Anaplerosis: Why Carbs Spare Protein in Ways That Fat Can’t | MWM 2.16


Which of these two foods has more
protein? Ah ha, it’s a trick question. The potato has carbs and carbs spare the need for protein in a way that fat can’t. A ketogenic diet has neurological benefits. Why do we have to eat such an enormous amount of food? Complex science Clear explanations Class is starting now. Hi. I’m Dr. Chris Masterjohn of chrismasterjohnphd.com. And you’re watching Masterclass with Masterjohn. We are now in our sixteenth in a series of lessons on the system of energy metabolism. And today we’re talking about a concept called anaplerosis, and why this concept means that carbohydrate spares the need for protein in a way that fat can’t. In the last lesson, we talked about how pyruvate, derived primarily from carbohydrate, can support the production of lactate. And how this can allow us to make ATP when oxygen is limiting in a variety of contexts. But the one we want to narrow down in is sports performance. In the next lesson we’re actually going to drill down into sports performance. But the reason we’re inserting this lesson here is because pyruvate also has several other fates. And to truly account for all of the potential differences between carbohydrate and fat in supporting sports performance, we really need to talk about those other fates. And to make those other fates interesting in their own right, we’re going to sprinkle in here another concept of how we can spare the need for protein. So let’s look at what those fates are.The two major fates of pyruvate (besides acetyl CoA and lactate) are conversion to alanine, which is an amino acid, and conversion into oxaloacetate, which is an intermediate in the citric acid cycle that we’ve talked a lot about so far. Shown on the screen is the interconversion of pyruvate and the amino acid alanine. Pyruvate is the simplest alpha-keto acid. And it’s called an alpha-keto acid because the keto group is alpha to the carboxyl group. Alanine is its corresponding alpha-amino acid because the amino group is now alpha to the carboxyl group. To go back and forth between pyruvate and alanine all we do is we swap the keto group for the amino group. However, we do that in the exact analogous way to how we did this for aspartate and oxaloacetate when we talked about aspartate aminotransferase earlier. What we do is, in a vitamin B6- dependent manner, the enzyme alanine aminotransferase, or ALT, is going to take an amino group from glutamate and add it to pyruvate, making pyruvate alanine, meanwhile taking the keto group from pyruvate, swapping it into that position yielding alpha-ketoglutarate. If we do that, we get alanine from pyruvate. Or we go in the exact opposite direction to yield pyruvate from alanine. Vitamin B6 plays the same role in swapping the amino group and keto group that we saw earlier when we looked at AST. Now this is a completely reversible reaction, and so it’s rate is going to be governed by the concentration of reactants. If we have more pyruvate and more glutamate or just one or the other, we’re going to get more alanine. And if we have more alanine and/or more alpha-ketoglutarate, we’re going to get more pyruvate. Like any reversible reaction they’re always going back and forth all of the time. But the net result that we get is when we need more alanine we make more alanine. When we have too much pyruvate, we make more alanine, and vice versa. If we don’t have enough pyruvate, or we have a lot of alanine, we’re going to go in that direction (leftward). As a result, the net balance between pyruvate and alanine is simply going to depend on what do we need more of in that given instant. The other major fate of pyruvate is conversion to oxaloacetate. If you look at the pyruvate molecule, it has one carboxyl group. We simply add a second one on the other end of the molecule to make oxaloacetate, which is a dicarboxylic acid. When we do that, we are using carbon dioxide. However, as we discussed previously, in energy metabolism usually when we engage in carboxylation reactions we’re using the vitamin biotin and we’re directly using bicarbonate. This is in contrast to the carboxylation reactions that largely happen outside of energy metabolism, where we’re usually using vitamin K. And we’re usually using carbon dioxide directly instead of bicarbonate. Now much could be said about this reaction and the role of biotin and why it happens this way. That is for another time. Our goal here is to talk simply about the role of pyruvate in being converted to oxaloacetate. So we’ll note briefly here that this does require biotin, that carbon dioxide is mixing with water in solution to produce bicarbonate, and that the