Archive for the ‘energy metabolism’ Category

Calories-in-calories-out, substrate cycles and diesel engines

Posted: April 1, 2017 in energy metabolism, TAG-fatty acid cycle, thermodynamics, triglycerides
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Discussions on energy balance and diet have not improved over the years. Most of social media and even the medical literature pretty much conform to what is called, in communications, half-duplex, and tends to generate, as they say, more heat than light. What remains interesting, however, are the scientific points associated with metabolic inefficiency,

Substrate-cycles

Many biological reactions function in a steady state cycle of synthesis and breakdown. In adipocytes (fat cells), for example, there is a continuous cycle of synthesis of fat (triacylglycerol, TAG) and lipolysis (break-down) that goes on all the time. The overall reversible reaction:    3 fatty acid + glycerol ⇌  triacylglycerol + 3 H2O

Fatty acid from the hydrolysis of TAG (or fatty acid from the circulation) is processed for energy(ATP is generated).BLOG_TAG-FA_CYCLE_OxidationFA

The lipolysis (breakdown) step goes by itself but to re-synthesize TAG constitutes an uphill reaction (requires energy) — it’s easy to break stuff. If you want to make things, it costs you. So to put the fat molecule back together, you have to transform the fatty acid and glycerol molecules to make them more reactive. The actual substrates are glycerol-3-phosphate and fatty acyl-CoA which are more chemically reactive but you have to get the energy from someplace, so the synthesis of these compounds requires ATP. This is how fat becomes stored from fatty acid coming into the adipocyte. Glycerol-3-phosphate can be made in the liver from the glycerol from a previous round of lipolysis but, in adipose tissue, the glycerol-3-phosphate comes indirectly from a series of reactions. It is currently believed that the main one is glyceroneogenesis, the truncated form of gluconeogenesis, although some may come from glycolysis.

“glycerol” (different sources of glycerol molecule) +  ATP  → glycerol-3-P + ADP + H2O.

fatty acid + CoA-SH +  ATP → fatty acyl-CoA  + AMP + 2 phosphate +  H2O.BLOG_FA-TAG_TAG_SynthThere is thus a steady-state that continuously readjusts levels of fat and fatty acid. The process will drift in the direction of oxidation when stored fat provides energy to other cells and will tend in the opposite direction — toward synthesis — when fat is stored. The important point is that the steady-state, like an equilibrium state, does not mean that everything has stopped. It means that the forward rate of breakdown is equal to the resynthesis rate. Every time there is a cycle, TAG → FA → TAG,  however, energy is wasted — synthesis of TAG requires ATP, lipolysis is spontaneous and no ATP is re-syntesized. Why would such a thing evolve? The common name of the process is substrate cycle but because each cycle wastes ATP and accomplishes nothing — you get back the substrate that you started with — it has been referred to as a “futile cycle.” Why would the adipocyte waste energy in this way?

The energy in the TAG-fatty acid cycle is not really wasted even though it looks that way on paper. It improves overall efficiency. The cycle regulates the availability of energy to the body. As such it must be able to respond to differing conditions rapidly. Regulation is easier if competing reactions are maintained in a cycling steady-state and then biased in one or another direction. This becomes, in the end, more efficient  than starts and stops in response to different conditions. The TAG-FA cycle :

BLOG_TAG-FA_LOOPDiesel engines

I usually describe, as an analogy, how, if you walk past a bus station, you might see that the buses are parked with their engines idling. Probably less common now than it used to be, the explanation was that it is difficult to start a diesel engine and it is more efficient to let it idle and then put it in gear. Fuel costs and engine designs have changed since the analogy first occurred to me so I checked on line. There is now some controversy and some of the discussion is reminiscent of Marissa Tomei’s testimony in My Cousin Vinny but it is still true that it is common to let diesel engines idle when parked for reasonable periods of time. Diesel engines don’t have spark plugs and depend on high compression and generate high temperatures. It is costly to start and stop the engine repeatedly.  The analogy is that is more efficient to run a cycle of metabolic reactions and then readjust which direction you want it to go in than to start and stop.

Soybeanbus.

The point is that you will store different amounts of fat depending on how many cycles you run in a given amount of time. For weight loss, of course, you hope to run as inefficiently as possible (relative to fat storage. The “wasted energy,” however, is less than if you had a lot of starts and stops.

To determine lipolysis in the adipose tissue, you can measure the appearance of fatty acid in the blood. If the process is simple, that is, if only lipolysis is going on, then the stoichiometry (balance of reactants and products) should be 3:1, three fatty acid molecules for every glycerol released.  If, however, the fatty acid is re-processed, more or less fatty acid will appear in the blood compared to the amount of glycerol that is produced. You can then calculate the rate of cycling = 3x (rate of glycerol appearance) – (rate of FA appearance).

The rate of cycling is increased by feeding, turned on by adrenergic stimulation (norepinephrine), turned on by glucagon and turned off by insulin.BLOG_FA-TAG_CYCLE_MAR_29

Whether, and to what extent this figures into metabolic efficiency and CICO seems like a good question. Anyway, here’s picture of the main inputs and outputs:

BLOG_FA-TAG-FINAL

Ketogenic Diets for Cancer III. More background and the Warburg Effect.

