Role of fatty acids and polyphenols in inflammatory gene transcription and their impact on obesity, metabolic. syndrome and diabetes.

Transcription

1 European Review for Medical and Pharmacological Sciences Role of fatty acids and polyphenols in inflammatory gene transcription and their impact on obesity, metabolic syndrome and diabetes B. SEARS, C. RICORDI* Inflammation Research Foundation, Marblehead, MA, USA and *Diabetes Research Institute, University of Miami, Miami, FL, USA 20012; 16: Abstract. Obesity, metabolic syndrome and diabetes represent multi-factorial conditions resulting from improper balances of hormones and gene expression. In addition, these conditions have a strong inflammatory component that can potentially be impacted by the diet. The purpose of this review is to discuss the molecular targets that can be addressed by anti-inflammatory nutrition. These molecular targets range from reduction of pro-inflammatory eicosanoids that can alter hormonal signaling cascades to the modulation of the innate immune system, via toll-like receptors and gene transcription factors. Working knowledge of the impact of nutrients, especially dietary fatty acids and polyphenols, on these various molecular targets makes it possible to develop a general outline of an anti-inflammatory diet that offers a unique, non-pharmacological approach for treating obesity, metabolic syndrome and diabetes. Key Words: Obesity, Metabolic syndrome, Diabetes, Inflammatory gene transcription, Fatty acids, Polyphenols. Introduction It is becoming more evident that inflammation plays an important role in the metabolic consequences of obesity, metabolic syndrome, and diabetes as well as other chronic degenerative conditions 1-7. However, the molecular mechanisms that control the inflammatory processes at the genetic level are only beginning to be understood. In particular, there is a growing knowledge of the role that gene transcription factors play in the inflammatory process. Pharmacology allows one to determine which components of the inflammatory response are important in treatment of obesity, metabolic syndrome, and diabetes. This same un- derstanding can illustrate how natural components of the diet alter the same molecular targets as pharmacological interventions and provide attractive, cost-effective alternatives to more traditional therapies. The purpose of this review is to establish linkages between diet, inflammatory hormones and gene transcription factors that affect inflammation and to propose dietary approaches for the reduction of chronic low-level inflammation, important in the development of obesity, metabolic syndrome and diabetes. An Inflammatory Perspective on Obesity, Metabolic Syndrome, and Diabetes The percentage of obese adults in the United States is approximately one-third of the adult population or about 72 million individuals 8. This number is similar to the number of individuals (estimated at 79 million) who have metabolic syndrome, a good indication of the number with pre-diabetes. Additionally, 26 million people in the United States have type 2 diabetes 9. A strong inflammatory linkage appears be involved with the current epidemics of obesity, metabolic syndrome, and diabetes 10. This would suggest that there may be epigenetic factors that when activated by the diet, could be responsible for the rapid increase of each of these conditions in the past 30 years. The mechanism may lie with the impact of diet on activation of gene transcription factors that are involved in the inflammatory process. Overview of Inflammation The inflammatory responses that developed over millions of years of evolution allow us to co-exist with microbes. The same inflammatory responses also make it possible for physical in- Corresponding Author: Barry Sears, Ph.D.; bsears@drsears.com 1137

2 B. Sears, C. Ricordi Innate Immune System Although the most profound linkage between diet and cellular inflammation can found with the imbalance of pro- and anti-inflammatory eicosanoids, the interaction of various dietary nutrients (fatty acids and polyphenols) with the innate immune system has a significant impact on the resulting inflammatory response. The innate immune system is the most primitive part of our overall immunological response and remains sensitive to nutrients 16. More importantly, its activation of the inflammatory response is based on primitive pattern recognition. When advances in molecular biology finally began to unravel the control mechanisms inherent in the innate immune system, a more detailed understanding of unexpected mechanisms for a variety of commonly used pharmacological drugs emerged 17,18. Likewise, these same advances also illustrated how the diet can affect inflammation induced by the innate immune system. Today the understanding of the linkage of diet to toll-like receptors, horjuries to heal. Most think of inflammation in terms of the pain associated with cellular destruction that comes as a result of these initial pro-inflammatory responses generated by the innate immune system. However, the complete inflammatory process is a complex interaction of both the pro- and anti-inflammatory phases 11,12. The pro-inflammatory phase induces pain, swelling, redness and heat, which are indicators that cellular destruction is taking place. Yet there are equally important anti-inflammatory