absolute amount of protein over this 12-hour period, but rather than ingesting smaller or more moderate amounts every three hours, they gave that same that same amount of protein every six hours. So this would be a higher overall rate of protein ingestion per meal, but the same absolute amount of protein. So if we reference that to our our body weight relative values, this would be something like 0.8 grams per kilogram of body weight per meal. So for these individuals, they were ingesting the same 80 grams of protein but doing it in two 40-gram sittings separated by six hours rather than four 20-gram sittings separated by three hours. And then the last condition that they had individuals perform was a condition known as a more pulsatile rate of protein ingestion. So he took that same 80 grams of protein, and now rather than feeding it every three hours or every six hours, they ingested it in even smaller servings. So this would be 10 grams for these individuals or a rate of about 0.2 grams of protein per kilogram of body weight per meal ingested at even more frequent intervals. So over the course of the 12 hours, they would get this smaller protein feeding every one and a half hours rather than every three hours or every six hours. And so if we look at the findings from this study, it looks like the intermediate ingestion of protein—so this protocol where individuals ingested 0.4 grams per kilogram per meal every three hours or so—it resulted in a much higher overall 12-hour rate of muscle protein synthesis compared to either a bolus or a less frequent and larger ingestion of protein or a more pulsatile pattern of protein ingestion where smaller amounts were ingested more frequently. And so why is this the case? Why is this more moderate approach or this intermediate pattern of protein ingestion apparently superior for improving muscle protein synthesis compared to these other strategies? Well, the researchers have suggested that in terms of the more pulsatile rate pattern of protein ingestion, it appears that when you are ingesting protein at these lower amounts per meal more frequently, this there's an insufficient stimulus or an insufficient amount of amino acids provided acutely from each meal to provide a maximal rate of synthesis. So then why did the bolus, why did the bolus protein ingestion pattern or the larger amount at a less frequent interval not result in a higher rate of synthesis?

The answer to that is that, um, in terms of the period of time or the latency period needed to maximize protein synthesis, um, the bolus didn't provide a long enough latency period for this to happen. And so, if we think about what's going on in terms of our maximal rate of absorption and uptake of the amino acids, it seems to peak at this, at this point: four grams per kilogram of body weight per meal. Any of the extra amino acids that were provided by this bolus, because sufficient time and latency wasn't provided for all of those amino acids to be ultimately uptaken, the remaining amino acids were either oxidized or excreted in the urine as waste.

So if we take a look at then what the recommendation would be for athletes, then we would say that ingesting, um, about a moderate amount of about 0.4 grams of protein per kilogram of body weight per meal, beginning as close to the period of exercise as possible and repeating that ingestion every three hours over the 12-hour period following the exercise bout, will produce the largest increases in protein synthesis and subsequent adaptations to the exercise training itself.

So we've established that there's an optimal daily amount of protein intake that is needed to increase muscle protein synthesis. We've also established that the timing and pattern of protein intake is important for increasing muscle protein synthesis, but what about the type of protein that is ingested? Now, first of all, we—I've already established that we want the sources of protein to be as high quality as possible and that those sources of protein should provide as many of, if not all of, the essential amino acids that are required or that can't be—that can't be naturally synthesized. But in particular, one specific amino acid, and that amino acid is leucine.

We've already discussed the importance of leucine in terms of its contribution to amino acid oxidation and how the branch-chain amino acids, of which leucine is a part, um, are heavily relied upon when protein oxidation is increased for any of the given situations that we've already described. However, leucine also has a, a powerful effect on hypertrophy and muscle protein synthesis. And the way that it does this is that, um, leucine actually, once it enters, um, once it enters the sarcoplasm, it then initiates or activates this transcription factor known as, um, known as mammalian target of rapamycin, or mTOR. And this mTOR factor is one of the main factors that is activated in order to increase muscle protein synthesis or hypertrophy.