Posted: December 4, 2016 in Cancer, Cell Signaling, energy metabolism, gluconeogenesis, glycolysis, ketogenic diet, low-carbohydrate diet, Warburg effect
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This series of posts is a followup to the project that Dr. Eugene Fine and I described in our campaign at Experiment.com. as follow-up to Dr. Fine’s pilot study of ten advanced cancer patients on ketogenic diets and the in vitro projects that we are carrying out in parallel.

The last post described the two major processes in energy metabolism, (anaerobic) glycolysis and respiration. Pyruvate is the product of glycolysis and has many fates. (Remember pyruvate and pyruvic acid refer to the same chemical). For cells that rely largely on glycolysis, pyruvate is converted to several final products like ethanol, lactic acid and a bunch of other stuff that microorganisms make in the fermentation of glucose. (The unique smell of butter, e.g., is due to acetoin and other condensation products of pyruvate). Rapidly exercising muscles also produce lactic acid.

The sudden interest in the metabolic approach to cancer stems from the work of Otto Warburg whose lab in the 1930’s was a center for the study of metabolism. (Hans Krebs was an Assistant Professor in the lab). Warburg’s landmark observation was that cells from cancer tissue showed a higher ratio of lactate to CO2 than normal cells, that is, the cancerous tissue was metabolizing glucose via glycolysis to a greater degree than normal even though oxygen was present. The Coris (Carl and Gerty of the Cori cycle) demonstrated what is now called the Warburg effect in a whole animal. Ultimately, Warburg refined the result by comparing the ratio of lactate:CO2 in a cannulated artery to that in the vein for a normal forearm muscle. He compared that to the ratio in the forearm of the same patient  that contained a tumor. Warburg claimed that this greater dependence on glycolysis was a general feature of all cancers and for a long time it was assumed that there was a defect in the mitochondrion in cancer cells. These are both exaggerations but aerobic glycolysis appears as a feature of many cancers and defects in mitochondria, where they exist, are more subtle than gross structural damage. The figure shows current understanding of the Warburg Effect.

kdforca_blog_iii_warburg_figure

What about this mechanism makes us think that  ketone bodies are going to be effective against cancer? We need one more step in biochemical background to explain what we think is going on. In normal aerobic cells, pyruvate is converted to the compound acetyl-CoA.  Acetyl-CoA represents another big player in metabolism and functions as the real substrate for aerobic metabolism. If you have taken general chemistry, you will recognize acetyl-CoA as a a derivative of acetic acid.

The reaction acetyl-CoA ➛ 2CO2 is the main transformation from which we get energy. Acetyl-CoA provides the building block for fatty acids and for ketone bodies. Conversely, fatty acids are a fuel for cells because they can be broken down to acetyl-CoA. Under conditions of starvation, or a low-carbohydrate diet, the liver assembles 2 acetyl-CoA’s to ketone bodies (β-hydroxy butyrate and acetoacetyl-CoA). The ketone bodies are transported to other cells where they are disassembled back to acetyl-CoA and are processed in the cell for energy. The liver is a kind of metabolic command center and ketone bodies are a way for the liver to deliver acetyl-CoA to other cells.kdforca_blog-iii_dec_4

Now we are at the point of asking how a cell knows what to do when presented with a choice of fuels? In particular, how does the input from fat dial down glycolysis so that pyruvate, which could be used for something else (in starvation or low-carb, it will be substrate for gluconeogenesis), is not used to make acetyl-CoA.  It turns out that acetylCoA (that is, fat or ketone bodies) regulate their own use by feeding back and directly or indirectly turning off glycolysis (in other words: don’t process pyruvate to acetyl-CoA because we already have a lot). The feedback system is known as the Randle cycle and appears (roughly) as the dotted line in our expanded metabolic scheme.

robin_map_2012-2Where we are going. In our earlier work Dr. Fine and I and our assistant, Anna Miller, found that if we grow cancer cells in culture, acetoacetate (one of the ketone bodies) will inhibit their growth and will reduce the amount of ATP that they can generate. Normal cells, however, are not inhibited by ketone bodies and the cells may even be using them. Our working explanation is that the ketone bodies are inhibiting the cancer cell through the Randle cycle. Now, normal cells can maintain energy, that is compensate for the Randle cycle, by running the TCA cycle (in fact, that is the purpose of the Randle cycle: to switch fuel sources). The cancer cells, however, have some kind of  defect in aerobic metabolism and can’t compensate.  How does this happen? That’s what we’re trying to find out but we have a good guess. (A good guess in science means that when we find out it’s wrong we’ll probably see a better idea). We find that our cancer cells in culture over-express a protein called uncoupling protein-2 (UCP2). We think that’s a player. To be discussed in Part IV.

Ketogenic Diets for Cancer. II. Background on why ketone bodies might help.