bicarbonate provides the CO2 that becomes the carboxyl group, which is the second carboxyl group of oxaloacetate. Notice here that we’re building up a larger molecule from two small molecules. So that’s energy intensive. To invest that energy we’re using the energy of ATP. We’ll go into the details of the reaction mechanism in a later lesson, but it’s sufficient to note here that although this isn’t a case of ATP hydrolysis it works out in largely the same way. In ATP hydrolysis you’d consume a molecule of water directly to break the bond between ADP and the free phosphate which is expressed here as P sub i for “inorganic phosphate.” And in a carboxylation reaction you’d take CO2, you’d add it and you’d add an electron, freeing up a hydrogen ion somewhere. Well that’s what we have here only the carbon dioxide and the water come together to make bicarbonate. Bicarbonate provides that CO2 and that electron. A hydrogen ion is left over, and that water is eventually indirectly consumed in the breaking apart of ADP and phosphate. Now the cell has to come to a decision. And that’s “if I can use pyruvate to make acetyl CoA, or I can make pyruvate into oxaloacetate, which one should I do?” And actually this is regulated quite simply. Under conditions of a mixed diet where carbohydrate is a major source of energy, overwhelmingly what you’re doing in most contexts is you’re using pyruvate to go through the pyruvate dehydrogenase complex to make acetyl CoA. That acetyl CoA doesn’t usually last very long because there’s a sharply negative delta G for this reaction where oxaloacetate and acetyl CoA are entering into the citric acid cycle. And that large negative delta G is driven by the ability to break the thioester bond of acetyl CoA, releasing the large amount of energy it carries. Now if acetyl CoA accumulates, it’s only accumulating because oxaloacetate is insufficient to condense with it. Otherwise that would be so favorable that it would just happen. Under conditions of relative oxaloacetate depletion, acetyl CoA will rise. Acetyl CoA allosterically inhibits the pyruvate dehydrogenase complex and it allosterically stimulates the pyruvate carboxylase enzyme that converts pyruvate to oxaloacetate. In fact, the pyruvate carboxylase enzyme will be completely inactive in the absence of acetyl CoA binding to it. So under ordinary conditions this pathway can be essentially off. But to the degree that acetyl CoA accumulates, because it’s not entering the citric acid cycle, that acts as the signal that oxaloacetate is insufficient. Oxaloacetate does not remain insufficient for long because suddenly when that happens pyruvate is redirected into pyruvate carboxylase to make the oxaloacetate keep the cycle running as normal. And so this will always be minor simply because pyurvate carboxylase is repleting what you have occasionally lost. Whereas pyruvate dehydrogenase is providing the energy that you constantly need. Well if the citric acid cycle is a cycle, why would you ever need oxaloacetate? Every time oxaloacetate is consumed in condensation with acetyl CoA, it goes through the cycle that ends in the regeneration of oxaloacetate. The reason is, as we’ve noted in previous lessons, the citric acid cycle acts as a metabolic hub that coordinates many different metabolic pathways. Oxaloacetate itself or other citric acid cycle intermediates can leave the cycle for biosynthetic reactions. Some of those that we’ve looked at so far include glucose, amino acids, and heme for hemoglobin. In fact, those molecules, particularly the amino acids, may undergo further conversions into other molecules, a topic that we’re not covering right now. But suffice it to say that whenever we need to synthesize any of these other molecules we lose citric acid cycle intermediates and that’s where we need to replete them. This concept that we need to refill the citric acid cycle intermediates whenever they’re lost for other purposes is an example of the concept of anaplerosis. Anaplerosis means “to fill up.” And it comes from the Greek, ana, meaning up, and plero, meaning to fill. Anaplerosis is to fill up a metabolic pathway, and compounds, substrates, that help you fill up the metabolic pathway are anaplerotic. Or reactions that help you fill up the metabolic pathway are anaplerotic. The citric acid cycle is not the only example where we have anaplerotic reactions. Pyruvate is not the only anaplerotic substrate. However, the citric acid cycle is the example most commonly invoked because it’s so central to metabolism. And its centrality invokes a constant need for anaplerosis because we’re constantly doing other things with those intermediates. And under standard conditions of a mixed diet that includes