So because leucine, um, seems to be the only amino acid—and that's really interesting—that leucine is the only amino acid that stimulates this mTOR pathway, it is a very important amino acid and probably the most important amino acid in terms of recovery from exercise and enhancements in muscle protein synthesis. So because it stimulates this mTOR pathway, um, it has been studied, um, it has been studied extensively in terms of how much leucine is needed to achieve peak rates of muscle protein synthesis in the recovery from exercise. And studies have shown us that this peaks at about 3 grams of leucine per meal. So that would be the optimal amount of leucine intake in order to facilitate muscle protein synthesis by activating this, this mTOR pathway.

So what does that look like, or what can we, um, where can three grams of leucine be found in protein-containing foods? So let's take a look at that. So if we take a look at the amount of food needed to supply 3 grams of leucine from various sources as well as per unit of energy, so per 100 calories of ingestion of any of these protein sources, we can kind of start to see why protein powders like whey protein and soy protein powder have become attractive options in terms of recovery from exercise. And the reason for that is that per every 100 calories of ingestion of something like whey protein—so this would be like one standard scoop of whey protein—that would provide about 25 grams of protein, nearly all of that three-gram of leucine, um, amount or optimal amount of leucine for muscle protein synthesis could be obtained from just 100 calories of energy coming from this particular source. Soy protein provides an added benefit as well, but it doesn't have as much leucine per unit of energy as something like whey does.

So one of the reasons that these have become popular is that they're more energetically efficient sources for providing the optimal amount of leucine needed for muscle protein synthesis. So that's not to say that you couldn't achieve that, as you can see from ingesting other food sources, but if we compare that to—let's do an easy comparison here—to something like eggs, well, given that per 100 calories of eggs you get about 1 gram of leucine as compared to about 3 grams of leucine per 100 calories of ingestion, you would need to consume three times as much energy in that form to get the same amount of leucine. So this may not be an issue, but if what you're trying to do is to maximize your energetic efficiency in the sources of protein that you are ingesting—maybe you're trying to get as much leucine as possible without ingesting as many calories—these sources of protein like whey protein powder are more energetically efficient means to do this.

So let's talk a little bit about protein powders, and I think that this market has grown again really due to the fact that, um, due to the fact that they are quick and efficient sources of protein that provide, uh, large amounts of essential amino acids, specifically leucine, that are really, really important for enhancing protein synthesis. And if we look at these, uh, the most common three protein powders that are available on the market today, the first is whey. And whey is derived from milk and it's actually derived from the process of making cheese. So as that cheese ferments, there is curd that is formed, and that curd is the whey protein component of the milk. And so milk is about 20% whey protein, and during the process of making cheese we can salvage that whey protein and concentrate or isolate it to make whey protein powder or whey protein supplements.

Now the other 80 percent of protein that is found in milk is in the form of casein, and this can also be condensed into powder form, um, and used as a source or supplement of protein, uh, or protein powder. And then we also have soy protein powders, which are derived from the concentration of soybeans. So these are the three most commonly used protein powders on the market today. There are also others available, but by and large in practical sport nutrition, these three are the ones that are the most prevalent.

So if these are the three most prevalent in usage, it would be good if we had some studies that compared the effects of these protein powders on, on amino acid availability and subsequent muscle protein synthesis. So if we look at studies that have investigated whey versus soy versus casein protein—I also want to point out that again, because milk is mostly casein protein, things like yogurt or Greek yogurt are, are by and large almost all, uh, casein protein in terms of sources where they come from—but if we take a look at what happens in terms of how these different powders are digested and absorbed and then contribute to the free amino acid pool, we see that when we look at whey protein—so whey is going to be our open squares here, soy is going to be our open triangles, and casein is going to be our open circles—we can see that, especially in the immediate period of time following ingestion or the, the hour following ingestion, which again is an optimal time to intake a meal in order to take advantage of the exercise-related increases and feeding-related increases in protein synthesis, we see that whey causes a lot—much larger, um, a much larger increase in amino acid availability when compared to casein or soy.