Posted: November 21, 2016 in Cancer, Cell Signaling, energy metabolism, ketogenic diet, lipid metabolism, Respiration, Warburg effect
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The series of posts Ketogenic Diets for Cancer  follows from the experiment.com campaign run by Dr. Eugene J. Fine and myself. The campaign is now over and we were most grateful for the support and wanted to keep the discussion going. Currently on this site we will try to summarize and organize some of the exchanges. Use the comments section if you have questions for me or Dr. Fine. We expect the discussion to be broad but the two key papers are Dr. Fine’s pilot study with ten advanced cancer patients which, though a small study, may still be the only prospective human study, and a related in vitro study.

Fine, et al. Targeting insulin inhibition as a metabolic therapy in advanced cancer: a pilot safety and feasibility dietary trial in 10 patients. Nutrition. 2012 28 (10):1028-35

Fine, et al. Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2 Cancer Cell Int. 2009; 9: 14.

To follow up on the previous post, the potential of the ketogenic diet derives from a change in basic outlook from the genetic approach to the metabolic approach. In our original discussion on experiment.com, several people thought that the explanation of the metabolism was too technical.  Here wepresent a simplified version that may allow easier access to the main ideas.

Energy exchange in biochemistry is represented in the interconversion of the molecules known as ADP and ATP, the former the “low energy” form and the latter, the “high energy” form. In essence, it costs you energy to make ATP from ADP and, if you have ATP, the energy from going back to ADP can be used to do work, usually chemical work, making something new like protein or DNA. (The quotation marks remind us that the energy is in the reaction not in the molecules as such). In a rough sort of way then the energy charge of the cell is identified with the level of ATP.

Two major processes, glycolysis and respiration, provide energy as ATP.  Glycolysis, common to almost all living cells, converts glucose into a three carbon compound pyruvic acid (or pyruvate — acids have two different forms and the names are used interchangeably in biochemistry). Glycolysis does not require oxygen and is referred to as anaerobic metabolism. Pyruvate is a key metabolite and can be converted to many substances. Some cells, rapidly exercising muscle, red blood cells and some microorganisms are restricted to anaerobic metabolism and the final product from pyruvate is lactate (lactic acid).

kdforca_blog-1_112116

The second method, respiration is aerobic and can convert all the carbons in pyruvate to CO2 and water. Most mammalian cells carry our respiration and process pyruvic acid aerobically.  Respiration is more efficient, produces more ATP than glycolysis, although glycolysis is faster — related to its role in rapidly exercising muslce. Respiration is dependent on oxygen and produces most of the ATP in aerobic cells. You probably know the punch line here: cancer cells are more likely to rely on glycolysis than the normal cells of which they are variants even if there is oxygen present. What Warburg original measured was the ratio of lactic acid to CO2 and this represents a good indication of the cancerous state.

The Warburg effect calls attention to the choice of fuel for cellular metabolism as a key in understanding  cancer. Closing in on the question of why we think ketone bodies are important, we have to look at other inputs to energy metabolism. Fat is obviously the major contributor. The fatty acids supplied by ingested and stored lipid goes directly into respiration. Under conditions of starvation or of carbohydrate restriction, the fatty acids can also provide the material for synthesis of ketone bodies. Ketone bodies, in turn, derived from fats provide an alternative fuel in place of glucose for many cells. Ketone bodies are made in the liver and transported to other cells, notably the brain, for energyy.  (Looking ahead to more detailed explanation, the derivative of acetic acid, acetyl-CoA is the actual input to respiration; the ketone bodies supply acetyl-CoA to other cells). The figure summarizes the basic ideas on energy metabolism.

kdforca-2_blog-2_112116

We found that if you grow cancer cells in culture, ketone bodies will inhibit their growth and the amount of ATP that they can generate. Next post will describe the experments and how we think they might be explained by the metabolic pathway in the figure.

“A Calorie is a calorie,” uncoupling and collateral estoppel.

Posted: May 16, 2016 in energy metabolism, low-carbohydrate diet, The Nutrition Story, thermodynamics
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One of my favorite legal terms, collateral estoppel, refers to procedures to prevent re-litigation of issues that have already been settled in court. From the same root as stopper, that is, cork, it prevents harassment and wasting of the court’s time. The context is the recent flap over a poster presented by Kevin Hall which has started re-trying the case of whether all diets have the same metabolic efficiency, a question which, in my view, has been adjudicated several times. I put it this way because frequently I have made an analogy between evidence-based-medicine (EBM) and evidence as presented in a court of law. My main point has been that, in the legal system, there are rules of evidence and there is a judge who decides on admissibility. You can’t just say, as in EBM, that your stuff constitutes evidence.  My conclusion is usually that EBM is one of the self-congratulatory procedures that allows people to say anything that they want without having to defend their position. EBM represents one of the many corruptions of research procedure now under attack by critics (perpetrators ?) as in the recent editorial by Richard Horton, editor of The Lancet. One thing that I  criticize medical nutrition for is its inability to be estopped from funding and endlessly re-investigating whether saturated fat causes heart disease, whether high protein diets hurt your kidneys, and whether a calorie is a calorie. It seems that the issue is more or less settled — there are dozens of examples of variable energy expenditure in the literature. It would be reasonable to move on by investigating the factors that control energy balance, to provide information on the mechanisms that predict great variability and, most important, the mechanisms that make it so small in biological systems — most of the time, a calorie is a calorie, at least roughly. Funding and performing ever more expensive experiments to decide whether you can lose more or less weight on one diet or another, as if we had never done a test before, is not helpful.

