Macrophages and Energy Metabolism in Cancer: The Ketogenic Connection

The notion that diet plays a pivotal role in the regulation of metabolic syndromes such as chronic inflammation has gained such wide acceptance that we now find the once accepted food pyramid established decades ago where fats and meats occupied a small percentage of total daily food intake is now replaced by one where carbohydrate intake has been dramatically reduced. Depending on the type of diet you are following, you may be consuming little to no carbohydrates and sugars, fruits and/or vegetables. You may also be consuming a high amount of animal protein from meats and/or fish, or your diet could comprise of mostly fats, as in the case of ketogenic (keto) diets.

The Ketogenic Diet is a high-fat/low-carbohydrate/adequate-protein diet. It has gained tremendous popularity because of proven anti-inflammatory actions via the suppression of glucose utilization. It has yielded impressive results in treatments for neuropathic pain, brain inflammation, or even weight loss accomplished via various mechanisms yet to be confirmed [1, 2, 3]. The ketogenic diet has been used since 1920 at John Hopkins to manage epilepsy in children. Recently, the ketogenic diet has been discovered to be effective against tumorigenesis in various cancer therapies [4]. It is believed to be effective against tumor progression because it is able to target the Warburg effect where cancer cells, in contrast to normal cells, mainly use aerobic glycolysis instead of oxidative phosphorylation (OXPHOS) in mitochondria to produce lactate. Lactate is the main energy substrate used by cancer cells for immune suppression and pH manipulation [28]. In an environment where circulating glucose levels are reduced under ketosis, cancer cells are hypothetically ‘starved’ of energy and unable to utilize lactate, while normal cells switch their metabolism to ketones generated by the ketogenic diet [5].

In preclinical evidence documenting the effects of ketogenic diets on tumor growth and progression, however, the association between ketogenic diets and tumorigenesis was not as straightforward as originally postulated. Data from multiple studies showed a range of effects on tumor growth. In some studies, the keto diet was anti-tumor, while in others there was no effect. In some cases, severe side effects or even pro-tumor effects resulted. It appears that whether the keto diet works or not depends upon the type of tumor being investigated and how the immune system responds to the diet. The best results were achieved in glioblastoma, and the worst in kidney cancer with a 50/50 outcome of pro-tumor/severe side effects. Melanoma treated with ketogenic diet also produced a 50/50 outcome of pro-tumor/no effect [5].

From Food to ATP: Understanding Mitochondrial Energetics and Cancer

When we eat different foods, they are broken down into smaller molecules like glucose, proteins and lipids before our cells can use them as fuel for energy, or as substrates for other molecules. When glucose is metabolized for fuel in our body, it is first converted into pyruvate in the cytosol, the liquid matrix within cell membranes. The classic view of pyruvate follows two paths: when there is a lack of oxygen, our body turns pyruvate into ATP via fermentation in the process of glycolysis. In the presence of oxygen, pyruvate enters the mitochondria and undergoes oxidative phosphorylation (OXPHOS) to create ATP.  Ketogenic diets supply our body with foods high in fats. Fatty acids are converted into acetyl-CoA in the mitochondria and undergo oxidative phosphorylation to create ATP.  Neither fatty acids nor protein undergo glycolysis directly in the generation of energy [15].

Cancer cells mostly derive their energy from glucose via glycolysis in the presence of oxygen. This unusual preference was first observed in 1956 by Otto Warburg who called this effect aerobic glycolysis, or what is now known ubiquitously as the Warburg Effect. Cancer cells thrive due to their ability to flexibly adapt their metabolism to increase their proliferation rate in the face of changing environments. The Warburg Effect refers to the unique trait of cancer cells to select glycolysis even in the presence of ample oxygen. This process of glycolysis used by cancer cells takes place in the cytosol of cells, where glucose is turned into pyruvate to yield ATP (energy). The process of glycolysis also results in the generation of nicotinamide adenine dinucleotide (NAD) in its reduced form NADH, as well as lactate [25]. How cancer cells manipulate these two important substrates to their advantage will be discussed later.