But if we look at the rate at which those things persist over time, we see that this increase in amino acid availability, because it is so rapidly digested and absorbed, that the fall is—or the increase is transient and it starts to kind of disappear over time. And if we were to extend this out beyond like a three-hour period, well, that increase in protein availability would be short-lived, and it would last about three hours, to which point those effects would, would go away. And this is due to the fact that whey protein is much more soluble in water. So its solubility in water allows for it to be more rapidly digested and absorbed, which is what causes this transient increase in—over in—protein availability and then in subsequent protein synthesis.

So if we look at the effects of this on protein synthesis at rest and during exercise, because of this rapid increase in amino acid availability from a rapidly digested and absorbed protein like whey, it does seem to provide, um, superior rates of protein synthesis following a bout of exercise. But if we look at something like casein, casein is not as soluble in water. When it's ingested in the stomach, it tends to coagulate with other molecules, and as a result of this coagulation, its digestion and absorption is delayed compared to a more rapidly absorbed and digested protein like whey, or even soy would be digested and absorbed faster than something like casein. So there's not as much of an increase in, in amino acid availability, and as a result, there's not as much of an increase in protein synthesis at rest or following a bout of exercise when ingesting this casein protein. However, because of its delayed, um, rate of absorption and digestion, if we were to again look beyond this three-hour time window—and you can kind of see this starting to show up here—this line wouldn't change as much over time. So if we were to extend this out, you know, several hours from the exercise session, greater than three hours from the exercise session, the casein protein, um, would sustain, and it would reduce protein breakdown to a greater degree over longer periods of time as opposed to a more rapidly digested protein, which will provide a larger increase in protein synthesis in the hours following an exercise session, um, but over time casein would facilitate a reduction in protein breakdown for a longer period of time.

So if we want to kind of translate this as to where these sources of protein have the most utility, it stands to reason that something like whey, given that it is rapidly digested and absorbed and has, um, significantly greater effects on protein synthesis, uh, in the hours following a bout of exercise, whey seems to be the best option as a pre- or post-workout recovery beverage. Now, in terms of casein, because casein causes smaller increases in protein synthesis but facilitates reductions in protein breakdown over longer periods of time, this could be a more attractive option throughout the hours of the day that are farther from the bout of exercise. And specifically, we might think about this as a good option if you were concerned with maintaining or decreasing the amount of protein that is broken down during an overnight fast or a bout of sleep. Something like casein would probably be a better option than whey, given that whey would be rapidly absorbed and cause an increase in synthesis, um.

Now we see here that the increase in synthesis, uh, at rest is still, is still achieved, but, um, in terms of staving off protein breakdown over a longer period of time, a slower digested and slower released protein like casein, which is found heavily in dairy products, might be a more attractive option in terms of sustaining protein or reducing protein breakdown over time. And then when we look at something like soy protein powder, well, at rest we see that the changes in overall protein synthesis are comparable, but during exercise, because it's not as rapidly absorbed and digested as something like whey protein is, we, um, we might not see that it provides comparable increases in, um, muscle protein synthesis when ingested, um, as a pre- or post-workout protein beverage. However, we do see that it would outperform casein protein in terms of protein synthesis following exercise, and we know that this particular source of protein might be an attractive option for individuals who are intolerant to milk protein such as whey or casein, or individuals who don't want to ingest sources of milk protein because of practicing a something like a vegan diet.

Now finally, let's take a look at whether it matters if you ingest carbohydrate along with the protein, um. So is there an effect of adding this other nutrient on protein uptake and protein synthesis? And if we look at studies that have compared ingestion of carbohydrate only versus only protein or only amino acids versus a mix of carbohydrate and protein and looked at the effects on protein uptake and protein synthesis, we see that adding some carbohydrates to the recovery beverage will result in higher rates of protein synthesis. And why does this happen? Well, if we take a look at the other effects that were observed in these studies, we can then see the effects of ingesting both carbohydrate and protein on insulin.