Several bloggers discussed Hall’s study which claims that either a calorie is a calorie or it is not depending on whether, as described by Mike Eades, you look at the poster itself or at a video of Kevin Hall explaining what it is about. Mike’s blog is excellent but beyond the sense of déja-vu, the whole thing reminded me of the old joke about the Polish mafia. They make you an offer that you can’t understand.  So, because this is how I got into this business, I will try to explain how I see the problem of energy balance and why we might want this trial estopped.

I have taught nutrition and metabolism for many years but I got into nutrition research because the laws of thermodynamics were, and still are, invoked frequently in the discussion. Like most chemists, I wouldn’t claim to be a real expert but I like the subject and I teach the subject at some level. I could at least see that nobody in nutrition knew what they were talking about. I tried to show that the application of thermodynamics, if done correctly, more or less predicts that different diets will have different efficiencies (from the standpoint of storage, that is, weight gained per calorie consumed).

But you don’t really need thermodynamics to see this. Prof. Wendy Pogozelski at SUNY Geneseo pointed out that if you think about oxidative metabolic uncouplers, that is all you need to know. “Coupling,” in energy metabolism, refers to the sequence of reactions by which the energy from the oxidation of food is converted to ATP, that is, into useful biologic energy. The problem in energy metabolism is that the fuel, as in many “combustion engines,” is processed by oxidation — you put in oxygen and get out CO2 and water . The output, on the other hand  is a phosphorylation reaction — generation of ATP from ADP, its low energy form. The problem is how to couple these two different kins of reactions. It turns out that the mitochondrial membrane couples the two processes (together called oxidative phosphorylation). A “high energy” state is established across the membrane by oxidation and this energy is used to make ATP. Uncouplers are small molecules or proteins that disengage the oxidation of substrate (food) from ATP synthesis allowing energy to be wasted or channeled into other mechanisms, generation of reactive oxygen species, for example.

BLOG_car_analogy_May_16The car analogy of metabolic inhibitors. Figure from my lectures. Energy is generated in the TCA cycle and electron transport chain (ETC). The clutch plays the role of the membrane proton gradient, transmitting energy to the wheels which produce forward motion (phosphorylation of ADP). Uncouplers allow oxidation to continue — the TCA cycle is “racing” but to no effect. Other inhibitors (called oxidative phosphorylation inhibitors) include oligomycin which blocks the ATP synthase, analogous to a block under the wheels: no phosphorylation, no utilization of the gradient; no utilization, no gradient formation; no gradient, no oxidation. The engine “stalls.”

In teaching metabolism, I usually use the analogy of an automobile where the clutch connects the engine to the drive train . The German word for clutch is Kupplung and when you put a car in neutral your car is uncoupled, can process many calories of gasoline ‘in,’ but has zero efficiency, so that none of the ‘out’ does the useful work of turning the wheels. Biological systems can be uncoupled by external compounds — the classic is 2, 4-dinitrophenol which, if you are familiar with mitochondrial metabolism, is a proton ionophore, that is, destroys the proton gradient that couples oxidation to ADP-phophorylation.  There are natural uncouplers, the uncoupling proteins, of which there are five, named UCP-1 through UCP-5. Considered a family because of the homology to UCP-1, a known uncoupler, it has turned out that at least two others clearly have uncoupling activity. The take-home message is that whatever the calories in, the useful calories out (for fat storage or whatever) depends on the presence of added or naturally occurring uncouplers as well.

This is one of many examples of the mechanisms whereby metabolic calories-out per calorie-in could be variable.  The implication is that when somebody reports metabolic advantage (or disadvantage), there is no reason to disbelieve it. Conversely, this is one of the mechanisms that can reduce variability.

In fact, homeostatic mechanisms  are usually observed. You don’t have to have a metabolic chamber to know that your intake is variable day-to-day but your weight may be quite stable. The explanation is not in the physics which, again,  predicts variation, but rather in the biological system which is always connected in feedback so as to resist change. However strong the homeostasis (maintenance of steady-state), conversely, everybody has the experience of being in a situation where it doesn’t happen. “I don’t understand. I went on this cruise and I really pigged out on lobster and steak but I didn’t gain any weight.”  (It is not excluded, but nobody ever says that about the pancake breakfast). In other words, biochemistry and daily experience tells us that black swans are to be expected and, given that the system is set up for variability, the real question is why there are so many white swans.

So it is physically predicted that a calorie is not a calorie. When it has been demonstrated, in animal models where there is control of the food intake, or in humans, where there are frequently big differences that cannot reasonably be accounted for by the error in food records, there is no reason to doubt the effect. And, of course, a black swan is an individual. Kevin Hall’s study, as in much of the medical literature, reported group statistics and we don’t know if there were a few winners in with the group. The work has not been reviewed or published but, either way, I think it is likely to waste the court’s time.

 

Hunger. What it is and what you can do about it.