mechanisms of the inflammatory cycle that are necessary for the resolution of the inflammatory response, thus allowing for cellular repair and regeneration. Only when these two phases are continually balanced can cells effectively repair the micro-tissue injury that results from inflammatory events. Yet, if the pro-inflammatory phase continues at a low, but chronic level below the perception of pain, its presence can become a driver of many chronic diseases. Understanding the role of diet in the generation of these same inflammatory responses had to wait for new breakthroughs in molecular biology. This allowed a more detailed understanding of the linkage between dietary factors and inflammatory gene expression mediated by gene transcription factors within the innate immune system. The innate immune response, the most primitive part of the immune system, has been conserved over hundreds of millions of years of evolution. It is non-specific, responding to conserved sequences, termed pattern recognition sites, and elicits an immediate response to various stimuli (microbes, injuries, burns, and the diet). The primary cellular components of the innate immune system include toll-like receptors and various gene transcription factors. These work together to activate the expression of inflammatory genes that can either amplify the pro-inflammatory attack phase of inflammation or inhibit the production of the same inflammatory mediators. Types of Inflammation There are two distinct types of inflammation. The first type is inflammation resulting in acute pain. This can be considered classical inflammation. A second type of inflammation is cellular inflammation that is described as low-level chronic inflammation below the threshold of pain Since there is no pain associated with this type of inflammation, nothing is done to stop it, and thus it can linger for years, if not decades, causing continual organ damage. As long as appropriate inflammatory resolution mechanisms and the regenerative/compensatory potential of organs and tissues are maintained, the development of chronic degenerative conditions can be prevented or delayed. Eventually, exhaustion or lack of activation of the necessary inflammatory resolution mechanisms necessary for regenerative potential will occur. This will result in subsequent organ damage, loss of function and the onset of overt chronic disease despite the fact that initiating pathogenetic events may have started decades earlier, triggered by the underlying and ongoing chronic cellular inflammation process. The primary mediator of the diet-induced inflammation response is ultimately driven by the production of pro-inflammatory eicosanoids derived from arachidonic acid (AA). AA is an omega-6 fatty acid, and its levels are entirely controlled by the diet. Anti-inflammatory drugs interact with molecular targets that are downstream from AA, primarily by either inhibiting the enzymes that convert AA into pro-inflammatory eicosanoids or inhibiting the release of AA from phospholipids in the membrane. A primary goal of anti-inflammatory nutrition is to go upstream by reducing the levels of AA in the target cell membranes. The overall goal is to reduce the excess production of pro-inflammatory eicosanoids, similar to pharmacological approaches, but using very different mechanisms to reach that goal. 1138

3 Fatty acids and polyphenols in inflammatory gene transcription monal second messenger signaling pathways, gene transcription factors and their resulting interactions allows anti-inflammatory nutrition to be considered a form of gene silencing gene therapy, especially the silencing of genes involved in the generation of cellular inflammation. Clinical Markers of Cellular Inflammation It is very difficult to discuss a concept of cellular inflammation that cannot be measured and evokes no pain. It is only recently that new clinical markers of cellular inflammation have emerged. The first of these clinical markers was high-sensitivity C-reactive protein (hs-crp). This is not a very selective marker since simple infections can raise its levels A much more selective marker of cellular inflammation is the ratio of two key fatty acids in the blood. The first is the omega-6 fatty acid arachidonic acid (AA), which is the precursor to pro-inflammatory eicosanoids. The other fatty acid is the omega-3 fatty acid eicosapentaenoic acid (EPA), which generates anti-inflammatory eicosanoids. The higher the AA/EPA ratio in the blood, the greater the level of the cellular inflammation that is likely to be found in various organs Dietary origin of Cellular Inflammation: the Perfect Nutritional Storm There has not been one dietary change alone in past 30 years that has increased the levels of cellular inflammation. However, there has been a convergence of four distinct dietary changes that can be termed as The Perfect Nutritional Storm 15. These dietary factors include: Increased consumption of refined vegetable oils rich in omega-6 fatty acids Increased consumption of refined carbohydrates Decreased consumption of long-chain omega- 3 fatty acids Decreased consumption of polyphenols The first two factors increase the production of AA, thereby increasing pro-inflammatory responses, whereas the last two factors are important in generating anti-inflammatory responses by their interaction with specialized gene