So if we take a look at the release of insulin or insulin concentration over time following a bout of exercise, we can compare the effects of ingesting these drinks at multiple time points on insulin concentrations. If we take a look at these square bars here, this would be our carbohydrate plus amino acid condition, um, these more diamond-shaped bars would be carbohydrate only, and our square bars would be—or excuse me, our triangles here would be the ingestion of amino acids alone. And we see that by far the ingestion of carbohydrate and amino acids together over time caused a larger increase in the concentration of insulin. And so why does this matter? Well, it matters because insulin has anabolic effects on protein turnover. So because insulin allows for more of the amino acids that are provided as the substrate for protein synthesis, the carbohydrate creates a more anabolic environment, and this is because of the effects of carbohydrate and insulin on the permeability of the muscle cell to these nutrients.

So when we increase the insulin effect of the beverage by adding carbohydrate, not only are we providing both the amino acids, um, we're—we are also providing a stimulus for more of those amino acids to be uptaken into the muscle and used for protein synthesis. So insulin has these sort of anabolic effects on protein turnover, and so by adding carbohydrate to, um, the protein beverage, we can then, um, we can then increase the response of insulin and allow more of those amino acids to enter the muscle cell and be used for protein synthesis. And so this has also been studied in relation to muscle glycogen synthesis. So given that protein has an effect on recovery from exercise, we may be interested in knowing whether or not adding protein to a carbohydrate beverage would improve the, the synthesis of muscle glycogen. And so if we think about, um, and look at this—is a study that had looked at muscle glycogen synthesis rates at any rate of carbohydrate ingestion—these studies looked at adding that standard amount of protein, so 0.4 grams per kilogram of body weight per meal, to a, a carbohydrate beverage that is intended to improve muscle glycogen synthesis. And what we see is that up to the optimal amount of carbohydrate ingestion that is recommended for enhancing glycogen synthesis—and we kind of established what that rate of intake would look like—if we look at this closer to 1.2 to 1.4 grams per kilogram of body weight per hour during that critical window of glycogen re-synthesis, we see that up to this optimal rate of carbohydrate ingestion, adding protein to the meal caused significantly higher rates of glycogen synthesis.

So why might this be the case? And if you remember back to our discussion of how amino acids can enter metabolic pathways, well, the first thing that you'll probably notice is that, well, less carbohydrate is going to be available at these sub-optimal rates of ingestion during this time, but we know that certain amino acids—many amino acids—are glucogenic and that they can be converted to glucose in the liver. So when we add these glucogenic amino acids to, um, these—or excuse me—when we add protein, um, to this beverage and provide, um, more glucogenic amino acids, those amino acids can be converted to glucose in the liver, and they can serve one of two functions. Once they get to the liver, they can either be—and they're converted to glucose—they can either be stored as glycogen or they can be released into the bloodstream and used to supply blood glucose, which can then be uptaken by skeletal muscle and stored as glycogen in the muscle itself.

So if we think about some situations where this might be practically useful, well, it would be useful in any situation where achieving the optimal rate of carbohydrate ingestion for improving glycogen synthesis wouldn't be possible. So we could apply this to athletes who are practicing ketogenic diets. So maybe they want to potentially ingest very little carbohydrate in order to not throw themselves out of ketosis, but they want to get the—some benefit in terms of improving their rate of muscle glycogen resynthesis. Adding some protein to that recovery beverage could help them do that. This may also be relevant for individuals who are practicing prolonged, um, periods of fasting, um, or exercising in a fasted state where they may also be, um, looking to, to maintain ketogenesis over longer periods of time. Ingesting some protein post-exercise might help them improve their glycogen re-synthesis in these situations where ingesting the optimal amount of carbohydrate may fly in the face of their current dietary practices that they are trying to adopt.