Posted: December 17, 2012 in energy metabolism, hunger, low-carbohydrate diet, Protein
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The reporter from Men’s Health asked me: “You finish dinner, even a satisfying low-carb dinner,” — he is a low-carb person himself — “you are sure you ate enough but you are still hungry. What do you do?”  I gave him good advice. “Think of a perfectly broiled steak or steamed lobster with butter, some high protein, relatively high fat meal that you usually like.  If that doesn’t sound good, you are not hungry.  You may want to keep eating. You may want something sweet.  You may want to feel something rolling around in your mouth, but you are not hungry.  Find something else to do — push-ups are good.  If the steak does sound good, you may want to eat. Practically speaking, it’s a good idea to keep hard-boiled eggs, cans of tuna fish around (and, of course, not keep cookies in the house).” I think this is good practical advice. It comes from the satiating effects of protein food sources, or perhaps the non-satiating, or reinforcing effect of carbohydrate. But the more general question is: What is hunger? (more…)

Suddenly last summer. The triumph of carbohydrate restriction.

Posted: September 3, 2012 in Cancer, energy metabolism, ketogenic diet
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It was in July of 2012 that I suddenly realized that we had won, at least scientifically. It was now clear that we had a consistent set of scientific ideas that supported the importance of insulin signaling in basic biochemistry and cell biology and that there was a continuum with the role of dietary carbohydrate restriction in obesity, diabetes or for general health.  The practical considerations, how much to eat of this, how much to eat of that, were still problematical but now we had the kernel of a scientific principle. In fact, it was not so much that we had the answer as that we had the right question.  In science, the question is frequently more important than the answer.  Of course, winning wasn’t the original idea. When my colleagues and I got into this, about ten years ago, coming from basic biochemistry, we hadn’t anticipated that it would be such a battle, that there would be so much resistance to what we thought was normal scientific practice.

(more…)

Introduction to Metabolism. The Black Box of Life.

Posted: February 2, 2012 in energy metabolism, glycolysis, Krebs cycle, low-carbohydrate diet
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I am currently teaching nutrition and metabolism to first year medical students.  The problem in this subject is the large number of individual reactions which leads students to think of the subject the way somebody described the study of history: just one damned thing after another.  I try to present the big picture and the approach is the systems or “black box”  strategy.  The method is to ask whether we can get some information just by looking at the inputs and outputs to a system even if we don’t know any of the details of what’s going on inside.  In other words, it is a way of organizing limited information.  The method is favored by engineers who are the people most unhappy with the idea that they don’t know anything at all.  First, the big principles.

Metabolism: two goals, two fuels.  

There are two major goals in human energy metabolism: First, to provide energy for life processes in the form of the molecule ATP and second, to provide glucose for those cells that require glucose (particularly brain and central nervous system) and to maintain blood glucose at a relatively constant level: too little is obviously not good but too much is also a problem in that glucose is chemically reactive and can interact with body material, particularly proteins when at high concentrations. Of course, metabolism does many things but these are the two major goals in providing energy.

A second big generalization is that in this process there two kinds of fuels: glucose and acetyl-Coenzyme A (abbreviated acetyl-CoA or sometimes written as acetyl-SCoA; the S, which is meant to show that the compound contains sulfur, is not pronounced).

The black box of life. 

You knew what we do in metabolism even before you started reading this. Putting it into black box terms, you knew: we take in food and we take in oxygen. We excrete CO2 and water.  Somehow this gives us the energy for life as well as the material to build up components of the body.  You don’t have to know too much chemistry to figure out the important conclusion that, inside the black box, living systems use oxidation, just like combustion in a furnace. Lavoisier’s whole animal calorimeter that I described in a previous post was a beautiful real demonstration of this black box.  More technically, this is an oxidation-reduction reaction.  Oxidation, in a biochemical context, means combination with oxygen or loss of hydrogen and reduction means loss of oxygen or gain of hydrogen; we say that the (carbons in the) food gets oxidized and the oxygen gets reduced (to water).  Like the common oxidation reactions you know (combustion in a furnace or an automobile engine), this produces energy which can be used to do work. Some work is mechanical work — moving muscles — but most of the energy is used for chemical: work making body material and keeping biological structures intact and generally keeping things running.  The medium of energy in metabolism is the chemical reaction of synthesis and breakdown of the molecule ATP.  Textbooks frequently refer to ATP as a “high energy molecule” but it is not the compound itself but rather the reaction (synthesis and breakdown (hydrolysis)) that is high energy.  For the moment, we can think of ATP as the “coin of energy exchange in metabolism.”  A heavy-duty thought concept: the challenge for biochemistry historically was to explain how the energy from an oxidation-reduction reaction could be used to carry out the synthesis of ATP which has a different mechanism (phosphate transfer).  The process is called oxidative phosphorylation and was only figured out about fifty years ago.

So again, our two goals in human metabolism: Make energy in the form of ATP and maintain a pretty much constant level of blood glucose for those cells, brain and central nervous system, that require glucose (the brain can’t use fatty acids as a fuel).