transcription factors. Increasing AA The primary cause of increased AA has been the increased consumption of refined vegetable oils rich in omega-6 fatty acids. The primary fatty acid in the most common vegetable oils is linoleic acid, an omega-6 fatty acid that is readily converted into AA. Prior to the last 80 years, linoleic acid was a relatively minor component of the human diet. For example, traditional cooking fats, such as butter, lard, and olive oil, contain less than 10% linoleic acid. Common vegetable oils, such as corn, soy, sunflower, and safflower, contain 50-75% linoleic acid. The use of these vegetable oils has increased by more than 400% since Accelerating the metabolic transformation of the increased intake of linoleic acid into AA has been the increasing consumption of refined carbohydrates that has significantly increased the glycemic load of the diet. The glycemic load of a meal is defined as the amount of a particular carbohydrate that is consumed at a meal multiplied by its glycemic index 23,24. Today, not only are high glycemic-index carbohydrates major components in virtually all processed foods, they are found in dietary staples, such as potato, rice, corn and wheat. As the cost of production of refined carbohydrates has dramatically decreased in the past 30 years, the availability of products made from these ingredients has dramatically increased 25. Increased consumption of high-glycemic food ingredients generates meals with a high-glycemic load. This results in the increased secretion of the insulin necessary to lower the resulting post-prandial rise in blood glucose 24. Since refined carbohydrates and vegetable oils are now the cheapest source of calories 25-28, it is not surprising that the combination of these two dietary trends has increased tissue concentrations of AA, thus leading to an epidemic of cellular inflammation. This can be understood from the metabolic pathway of linoleic acid conversion to AA as shown in Figure 1. The two rate-limiting steps in this metabolic cascade of linoleic acid to arachidonic acid are the delta-6 and delta-5 desaturase enzymes. These enzymes insert cis-double bonds into unique positions in the omega-6 fatty acid molecule. Normally, these steps are rate limiting, thus controlling the production of AA. However, insulin is a strong activator of each of these enzymes This means that a high glycemic-load diet coupled with increased intake of vegetable oils rich in linoleic acid will lead to increased production of AA and a corresponding increase in cellular inflammation. 1139

4 B. Sears, C. Ricordi Linoleic Acid Gamma Linolenic Acid Dihomo Gamma Linolenic Acid Arachidonic Acid (AA) Delta-6 Desaturase Activated by insulin Delta-5 Desaturase Activated by insulin Inhibited by EPA Figure 1. Metabolism of omega-6 fatty acids. Decrease in Anti-Inflammatory Nutrients Intensifying the inflammatory damage caused by increased production of pro-inflammatory AA has been a corresponding decrease in the dietary intake of anti-inflammatory nutrients, specifically the long-chain omega-3 fatty acids and polyphenols. The omega-3 fatty acid EPA has a significant inhibitory effect on the metabolic cascade that results in the synthesis of AA and its subsequent effect on cellular inflammation. In high enough concentrations, EPA can partially inhibit the activity of the delta-5-desaturase enzyme, thus reducing AA formation by acting as a weak feedback inhibitor because both fatty acids use the same enzyme (delta 5-desaturase) for their production. Equally important, an increased EPA content in membrane phospholipids decreases the release of AA that is necessary to make proinflammatory eicosanoids. In this regard, increased consumption of EPA dilutes out existing AA, thus decreasing the potential production of pro-inflammatory eicosanoids. Finally, EPA is the molecular building block to powerful anti-inflammatory eicosanoids known as resolvins Unfortunately, the consumption of long-chain omega-3 fatty acids, such as EPA, has dramatically decreased over the past century 37. Another dietary change has been reduced intakes of polyphenols. Polyphenols are the chemicals that give fruits, vegetables, and whole grains their color. It was originally thought that their primary role was as anti-oxidants, but now it is realized they have important anti-inflammatory effects on gene transcription factors. The increased use of refined carbohydrates has correspondingly reduced the dietary intake of these anti-inflammatory polyphenols to extremely low levels compared to previous times. How Cellular Inflammation Induces Chronic Disease Cellular inflammation can be insidious since it causes no pain, yet the long-term damage to organ function is significant. This is a combination of two factors: (1) disruption of hormone-induced second messenger signaling within the cell and (2) disturbances in the homeostasis between pro-inflammatory and anti-inflammatory gene transcription factors. Successful hormonal cellular communication involves fidelity of the signals initially generated by a hormone interacting with its receptor on the cell surface. The corresponding transfer of that interaction via second messengers into the interior of the cell elicits the appropriate biological response. The classic case of disruption of this hormonal communication is insulin resistance. Insulin