carbohydrate, the primary anaplerotic reaction is going to be the conversion of pyruvate to oxaloacetate. Well what happens if you restrict carbohydrate? If you restrict carbohydrate two things are happening. One is that you are getting less pyruvate and that means less pyruvate is available for anaplerosis. But also you’re using citric acid cycle intermediates for gluconeogenesis because you need to compensate for the glucose that you’re not getting in the diet. And in gluconeogenesis, which we’ll talk about in much more detail in later lessons, oxaloacetate is the exit point from the citric acid cycle for that process. So every time you need to make a new molecule of glucose that you didn’t get in your diet oxaloacetate is leaving the citric acid cycle to do that. Meanwhile you have two pyruvate for that glucose that didn’t come in from the diet that you don’t have to make oxaloacetate. That’s clearly a net loss of oxaloacetate. So what happens to replace the anaplerotic role of carbohydrate in that context? Well, as shown on the screen, the anaplerotic role of protein takes over in importance. As we talked about in the beginning of this lesson, alanine can become pyruvate. If alanine can provide pyruvate and pyruvate levels are low, meanwhile alanine concentrations are high from dietary protein, then alanine will be converted to pyruvate. And pyruvate will then become anaplerotic through its conversion to oxaloacetate in the ways that we discussed before. Furthermore, glutamate is a source of alpha-ketoglutarate. Branched-chain amino acids are sources of succinyl CoA. Aspartate is a source of oxaloacetate. This is one layer of the onion. If we were to peel back the next layer of the onion we would find other amino acids that can be converted into these amino acids. Or indeed other amino acids that can eventually yield pyruvate. And therefore we would see that, even beyond what’s shown in this screen, protein has multiple ways in which it can become the important source of anaplerosis during carbohydrate restriction. This is in striking contrast to the primary metabolic fate of fatty acids, which is, through the process of beta-oxidation, to yield purely acety CoA. If you’re burning more fat, it’s likely because you’re restricting carbohydrate. So there’s likely some degree to which oxaloacetate is leaving the cycle for gluconeogenesis and needs to be replaced. Acetyl CoA cannot generate oxaloacetate. Acetyl CoA demands oxaloacetate. And as such the primary metabolic pathway that we use to metabolize fat does not yield anaplerosis for the citric acid cycle. Therefore it is primarily carbohydrate and protein that are anaplerotic. And fat stands out as the least anaplerotic of the macronutrients. The role of pyruvate in anaplerosis is the basis of the old saying within biochemistry, “fat burns in the flame of carbohydrate.” And what that means is that, if fatty acids are primarily generating acetyl CoA, they need to enter the fire of the citric acid cycle through the flame of oxaloacetate. And under normal conditions of a mixed diet, the fat that you burn in the citric acid cycle is burned with the help of glucose, which, through glycolysis, generates pyruvate, and through pyruvate carboxylase it generates oxaloacetate and provides that flame. That flame is the flame of anaplerosis. In fact as we’ll cover in later lessons, inadequate anaplerosis in the liver is the biochemical event that initiates the process we know as ketogenesis. Under conditions of fasting or a primarily fat-based diet, fatty acids are entering the liver to undergo beta-oxidation and yield acetyl CoA. Since the liver carries the demand for metabolism when the rest of the body can’t meet its own needs, the fact that the fatty acids are all shifted toward the liver for metabolism, means that the acetyl CoA in the liver is rising to a much higher degree than it would if the liver were just metabolizing energy for its own needs instead of trying to help out the rest of the body. So in the liver cells you have a large increase in the amount of acetyl CoA. Simultaneously the same thing that makes you run more on fat also makes you need more glucose. Carbohydrate restriction is going to mean that oxaloacetate, in the liver, the same place where acetyl CoA is provided to a greater degree, oxaloacetate is leaving to make glucose in gluconeogenesis. Therefore you have this striking amount of acetyl CoA that can’t enter the citric acid cycle because oxaloacetate is leaving at the same time that it’s coming in. The accumulation of acetyl CoA is what initiates ketogenesis, where that acetyl CoA is turned into ketones. Under these conditions, the anaplerotic affect of protein is incredibly