Let’s look at energy production first because it is a little easier to understand.  As we look inside the black box, each of the processes uncovered will have its own degree of complexity.  In reading this you have to do what scientists do: hang in there.  Skip over the parts that seem complex and see if you can come back to them later.

The role of redox coenzymes

So, breaking into the black box, the first thing to notice is that the oxidation of food is done in steps, and that there is another player that mediates the process by coupling separate pieces: the food never sees the oxygen.  The intermediaries are called coenzymes or cofactors.  The most important oxidative coenzyme is known as NAD.  It’s always referred to by the acronym, but if you’ve had some organic chemistry and you’re curious, NAD stands for nicotinamide-adenine-dinucleotide; the structure is shown in the figure and the action end of the molecule is indicated. NAD coenzymes are derived from the vitamin niacin.  So   what happens in metabolism is that food is oxidized by NAD+ (the oxidized form of NAD) and the product, NADH (the reduced form) is re-oxidized by molecular oxygen. Although it is still just as we thought (food+oxygen-in, CO2+water-out), the oxygen never sees the food.   Why do we do it this way?  If we did it all in one big blast like an automobile engine, we would have little control over it and we would not be able to capture the energy in a usable chemical form.

It’s easiest to start with glucose, a six-carbon compound. The early steps in metabolism involve a process known as glycolysis (sugar splitting) that ultimately gives you two molecules of a three-carbon compounds known as pyruvic acid. Pyruvic acid is oxidized to a derivative of acetic acid, known as acetyl-CoA. The CoA is short for Coenzyme A, a complicated molecule but, like many coenzymes is always referred to in this way so it is not important to know the detailed structure.  The compound is frequently written acetyl-SCoA to emphasize that it is a thioester (sulfur ester); again, the “S” is not pronounced.

Acetyl-SCoA is the fuel for the major NADH-producing process, known as the Krebs cycle after the major player in its discovery. Without looking into that black box too much the key compound is citric acid, which is, chemically a try-carboxylic acid (TCA) so the Krebs cycle is also called the citric acid cycle or TCA cycle; Krebs called it the TCA cycle so I will generally use that term.  The process whereby NADH is finally re-oxidized by oxygen is known as electron transport.  So, The big black boxes of metabolism:

Where do we get Glucose and Acetyl-CoA?

So far we know: most energy comes from the oxidation of acetyl-CoA and most of the glucose that provides energy does so by first being converted to acetyl-CoA. Where else can we get acetyl-CoA? We’ve taken glucose as synonymous with food but where else can we get glucose from besides the diet?

Looking ahead, the big results that will come out of opening up the black box of metabolism: 1) Acetyl-CoA also comes from fat and to a smaller extent from protein.  2) Glucose can also be formed from protein. 3) Under conditions where there is no dietary glucose (starvation, low carbohydrate diet), glucose will be made from protein or released from stored glycogen, and an alternative fuel ketone bodies will provide acetyl-CoA; ketone bodies are essentially a dimer of acetyl-CoAs and the liver makes and exports ketone bodies to other cells.  Acetyl-CoA and, therefore, glucose can be converted to fat but a major asymmetry that will have profound significance is that 4) glucose cannot be formed from acetyl-CoA.  The significance of the last statement is that: we know all too well that fat can be formed from glucose but, with minor exceptions, 5) glucose cannot be formed from fat. (Chris Masterjohn’s post “We Really Can Make Glucose From Fatty Acids After All!”
indicates the extent to which the exceptions become important but the overriding principle that has the most impact on metabolism is that you cannot make glucose from fat).
So that’s it.  You now have a blackbox view of metabolism.  I will try to open some of the boxes in future posts.

Summary of fuel sources and synthesis and looking ahead.

  1. There are, roughly speaking, two kinds of fuels: glucose and acetyl-CoA.
  2. Carbohydrates and other nutrients, fat (that is, fatty acids) and protein (amino acids) can supply acetyl-CoA.  Glucose is not required for acetyl-CoA and under conditions of low carbohydrate or low total food, fatty acids become the major source of acetyl-CoA.
  3. Not all tissues can use all fuel sources. Brain, CNS and red blood cells, for example cannot use fatty acids. Brain and CNS can use acetyl-CoA but cannot get it from fatty acids.  Red blood cells only use glucose and, to a first approximation, brain and CNS are also dependent on glucose for metabolism.
  4. Under conditions of starvation or carbohydrate restriction, acetyl-CoA can be effectively transported from the liver in the form of  ketone bodies. Ketone bodies, then, are a source of acetyl-CoA that can be used by brain and CNS.  Red blood cells are still dependent on glucose but the brain’s demand for glucose is reduced by the availability of ketone bodies.
  5. There is no dietary requirement for carbohydrate and amino acids can also supply glucose through the process of gluconeogenesis.
  6. Fat as a source of acetyl-CoA also works the other way: acetyl-CoA can be converted to fat.
  7. Whereas glucose can be converted to fat, with a few exceptions, fat cannot be converted to glucose. This will be a key idea behind carbohydrate restriction.
  8. Glucose can also be stored as the polymer glycogen.
  9. Bottom line is the limitation of “you are what you eat.” Metabolism means the interconversion of food and metabolites. Conversely, it will be critical that not everything is interconvertible. In particular, we will emphasize that you can make fat from carbohydrate but, to a large extent,  you cannot make glucose from fat.