resistance and the resulting hyperinsulinemia is often a driving force behind both obesity and metabolic syndrome. It is amplified in type 2 diabetes leading to the beta cell burnout resulting in chronic hyperglycemia. There are a number of control points in the insulin signaling pathways within a cell that can be disrupted by cellular inflammation and inflammatory mediators that are genetically expressed in response to cellular inflammation 38,39. This helps explain why for more than a century there have been reports in the medical literature about the ability of high-dose anti-inflammatory drugs to reverse insulin resistance and partially restore normal glycemia in diabetic patients 40,79. These anti-inflammatory drugs appear to work at the level of inhibition of pro-inflammatory gene transcription factor known as NF-κB. Once activated, NF-κB will prompt the genetic expression of a wide variety of inflammatory mediators including the inflammatory cytokines (including IL-1, IL-6, and especially TNF-α) that are implicated in disrupting insulin signaling and the COX-2 enzymes that can amplify the conversion of AA into inflammatory eicosanoids, such as prostaglandins and leukotrienes. The inflammatory cytokines generated by the activation of NF-κB can interact 1140

5 Fatty acids and polyphenols in inflammatory gene transcription through receptors on the cell surface to amplify the inflammatory response both in the same cell (i.e. autocrine) or nearby cells (i.e. paracrine) thereby spreading the inflammatory signals. There are a number of dietary factors that can activate NF-κB transcription, including saturated fatty acids and AA. Saturated fatty acids activate of NF-κB by stimulating the toll-like receptors, primarily TLR-4 and potentially TLR AA has both a direct effect on the activation of NFκB 41 and an indirect effect via its leukotriene metabolites 42,43. A secondary inflammatory mechanism by which NF-κB can be activated is via the interaction of saturated fatty acids interacting with toll-like receptors, primarily TLR-4 and potentially TLR At the same time it is known that nutrient activation of anti-inflammatory gene transcription factors, such as PPARγ can inhibit cellular inflammation and cause reversal of diabetes. Nutrients that activate this anti-inflammatory gene transcription factor include omega-3 fatty acids as well as polyphenols 53,54. In addition, polyphenols can activate AMP kinase, which acts as an upstream activator of gene transcription factors SIRT1 and FOX leading to a reduction in the levels of cellular inflammation 55. A simplified overview of these diet-induced cellular inflammation pathways is shown in Figure 2. An industrially manufactured form of fat, trans fats, also can generate insulin resistance; however, animal experiments using knockout models of TLR-4 indicate that trans fatty acids still cause inflammation via a TLR-4 independent mechanisms 56. How Obesity Induces Cellular Inflammation Obesity can be defined as accumulation of excess body fat. However, it is the location of that excess body fat that determines whether or not obesity leads to acceleration of chronic diseases. If the extra body fat is constrained to the adipose tissue and there is no compromise of metabolic function, this obese individual would be considered metabolically healthy 57. A significant number of obese Americans fall into this category 58. On the other hand, if increasing amounts of the excess fat become deposited in other organs (muscles, cardiac tissue, liver, pancreas), this is known as lipotoxicity With lipotoxicity comes an acceleration of the chronic diseases (e.g., metabolic syndrome, fatty liver type 2 diabetes, heart disease, cancer) associated with obesity. The extent of lipotoxicity is determined by the health of the fat cells in the adipose tissue. Adipose Tissue as a Staging Area for Systemic Cellular Inflammation The adipose tissue is the only organ in the body that can safely store triglycerides. As a consequence, it occupies the central position in controlling cellular inflammation by acting as a fatbuffering system, especially by controlling the Toll like receptor-4 AA PPARγ NF-κB DNA Inflammatory Enzymes (COX-2) and Cytokines (IL-1, IL-6, TNF) Cytokine receptors Figure 2. Simplified version of diet-induced activation of NF-κB. 1141

6 B. Sears, C. Ricordi blood levels of AA. Healthy fat cells have the ability to extract any excess fatty acid (including AA) in the blood and to safely store it as a triglyceride. In addition, the adipose tissue can readily induce the formation of new fat cells from internal stem cells to increase the storage of increasing levels of circulating fat in blood coming from either ingestion of dietary fat or metabolism of excess carbohydrates and protein that have been converted into circulating fat by the liver 62. The Life Cycle of a Fat Cell The definition of a healthy fat cell is one that can easily expand to sequester incoming fats, in particular for long-term storage and effectively govern the controlled release of stored fat for ATP production in the peripheral tissues. The ability to sequester circulating fat into the fat cell depends upon the integrity of insulin signaling that brings adequate levels of glucose into the fat cell that can be converted to glycerol. This necessary step is required to convert incoming free fatty