important. Let’s get an overall big picture of what’s happening to our use of fuel here. Fatty acids are coming from the blood into the liver. Or maybe the liver is taking up triglycerides and fatty acids are being yielded in the liver in that way. And those fatty acids undergo beta-oxidation yielding acetyl CoA that isn’t going to enter the citric acid cycle because the oxaloacetate is not there in adequate quantities. So it undergoes ketogenesis and the ketones go back into the blood. Meanwhile, protein through aspartate being transaminated to oxaloacetate, or through yielding glutamate that gets transaminated to alpha-ketoglutarate and enters the citric acid cycle, is going to generate oxaloacetate. The oxaloacetate is going to be used for gluconeogenesis. That’s going to provide glucose that then goes out into the blood. The glucose is under high demand in carbohydrate restriction. And it’s primarily going to go to cells that have an obligate need for anaerobic glycolysis, as we discussed in the previous lesson. As the ketones enter the ketone-utilizing tissues, they provide energy by being metabolized to acetyl CoA that enters the citric acid cycle. And so this is in a sense a way of getting acetyl CoA from one place to another. But acetyl CoA is not anaplerotic. So to the degree that there’s any need for oxaloacetate or for any of the other citric acid cycle intermediates in the ketone-utilizing tissues, there’s a need for anaplerosis. And in that proportion, protein is going to be important to provide that anaplerosis to allow the metabolism of the ketones. So under the conditions of carbohydrate restriction, protein is important as the primary source of glucose in the liver. Meanwhile protein is important in ketone-utilizing tissues as the source of anaplerosis to allow the metabolism of the ketone bodies that were derived from fat. The waters of this story are slightly muddied by considering some minor anaplerotic roles of fat. When you burn fatty acids for fuel, you first get them through lipolysis and lipolysis takes triglycerides and breaks three fatty acids off of the triglyceride molecule. When you break those fatty acids off, you leave the glycerol backbone. The glycerol backbone of a fat of a triglyceride is actually a carbohydrate. It has the ability to enter glycolysis to yield pyruvate. Now under those conditions we have carbohydrate restriction needing increased production of glucose. Also the liver which is making the glucose is the primary organ that’s able to metabolize the glycerol. So although in theory yielding of glycerol from fat allows some degree of anaplerosis, it’s mostly going to the liver where that pyruvate is going to make oxaloacetate that’s going to be used for gluconeogenesis to yield glucose for the rest of the body, primarily going to the tissues with obligate needs for that glucose. Further, the glycerol is a small proportion of the triglyceride. Most of the energy generates fatty acids, only a small amount generates glycerol. So this is a minor pathway to begin with. And given the need to make glucose in the liver it probably has very minor ability to contribute to anaplerosis. As we talked about in previous lessons, odd-chain fatty acids generate mostly acetyl CoA. But each odd-chain fatty acid, because it has an odd number of carbons, will generate one molecule of propionyl CoA, which is then metabolized to succinyl CoA. And so odd-chain fatty acids have a minor ability to contribute to anaplerosis by providing succinyl CoA that can enter the citric acid cycle. However, odd-chain fatty acids are mostly produced by bacteria. And in the diet they’re mostly consumed in trace amounts from dairy products. So this is a minor supply of fatty acids overall. And even in their metabolism it’s only one molecule of succinyl CoA generated for the entire fatty acid which is generating many other molecules of acetyl CoA. So this can only contribute to anaplerosis in very trace amounts. Finally, acetyl CoA, when it generates ketones through ketogenisis, is going to generate acetone. Through pathways that we will talk about later, which are pretty much absent from any biochemistry textbook that I’ve ever seen, acetone can be metabolized into