Looking ahead on sources of blood glucose:

  1. Glucose from dietary input (referred to as the fed state; in nutrition, as the postprandial period), is depleted after about 8 hours.
  2. Glycogen is a storage/supply source of glucose.  Liver glycogen can supply export glucose to the blood, thereby supplying other tissues.  Muscle glycogen supplies glucose only for the muscle itself.  Glycogen may become largely depleted after 24 hours, depending on the conditions (exercise, for example).
  3. The third source of blood glucose is gluconeogenesis (GNG) which, as the name implies, makes glucose anew from existing metabolites. Depending on the conditions, the source of carbon may be amino acids, lactic acid or glycerol from fat metabolism.  Whereas it is sometimes indicated to be a “last ditch” source of glucose in the textbooks, it goes on all the time. The glucose it synthesizes in GNG may be used to replenish glycogen and only appear in the blood at a later time.

I Told George Bray How to do it Right.

Posted: January 19, 2012 in energy metabolism, low-carbohydrate diet, Protein, The Nutrition Story
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“Despite the claims of various diet gurus, excess calorie consumption alone and not the amount of protein in an individual’s diet contributes to the accumulation of unwanted fat….” That’s the tendentious and pretty much inaccurate first line of the press release from JAMA on George Bray’s over-feeding study “Effect of Dietary Protein Content on Weight Gain, Energy Expenditure, and Body Composition During Overeating.”  “Amount of protein?”  What’s going here?  It hasn’t really been about protein.  Most of us “diet gurus” have claimed that carbohydrate, not protein, in the diet was the key macronutrient in regulating metabolism, consistent with the basic biochemistry of the glucose-insulin axis, or as Dr. Bray described Gary Taubes’s position in a review of Good Calories, Bad Calories:

“The problem is the carbohydrates in the diet, their effect on insulin secretion, and thus the hormonal regulation of homeostasis – the entire harmonic ensemble of the human body.”

Reduction in dietary carbohydrate puts increased demands on protein for gluconeogenesis and other processes but the controlling variable is the carbohydrate. The controversy in nutrition has been largely about fat vs carbohydrate.  Should we be on a low-carbohydrate diet or a low-fat diet?

The quotation in the press release says accurately that “Earlier studies in human beings suggested that diets containing either high or low [levels of] protein are less ‘metabolically efficient’ than diets with normal protein levels.”  Accurate, but written as if metabolic efficiency had always been recognized for its importance in weight loss, as if there had not been a dispute over whether the costs of processing protein were important in energy balance, indeed, written as if Bray and coworkers had not maintained that only calories count in weight gain or loss.  The idea of metabolic advantage, that one diet could be more efficient — more weight gained/calorie — has been largely resisted by the nutritional establishment.  Is this slouching toward Metabolic Advantage? (“Who knows not [the Duke] is dead?  Who knows he is?”)

The debate is also about calories.  Should you cut calories or just cut out carbs?  Is it really “excess calorie consumption” and not the effect of excess carbohydrates ? “A calorie is a calorie” or not. Many of the gurus have gone beyond “claiming” to demonstrating that when carbohydrates are low, weight loss is greater than when carbohydrate is high and that the weight loss on a low-carbohydrate diet is primarily in fat stores rather than lean mass.  In head-to-head comparisons, for however long they are compared, low-carbohydrate diets generally out-perform low-fat diets on other parameters as well, glycemic control, the features of atherogenic dyslipidemia. This has been the major challenge to traditional nutrition and the general approach has been to simply ignore this data and dismiss the researchers with innuendo as above.

In some sense, Bray, et al. answered a question that we weren’t asking, but protein is important if more complicated than carbohydrate and fat. So what did the study find? Bray and coworkers compared three diets of 5 %, 15 % and 25 % protein at an excess of calories, that was nominally the same in each group. The study was a random controlled study and was carried out in a metabolic ward so the results are more accurate than the usual diet study that relies on dietary records.  There is something odd about this study, though, in that if you want to say that only calories are the independent variable, you can’t keep calories constant.  What was actually done was to determine the energy requirements for weight maintenance over a run-in period of 2-3 weeks on a maintenance and then an additional 40 % of calories was added.  So although the calories are constant relative to initial energy expenditure, they are not absolutely the same and this is a study of the effect of varying calories while keeping calories constant. The figure below, re-drawn from Figure 6 of the paper comparing intake of absolute energy to protein intake makes you stop and think.

When you have a small number of subjects, a single outlier can bias the results.  If you remove the single highest point (circled in red), the correlation is likely to get much weaker and the normals and low begin to separate.  In other words, the individual variation (the relative efficiency) is sufficient to make it hard to see the effect of variable energy or, perhaps, as the authors themselves set it up, it is energy normalized for baseline that is the key variable.  Then the authors are right (at least by inspection) that the protein intake does not effect the change in body fat but you have only a single value for the energy. In this case, you cannot say “calories alone account for the increase in fat” (Conclusion in Abstract) because you have only a single point.  If you keep constant the variable (carbohydrate) that is most likely to bring out differences, you shouldn’t be surprised in there are no big differences.