acids into triglycerides for long-term storage. The problem begins to arise when AA levels become too great in a particular fat cell. As an initial defensive mechanism, the generation of new fat cells is induced by metabolites of AA 63,64. Although this is associated with greater adiposity 65, the creation of new healthy fat cells maintains the capacity of the adipose tissue to prevent potential lipotoxicity. However, as the AA levels continue to increase in any particular fat cell, the cellular response to insulin signaling becomes compromised due to internal cellular inflammation that interrupts the flow of glucose into the fat cell to provide the necessary glycerol for fatty acid storage 41. It appears this may be a consequence of the generation of pro-inflammatory eicosanoids (leukotrienes) derived from AA 42,43. As a result, the fat cell has a more difficult time sequestering newly formed AA as well as other fatty acids circulating in the blood. At the same time, insulin inhibition of the hormonesensitive lipase in that particular fat cell becomes compromised because of the same disruption in the insulin-signaling cascade. Consequently, more free fatty acids are being released into circulation 67,68, the hallmark of classical insulin resistance. It appears that insulin resistance due to increased AA levels may arise in the fat cell prior to developing in the muscle cells 69,70. The greater amounts of AA in circulation are taken up by other cells potentially leading to the acceleration of insulin resistance in the muscle cells and marked hyperinsulinemia. To further compound the situation, a compromised fat cell is releasing greater amounts of previously sequestered AA into circulation 71. As the levels of AA increase in any one particular fat cell beyond a critical threshold barrier, cell death may occur 72. The necrosis of that particular fat cell causes a migration of macrophages into the adipose tissue 73,74. This increase in macrophage accumulation in the adipose tissue is clearly seen in both animal and human models of obesity 75. These newly recruited macrophages cause the secretion of additional inflammatory mediators, such as IL-1, IL-6 and TNF-α, which increase inflammation within the adipose tissue These newly released inflammatory cytokines can interact with their receptors at the surface of nearby fat cells to signal a further activation of NF-κB, the key gene transcription factor that drives the inflammatory responses of the innate immune system. Support for this hypothesis of AA-driven inflammation in the fat cells comes from the observations that the amount of macrophage accumulation can be significantly reduced upon supplementation with high-dose fish oil rich in omega-3 fatty acids to reduce the inflammation in the adipose tissue This effect of omega-3 fatty acids may be mediated through GPR-120, which binds specifically to macrophages and reduces their inflammatory potential in the adipose tissue 92. As cellular inflammation in the adipose tissue increases, inflammatory cytokines, such as IL-6 derived from the macrophages attracted to the inflamed fat cells, can exit into the circulatory system to cause an increase in CRP formation in the liver. Hence the correlation between obesity and CRP levels 93. Likewise TNF-α generated by the same macrophages causes further insulin resistance in the surrounding fat cells, thus decreasing their ability to sequester newly formed AA as well as causing even more stored AA to be released into the circulatory system. In many ways, the staging area for insulin resistance in other organs (muscles, liver, and eventually the pancreas) can be considered to start in the adipose tissue. As insulin resistance spreads to other organs, the end result is lipotoxicity marked by the accumulation of lipid droplets in the muscle cells (both smooth muscle and cardiac muscle), liver, and the beta cells of the pancreas. As long as the adipose tissue is composed of healthy fat cells, the increased production of dietary-induced AA can be handled by rapid re- 1142

7 Fatty acids and polyphenols in inflammatory gene transcription moval from the blood and safe storage in the fat cells. In the absence of a large percentage of healthy fat cells in the adipose tissue, the combination of the growing lack of ability to sequester AA from the blood coupled with the accelerated release of stored AA from the fat mass into the circulation is similar to the metastatic spread of a tumor, only now it is cellular inflammation that is spreading. The metastasis of cellular inflammation is characterized by deposition of lipid droplets in non-adipose cells that cause lipotoxicity. If these accumulated lipid droplets are also enriched in AA, then the development of inflammatory diseases, such as metabolic syndrome and type 2 diabetes, will be accelerated. Understanding the role of healthy fat cells may explain why approximately one-third of obese individuals are actually quite healthy 58. As discussed, these individuals known as the metabolically healthy obese 57 appear to have higher levels of the adipose-derived hormone adiponectin 94. Adiponectin is an adipose cell generated hormone that is associated with decreased insulin resistance. This is confirmed by studies of the over-expression of adiponectin in diabetic animals 95. It should be noted that adiponectin levels can be