Archive for the ‘Cell Signaling’ Category

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.

Ketogenic Diets for Cancer. I. “What makes you think that ketone bodies will help?”

Posted: October 3, 2016 in Cancer, Cell Signaling, diabetes, evolution
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Dr. Eugene Fine and I will described the problem as laid out in our campaign at Experiment.com. The campaign intends to follow-up 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.We got good feedback and some good questions and we want to continue the scientific interaction and keep the community intact that was started on the “lab notes” at Experiment. We will recapitulate some of the points made during the  campaign and you can “ask the researchers” in comments.

“What makes you think ketone bodies will help?”

We and others have carried out experiments that show the effects of ketone bodies on cancer cells in culture, as diet for patients with advanced cancer or as adjuncts to other modalities. Most direct experimental studies, however, must be considered preliminary and it is reasonable to ask why we thought ketone bodies might help.

The evidence supporting carbohydrate restriction, or specifically ketogenic diets in cancer remains largely indirect and speculative. Our recent perspective  summarized some of the relevant evolutionary and mechanistic factors: the central theme rests with the role of the glucose-insulin axis in promoting growth and proliferation, the predominant characteristic of cancer sells. So it has been observed for some time that patients with diabetes have higher risk of cancer. Epidemiological and other kinds of studies are generally consistent with the idea although different cancers are more or less closely associated with diabetes. Drugs employed as diabetes therapy, particularly metformin, have been found to have beneficial effects in cancer as well. Metformin reduces the risk of developing cancer although the effects on mortality are not clear cut. We made the case, in our critical review that dietary carbohydrate restriction is the first line of treatment for type 2 diabetes and the best adjunct for pharmacology in type 1 diabetes.

cancer-diabetes

The association between cancer and diabetes in combination with the benefits of carbohydrate restriction in diabetes constitute one big connection. In dietary approaches, however, it is total caloric reduction that has received the most attention and, in fact, experiments show that if implemented as stated, calorie restriction represents a reliable approach to prevention and treatment of cancer, particularly in animal models. It is unknown how much of the effect is due to de facto reduction in particular macronutrients but when tested, carbohydrate reduction as the means of reducing calories prove most effective. We cited an important study by Tannenbaum. He found, in 1945 (!) that a carcinogen-induced sarcoma in mice was repressed by reduction in total calories but if  reduced by specifically lowering the carbohydrate intake, there was an enhanced response.

tannenbaum_low-carb_cr

Impressive cancer prevention with calorie restriction in animal models has been repeated many times. Oddly, the protocol is most often presented as caloric restriction.  Odd in that this appears in sophisticated scientific papers where the downstream effects of the stimulation may pinpoint twenty molecular components and where the molecular targets of the “nutrients” are characterized and may specifically be the insulin receptor and the related IGF-1 (insulin-like growth factor -1) receptor. (Insulin is probably most important in that it stimulates IGF-1 activity by reducing the levels of the associated binding proteins). In these studies, where total caloric reduction is the independent variable, the involvement of insulin and the insulin-dependent downstream pathways have been shown to be involved.

It is now appreciated that the Warburg effect, the apparent reliance of tumors on glucose for fuel, is a key observation that has been insufficiently explored. The effect provides motivation and clues for exploring the metabolic approach to cancer. Warburg thought that all cancers showed this phenotype which is not true but a large number do; of significance is that one that does not, prostate cancer, is the outlier in the figure above on relation to diabetes. The next post will start from some basic biochemistry and explain why (and how) we think that the Warburg effect points to the potential value of ketogenic diets.

 

Targeting insulin inhibition as a metabolic therapy in advanced cancer. Part 2. The hypothesis.

Posted: November 30, 2012 in Cancer, Cell Signaling, evolution, ketogenic diet, low-carbohydrate diet
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Guest post: Dr. Eugene J. Fine

Last time I discussed our pilot study showing the effects of carbohydrate (CHO) restriction & insulin inhibition (INSINH) in patients with advanced cancers.  We described how the molecular effects of INSINH plus systemic (total body) effects like ketosis might inhibit cancer growth. My goal now is to present the underlying hypothesis behind the idea with the goal of understanding how patients with cancers might respond if we inhibited insulin’s actions? Should all patients respond? If not, why not? Might some patients get worse? These ideas were described briefly in our publication describing our pilot protocol. (more…)

Targeting insulin inhibition as a metabolic therapy in advanced cancer

Posted: October 15, 2012 in Cancer, Cell Signaling, ketogenic diet, low-carbohydrate diet
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Dr. Eugene J. Fine.   Dr. Feinman invited me to contribute a guest blog on our recently published cancer research study: “Targeting insulin inhibition as a metabolic therapy in advanced cancer: A pilot safety and feasibility dietary trial in 10 patients” which has now appeared in the October issue of the Elsevier journal Nutrition, with an accompanying editorial.  Today’s post will focus on this dietary study, and its relation to the general problem of cancer and insulin inhibition. Part II, next week, will discuss in more detail, the hypothesis behind this study. Richard has already mentioned some of the important findings, but I will review them since the context of the study may shed additional light. (more…)

Suddenly last summer. Part II

Posted: October 2, 2012 in Cancer, Cell Signaling, low-carbohydrate diet
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In the last post, I had proclaimed a victory for dietary carbohydrate restriction or, more precisely, recognition of its explicit connection with cell signaling. I had anointed the BMC Washington meeting as the historic site for this grand synthesis. It may have been a matter of perception — many researchers in carbohydrate restriction entered the field precisely because it came from the basic biochemistry where the idea was that the key player was the hormone insulin and glucose was the major stimulus for pancreatic secretion of insulin. We had largely ignored the hook-up with cell-biology because of the emphasis on calorie restriction, and it may have only needed getting everybody in the same room to see that the role of insulin in cancer was not separate from its role in carbohydrate restriction. (more…)