pyruvate. But metabolism of pyruvate from acetone is extremely small, and again under conditions of carbohydrate restriction it’s probably going to be mostly used to make glucose. Acetone further is very volatile and it’s famous for creating ketone breath in people on ketogenic diets. The more acetone you make from your ketones, the more you breathe it out. So acetone is poorly utilized in that sense. And when it’s been measured under extremely ketogenic conditions, acetone is yielding about 10% of the requirement for gluconeogenesis. Is there some acetone that slips into the ketone- metabolizing tissues and acts as a source of pyruvate that can then yield oxaloacetate for the purposes of anaplerosis? Probably, but we’re talking about a minor pathway here of a poorly utilized ketone body where we need more anaplerosis than usual. And we’re primarily using these things to make glucose. So it’s very unlikely that through any of these very minor pathways that fat is going to provide enough anaplerosis to wipe out the need for protein under conditions of carbohydrate restriction. And when I say the need for protein, I mean the need for protein for anaplerosis. Of course you always need protein for your muscle mass, to make all your enzymes and all those other things. But here under conditions of carbohydrate restriction you have increased reliance on that protein for anaplerosis so that you can metabolize acetyl CoA from fatty acid beta-oxidation in the tissues running mostly on fatty acids. And so you can utilize ketone bodies in the tissues that are mostly utilizing ketones. The implication of this is that carbohydrates spare the need for protein in a way that fat can’t. And what that implies is that if you’re mostly running on fat and you’re restricting carbohydrate, your need for protein is going to be higher because you need to cover the normal functions of protein and you need to cover the increased need for anaplerosis and the fact that the anaplerosis is less being met by carbohydrate. Shown on the screen is some evidence
supporting this. In this study, 16 morbidly obese women were randomized to one of two weight loss diets. The non-ketogenic diet and the ketogenic diet were approximately matched for calories. And they were approximately matched for protein. In fact the calories and protein were slightly higher on the ketogenic diet. The difference in calories and protein was incredibly minor between them. And their main difference was that the non-ketogenic diet had 10 grams of fat and the remainder as carbohydrate. Whereas the ketogenic diet had 38 grams of fat and the remainder as carbohydrate. These are matched for calories. The fact that the grams don’t add up reflects the fact that a gram of fat has almost twice as many calories as a gram of carbohydrate. In the panels on the left you see urinary nitrogen excretion on the top and nitrogen balance on the bottom. Nitrogen balance is to what degree does the nitrogen coming into your body equal or exceed the nitrogen going out of your body? Nitrogen balance should be zero when nitrogen coming in equals nitrogen going out. The difference between urinary nitrogen on the top and nitrogen balance on the bottom is that nitrogen balance also covers net absorption of nitrogen. You’re always releasing protein and other sources of nitrogen into the bile. Some of that leaves into the feces. And there are other losses of nitrogen, but it’s mainly urine and feces. In both panels, the ketogenic diet has the filled in circles and the non-ketogenic diet has the open circles. You can see on the top that urinary nitrogen was high as the diets first started out. And in both groups, it tended to decrease over time as they were better able to conserve their nitrogen balance. This was also seen with actual nitrogen balance, which was very negative at first and eventually reached zero. Now you can also see that despite matching for calories and matching for protein the non-ketogenic diet had less urinary nitrogen at all time points through the entire period of this study, which lasted 27 days. Nitrogen balance was better for almost the entire study period in the non-ketogenic group than it was in the ketogenic group. Nitrogen balance reached close to zero, meaning close to balance, as early as around day six in the non-ketogenic group. And it took much longer to get close to zero in the ketogenic group. The difference in nitrogen balance between the two groups is represented by the gap between these two lines. And that gap equates to a greater loss of two pounds of lean mass in the ketogenic group. What that indicates is that carbohydrate, when matched for calories and protein, was able to spare two pounds of lean mass worth of nitrogen over the course of about four weeks. Now there are some questions left unanswered. For example, what happens to nitrogen balance if this carries on for eight weeks? It looks, from this figure, like if you extrapolated it out, nitrogen balance would be the same between the two groups over time. If that were the case then that two pounds of lean mass might be the only nitrogen that you lose during that period and the role of carbohydrate may be primarily to help you adapt to the low