It is accepted that glycolysis yields two molecules of ATP per glucose molecule, and oxidative phosphorylation yields up to 38 molecules of ATP per glucose molecule. Whereas depending on the type of fatty acid, fatty acid oxidation can yield over 100 ATP molecules per fatty acid. The incorporation of ketogenic diet to curtail the growth of tumors seems like an excellent strategy, as it could sidestep the Warburg Effect, while providing ample units of cellular energy. However, fatty acid oxidation in the ketogenic diet also initiates an immune response, one that can be either pro- or anti-inflammatory, or both.

Ketogenesis, Inflammation, and Cancer

One of the main attractions of the ketogenic diet is the ability to reduce the symptoms or even reverse chronic inflammatory diseases. Injury triggers inflammatory responses, and recovery is the successful healing conclusion achieved via anti-inflammatory responses. Chronic inflammatory diseases are basically the result in the failure to resolve initial inflammatory responses. The traditional understanding is that glycolysis drives inflammation, and fatty acid oxidation (FAO) is always anti-inflammatory. In general, non-resolving inflammation in a tumor microenvironment is the hallmark of cancer [26]. In fact, classic healing cycles begin with injury and end with recovery [27] .  Proinflammatory responses are associated with enhanced glycolytic activity and breakdown of the mitochondrial tricarboxylic acid cycle (TCA). Hence, ketogenic diet is considered anti-inflammatory because it mainly uses fatty acid oxidation in the generation of energy in the mitochondria without the involvement of glycolysis [7].

Macrophage Metabolism, Inflammation, and Tumorigenesis

An interesting ongoing development in the study of immunology is the role played by intracellular metabolism as the regulator of the fate and function of cells of the immune system. The latest studies are now focused on how the polarization of macrophages and the modulation of their properties are affected by energy metabolism[22]. Macrophages are phagocytic cells present in almost all tissues. Macrophages are produced from bone marrow-derived blood monocytes, and are very much a part of our innate immune system. Macrophages play an important role in the initiation and resolution of inflammation. Depending on the signals they receive, macrophages can be pro-inflammatory, classified as M1, or anti-inflammatory, classified as M2. In cancer, macrophages can both increase and decrease tumor growth depending upon the type of tumor and type of macrophage involved. Interestingly, diet plays a role in determining the type of macrophage likely to be activated. There are two types of macrophages.

• M1 macrophage metabolism is characterized by glycolysis, the pathway associated with cancer and often with the consumption of high carbohydrate diets. M1 macrophages have been associated with high levels of pro-inflammatory cytokines such as IL6, IL12, IL23, and tumor necrosis factor-α (TNF-α). M1 macrophages are able to produce substantial levels of reactive oxygen and nitrogen species, and are therefore, highly microbicidal and tumoricidal. Mitochondrial reactive oxygen species (mROS) are the key regulators of these M1 classically activated macrophages [8].
• Conversely, M2 macrophage metabolism is characterized by fatty acid oxidation, producing ATP (energy) via oxidative phosphorylation. Unlike M1, M2 macrophages are associated with anti-inflammatory, tissue repair and resolution of inflammation, and are classified as alternatively activated macrophages. M2 macrophages are activated by Th2 cytokines like interleukin IL-4, IL-10, and IL-13 [9].

Recent research has discovered that these metabolic pathways are closely interconnected. Any attempt to define glycolysis as pro-inflammatory or fatty acid oxidation (FAO) as anti-inflammatory may be an oversimplification. Studies now demonstrate the need for glucose metabolism in anti-inflammatory as well as inflammatory macrophages, and that fatty acid oxidation (FAO) supports not only anti-inflammatory responses but also drives the activation of inflammatory macrophages [8].

M1 macrophages, being ‘pro-inflammatory’ are now identified as critical components involved in anti-tumor immunity, whereas M2, the macrophages traditionally viewed as anti-inflammatory, are now associated with tumor growth responses. Research now demonstrates that the infiltration of M2 macrophages can account for more than 50% of the tumor mass in some cancers. M2 type macrophages aid in metastasis by inducing angiogenesis (the development of new blood vessels), and the presence of M2 macrophage usually signify a poor prognosis for cancer survival. These macrophages that migrate to tumor sites and aid in angiogenesis and metastasis are called tumor-associated macrophages (TAMs) and they express a distinct M2 phenotype [10].