Even taking the conclusions at face value, the authors found, as other diet comparison studies have, that weight loss or, in this over-feeding study, weight gain, was not dependent on calories alone: “a calorie is a calorie” not.  It is likely that this was what the study was originally trying to disprove and the results must have been a disappointment.  The way out was that, in this particular case, the differential weight loss showed up in difference in lean mass, rather than in fat mass as has been found in other studies showing variable efficiency.  Since 5 % is very low protein it is probably not surprising that the diet could not provide enough protein for an increase in lean mass this group.

So what are the other diet studies that have found variable efficiency. The reduction in weight found in studies comparing low-carbohydrate diets and low-fat diets not only shows a difference favoring carbohydrate restriction but the improved weight loss is preferentially fat over lean mass. For example, Volek, et al. compared a low fat with a VLCK and the results are as shown below.  In their study, subjects were randomized to one of two hypocaloric diets, a very low-carbohydrate ketogenic (VLCK) diet (carbohydrate <10% of energy) or a low fat (LF) diet and after 8 weeks switched to the other diet. Reported energy was slightly higher during the VLCK but the VLCK group lost more weight and as shown below predominantly in fat, total fat loss, and trunk fat loss for men (despite significantly greater energy intake). The majority of women also responded more favorably to the VLCK diet, especially in terms of trunk fat loss the ratio of trunk fat/total fat was also significantly reduced during the VLCK diet in men and women.  These studies depend on diet recall so are less accurate than the JAMA study but because of the better experimental design, the changes are bigger and with appropriate correction make a less ambiguous case than the JAMA study. The more accurate measurements in the metabolic chamber suggest that individual variation is real and not just due to random error.

So what do we know from Bray, et al.? As described above, there is some ambiguity in what constant energy means. Still, nobody questions that under many conditions, a “calorie is a calorie,” but they actually found that weight gain was different so when metabolic advantage is “claimed” it cannot be dismissed out of hand.  This is different than widely cited studies in the literature that claim macronutrients do not effect weight loss, since if weight gain depends on macronutrient, it is reasonable that weight loss does too.  Similarly, if tissue distribution affects lean mass in this case, then studies where the tissue distribution shows preferential loss of fat can’t be dismissed — again, it is certainly not surprising that a low protein diet will lead to less storage of protein; generally, while it is just as bad a generalization as “a calorie is a calorie,” there is some truth in “you are what you eat.” Also, in the JAMA study, protein was exchanged for fat so a reduction in fat did not have an effect on fat which may or may not be a surprise to many people. Tom Naughton raised a few other questions about Bray, et al. but in the end, the paper reminds me of the joke about the Polish Mafia: they make you an offer you can’t understand.

How to do it.

But  I told George how to do it. A couple of years ago, he and I had a brief correspondence. I made the following proposal. I suggested we could apply for a joint grant and publication to get the answer.  The following is from my email to him in 2008  (I have added some highlights):

 “A modest proposal

 Proponents of carbohydrate-restricted diets (CRD) and critics of such diets cooperate to design a long-term comparison of CRD and low-fat diets.  The groups agree on methods of procedure, make-up of the diets, how compliance will be effected, and what parameters will be measured.

We write the paper first, leaving room for the data, that is, we agree in advance on what the possible outcomes are and what conclusions could be drawn from them.  The final MS can only be edited for language usage. There are no disclaimers, no Monday-morning-quarterbacking, no excuses.

The paper could be submitted while the grant application is being written and would have to be accepted because any objections could be incorporated in the plan.  The grant itself would surely be funded since it incorporates everybody’s specific aims.”

 George hasn’t answered and he obviously has a different approach to the problem but my offer still stands.

In the end, that is what it will take to solve the problem.  Unless we agree on what the question is, how it can be tested and work together to do the experiment, the lipophobes will ignore the low-carbohydrate studies and we will criticize their studies. The real losers, of course, will be the people suffering from obesity and diabetes.  The question everybody always asks me, is why can’t there be a meeting of the minds?  In the current case, why was the JAMA study done?

Why was this study done? 

 “Dr Bray discussed the results with news@JAMA via e-mail.

news@JAMA: What are the practical implications of these findings for patients trying to lose weight or for the physicians trying to counsel them?

 Dr Bray: The first practical implication is an old one: calories count. We showed very clearly that the increase in body fat was due to the increased intake of calories and that the amount of protein in the diet did not change it.

 To avoid that slow weight gain that many adults experience in their middle years, people need to watch their weight and increase activity, decrease food intake, or both; changing the diet alone will not do it.”

This sounds like the the same recommendations we’ve had for years.  Writing this, I suddenly realized that, as they say in German: that’s where the dog is buried.  It is about recommendations.  This research is following the recommendations.  It used to be (should be? assume it must be?) that recommendations follow from the research. Now, it’s the other way around.  Committees make recommendations and then research (sometimes by members of the committee) tries to support the recommendations. Something about this bothers me.