calorie diet faster by conserving your nitrogen faster. We don’t know the answer to that for sure. We also don’t know how this would play out in other contexts. When it comes to lean body mass there’s really three big factors. One is calories. One is protein. And one is exercise. Calories are important because muscle is energetically expensive. If your body perceives that it doesn’t have enough energy to support that muscle mass, it’s going to economize and get rid of it. Exercise is important because it’s the thing that tells your body that that muscle mass is important and is worth investing the energy in. So if you want to conserve muscle mass while you’re on a hypocaloric diet, the most important thing to do is have an exercise program that provides an anabolic stimulus. Now if you’re not athletically trained, probably any exercise can provide that anabolic stimulus. But if you’re talking about what stimulus is most effective it’s going to be body-building exercises that are aimed at hypertrophy. The same exercise stimulus that tells your muscles to grow when you have a caloric excess, is going to tell your muscles to stay there when you have a caloric deficit. Finally, protein is super important because protein is what you build muscle from. It doesn’t matter if your body thinks that that muscle is important. If it doesn’t have the raw materials to make it, it can’t make it. So if we were to view carbohydrate sparing the protein requirement in the context of different regimes of calorie intake and exercise, we would find that calories spare the protein requirement and exercise provides the anabolic stimulus that prevents you from degrading that protein. Both of those would be incredibly protective. And they might make the degree of carbohydrate or the importance of carbohydrate much less. My point here is the principle that carbohydrate and protein are far more anaplerotic than fat. And when you have conditions of carbohydrate restriction, your need for anaplerosis rises and your supply of pyruvate as the normal means of anaplerosis declines, which means that gap in anaplerosis has to be met by protein. If that’s the case, then a small amount of glucose in a diet may make its way into those anaplerotic reactions. And the protein that’s used to make the glucose may be the ultimate source of a lot of that anaplerosis. But if you’re really efficient with your carbohydrate then maybe the protein that you’re getting in your diet is enough to support the normal requirements of protein, plus the requirements for glucose for anaerobic glycolysis, plus the requirements of glucose to provide some anaplerosis to the ketone-metabolizing tissues. Those same efficiencies may also help you better harness the anaplerosis that you get from glycerol and that you get from the minor conversion of acetone to pyruvate. Studying the best ways to maintain body composition under different caloric intakes is something that we’ll be in a much better position to study and discuss when we’ve fully talked about the metabolic pathways of the three major classes of macronutrients and the effects of diet on hormones and regulation of those pathways. So we’ll get there and talk about this in much more exhaustive detail.The purpose for today is just to illustrate the general principle and why anaplerosis is relevant in its own right. But in the overall context of the series of these lessons, the purpose of covering this here is that in the next lesson we’re going to take what we learned about lactate in the previous lesson. We’re going to take what we learned about the conversion of pyruvate to alanine and oxaloacetate in this lesson. And we’re going to apply that to the importance of carbohydrate, through these multiple fates of pyruvate, to sports performance. The audio of this lesson was generously enhanced and post-processed by
Bob Davodian of Taurean Mixing, giving you strong sound and dependable quality. You can find more of his work at taureanonlinemixing.com. If you want to keep watching these lessons, you can find them on my YouTube channel at youtube.com/chrismasterjohn. Or on my facebook at facebook.com/chrismasterjohn. Or you can sign up for MWM Pro. MWM Pro gives you premium features
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reading materials, and a community with a forum for each lesson. So if you want to get the most out of these, and you really want to own them, sign up for MWM Pro at chrismasterjohnphd.com/pro. All right, I hope you found that useful. Signing off, this is Chris Masterjohn of chrismasterjohnphd.com. You’ve been watching Masterclass with Masterjohn. And I will see you in the next lesson.

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