The Role of the Ketogenic Diet in Cancer

Two fundamental keys to understanding how the ketogenic diet affects tumorigenesis are the NADH:NAD+ ratio, and the access to lactate.

Lactate and Cancer

Cancer cells rely on glycolysis for energy even in the presence of oxygen, forsaking increased ATP energy production via oxidative phosphorylation (OXPHOS) in the mitochondria, in exchange for just 2 molecules of ATP and lactate. Lactate is a critical substrate for cancer cells, because it is used to generate lactic acid. Cancer cells remove one proton (H+) from lactate to produce lactic acid. Lactic acid produced by cancer cells has been found to play critical roles in their growth and proliferation. Cancer cells use lactic acid as an immunosuppressant as well as the promoter for angiogenesis, cell migration and metastasis. Lactic acid is also used by cancer cells to change the phenotype of M1 macrophages into the tumor-associated macrophage M2 [20, 21].

The level of lactate in tumor cells has been found to be highly correlated to the malignancy of tumors. Researchers discovered that some of the most malignant tumor lines tested were the ones that yielded the highest concentrations of lactate. Lactic acid produced by tumor cells during glycolysis is capable of generating signals that can induce the expression of the potent angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the M2-like polarization of tumor-associated macrophages (TAMs) [21]. In addition, lactic acid from tumor cells has been found to inhibit pro-inflammatory cytokines, and the activation of T-cells.

“Lactic acid suppressed the proliferation and cytokine production of human cytotoxic T lymphocytes (CTLs) up to 95% and led to a 50% decrease in cytotoxic activity” [32].

Ketones and Lactate: A Misunderstood Relationship

The main premise for using Keto diets in cancer therapies is its ability to simulate the natural state of fasting, where the body breaks down stored body fat to produce ketones for energy when food is not available. Hypothetically, the burning of fats in Keto diets will reduce the flux of glucose through glycolytic pathways that are favored by cancer cells in the generation of energy and critical substrates like lactate [29].

We already know that lactate is an important substrate for cancer cells. But how effective are Keto diets in suppressing lactate formation? One would think that since glucose consumption is significantly reduced in Keto diets, lactate production levels should also decline.

Let us take a look at glioblastoma, where Keto diets have been reported to have a high success rate. Our brains are able to use ketones as fuel. Keto diets can supply adequate fuel to our brains via ketones when glucose is not available. Keeping the level of blood glucose low may sound quite attractive as it has been reported that “hyperglycemia is associated with adverse prognosis and postoperative function loss in GBM (glioblastoma multiforme) patients” [30]. Does that mean that if one can artificially reduce blood sugar levels by implementing Keto diets, the prognosis for glioblastoma patients will improve? The answer may surprise you, but not if you understand that lactate is also an important substrate for cell survival in normal non-cancerous cells.

A study on the effects of low carbohydrate/high fat diets and high carbohydrate/low fat diets in ultra-endurance athletes revealed that plasma glucose and serum insulin were not significantly different between the two groups when they were at rest and also during exercise. There was no significant difference between the two groups in insulin resistance as determined by HOMA (a homeostatic model assessment of beta cell function and insulin resistance). However, serum lactate increased by twofold during the last hour of exercise training in the group of athletes consuming a low carbohydrate/high fat diet. The researchers found that all athletes in the low carbohydrate/high fat group broke down substantially more glycogen in their muscles than the total amount of carbohydrate oxidized during the 3 hour run. Why were muscles broken down for glycogen when substrates like fatty acids were readily available to the athletes on Keto diets? Obviously these glycogen were not used for energy production because they were not oxidized. The researchers believed the glycogen were a necessary source for the production of lactate [31].

Our bodies are very adept at maintaining homeostasis. When glucose availability is limited by diet, our bodies can produce glucose from other sources like proteins and fatty acids. Gluconeogenesis is one of several main mechanisms used by humans and many other animals to maintain blood glucose levels.  Gluconeogenesis uses non-carbohydrate substrates from lipids such as triglycerides to produce glucose in the liver [19]. Amino acids from protein, such as the glucogenic amino acids can also be converted into glucose through gluconeogenesis [18].

It is highly likely that the long-term consumption of a low-carbohydrate/high-fat diet leads to adaptations in our body to maintain homeostasis in the regulation of glycogen so as to preserve an environment that is similar to one maintained under high carbohydrate consumption [31]. Based upon these findings, it is perhaps necessary to re-examine the ability of the Keto diet to truly reduce access to lactate in cancer cells.

Is NAD+ the Double-edged Sword in Ketogenic Diets?

In recent years, NAD+ has been recognized as having neuroprotective qualities in addition to anti-inflammatory and anti-aging benefits [34].

NAD, or Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. It exists in two forms, NAD+, the oxidized form, and NADH, the reduced form. The main functions of NAD is to transfer electrons.  NAD+ accepts electrons from other molecules to produce NADH, which then can donate electrons again to other molecules [24].

In the process of glycolysis, electrons are added to NAD+, resulting in the reduced form of NADH. Two molecules of NADH are formed per molecule of glucose during glycolysis. During oxidative phosphorylation (OXPHOS) these NADH molecules donate their electrons to form NAD+, which can then be used again for glycolysis [15]. When the metabolic pathways used is predominantly glycolytic, there will be an NADH excess because more NAD+ are consumed in the process.

A high NADH:NAD+ ratio in the cytosol is an indication of high glycolytic activity. Higher levels of NADH results in an increase of pro-inflammatory gene expression of macrophages with the M1 phenotype. Conversely, limited glucose availability as a result of diet restrictions can lower the NADH:NAD+ ratio. When there is more NAD+ to NADH, pro-inflammatory gene expression and responses are suppressed as a consequence [16].

Ketogenic diets are believed to be able to increase NAD+.  Ketogenic diets have been found to significantly raise hippocampal NAD+ levels in studies on rodents, conferring neuroprotective benefits [17]. Having more NAD+ than NADH also leads to the suppression of glycolysis, which in turn will increase anti-inflammatory macrophage responses that can reduce brain inflammation, tissue loss, and functional impairment after brain injury [16]. In terms of understanding how the NADH:NAD+ ratio relates to the two macrophage phenotypes, a simple way is to remember that:

• Low NADH, high NAD+ = Anti-inflammatory, tumor-promoting macrophage (M2)
• High NADH, low NAD+ = Pro-inflammatory, tumor-suppressing macrophage (M1)

A low NADH:NAD+ ratio (low level of NADH but high level of NAD+) is pro-tumor. Cancer cells thrive when there is an abundance of NAD+.  NAD+ is an essential coenzyme for aerobic glycolysis. A higher rate of glycolysis will reduce the amount of NAD+ available. Fewer NAD+ in turn slows down the rate of aerobic glycolysis. So the more cancer cells rely on glycolysis, the more NAD+ they require. It has been demonstrated that NAD+ levels are much higher in cancer cells compared to normal cells, likely due to an upregulation of NAD+ synthesis by cancer cells, [33] and the inhibition of the NAD+ metabolic pathways leads to enhanced autophagy and decreased survival rate of cancer cells [35].  When one employs a high fat diet to inhibit cancer growth via the limitation of access to glucose, the inadvertent generation of NAD+ is a factor that must be taken into consideration.

The Future of Macrophage Polarization

Cancer cells are able to circumvent our body’s natural immune defenses via many mechanisms. By manipulating lactate produced during aerobic glycolysis, lactic acid is used by cancer cells to induce the polarization of macrophage into the tumor-associated macrophage with distinct M2 phenotypes for growth and proliferation, chemotherapeutic resistance and immune evasion[37]. Ketogenic diets may limit the substrates available to tumors for use in glycolysis, but they are not able to suppress all the tactics used by cancer cells to create environments that are hospitable to their proliferation.

The type of diet we choose exerts significant influence on how macrophages are activated in our body. Depending on their activation status, macrophages can either facilitate tumorigenesis by antagonizing the cytotoxic activity of immune cells or suppress tumor progression by enhancing anti-tumor responses. Current strategies in the fight against cancer development show promise in the manipulation of macrophage responses via therapies that either block the recruitment or depletion of macrophages from the tumor; affect the polarization of the tumor-associated macrophage to an anti-tumorigenic phenotype; or the reactivation of immunostimulation[36]. Whichever therapy is employed in the manipulation of macrophage polarization, the incorporation of energy metabolism as part of the strategy may prove truly invaluable.

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