increased by high levels of fish oil rich in EPA possibly acting through the PPARγ transcription factor One of the first indications that lipotoxicity is taking place is the appearance of metabolic syndrome. Metabolic syndrome is a cluster of metabolic disorders that parallels the development of pre-diabetes and is associated with the spreading lipotoxicity in both the liver and muscle cells. It is characterized by a combination of clinical markers, including a high fasting TG/HDL ratio (greater than 3.5), increased abdominal fat, and hyperinsulinemia. Recent data indicate that there is a strong correlation between metabolic syndrome and levels of AA in the adipose tissue 69. Left untreated, metabolic syndrome will usually result in the development of type 2 diabetes within 8-10 years. During this latency period, the insulin resistance of the individual is continually increasing. This will cause even more AA formation, especially if consumption of omega-6 fatty acids remains high, as the hyperinsulinemia will further activate the delta-6 and delta-5-desaturase enzymes that convert linoleic acid into AA. Since the fat cells are now compromised in their ability to sequester this increased AA production, the increased AA levels induced by the hyperinsulinemia in the blood are more likely to be picked up by other organs. The onset of type 2 diabetes occurs when the lipotoxicity has metastasized to the pancreas, causing a decreased output of insulin 99. With insulin secretion decreased, there is a rapid rise of blood sugar levels. The development of type 2 diabetes indicates that the metastasis of cellular inflammation from the adipose tissue to the pancreas is now complete. Ironically, even extreme lipotoxicity can be reversed by the creation of new healthy fat cells. This has been demonstrated in transgenic obese, diabetic mice that over-express adiponectin, an adipocyte-derived hormone that reduces insulin resistance 95. It is hypothesized that this increased production of adiponectin activates PPARγ, which causes the proliferation of adipose stem cells to produce new healthy adipocytes. These transgenetic obese mice become even more obese, but there is a normalization of blood glucose and lipid levels 95. This is similar to the elevated levels of adiponectin found in metabolically healthy obese individuals 94. One mechanism of protection against lipotoxicity might be that the new healthy fat cells in the adipose tissue can now sequester circulating fatty acids (including AA) more effectively to allow the resolution of the inflammatory lipid droplets in the muscle, liver and beta cells of the panaceas. Essentially this resolution process represents a reverse flow of the lipotoxic lipid droplets in other organs back to the adipose tissue and reverses insulin resistance in the muscle and liver cells as well as decreases the inflammation in the beta cells of the pancreas. Support of the hypothesis regarding the impact of AA on fat cell metabolism comes from studies on AA levels in fat cells on various chronic disease conditions. In particular, increased AA levels in the fat cells are significantly associated with increased body fat, development of metabolic syndrome, and incidence of non-fatal heart attacks 65,69,100. Pharmacological Targets for Reducing Cellular Inflammation Gene transcription factors that control inflammatory gene expression have been highly conserved for hundreds of million of years of evolution. Many of these gene transcription factors were initially identified as orphans meaning even though the gene transcription factor itself could be studied; it wasn t clear what it actually did. Many of the recent advances in pharmacology have been to find drugs that interact with such orphan gene transcription factors to provide a 1143

8 B. Sears, C. Ricordi measurable biological effect. By working backwards, it could then be hypothesized how that orphan transcription factor had a significant biological function. Anti-Inflammatory Drug Targets There is a growing awareness that obesity, metabolic syndrome and diabetes are inflammatory conditions, hence they theoretically could be treated by the chronic use of anti-inflammatory drugs. Unfortunately, the drug doses required also generate significant toxic side effects. Our working hypothesis is that chronic low-level inflammation driving the development of these conditions can be affected by anti-inflammatory nutrition to interact with the same molecular targets as anti-inflammatory drugs without the same side effect profile. The classical pathways of anti-inflammatory drugs have been focused on the inhibition of the COX and LOX pathways of eicosanoid production. Inhibitors of the COX enzymes, such as aspirin and non-steroidal anti-inflammatory drugs (NSAIDs), have different modes of action. Aspirin is a suicide inhibitor of the PGH 2 synthase enzyme that is the rate-limiting step of the formation of pro-inflammatory eicosanoids. NSAIDs, on the other hand, are competitive inhibitors of the same enzyme. However, neither class of antiinflammatory drugs has much effect on the LOX pathways that generates leukotrienes. Corticosteroids inhibit both the COX and LOX pathways by inhibiting the release of AA from the phospholipids in the cell s membranes thus making it a more powerful anti-inflammatory drug with correspondingly increased side effects. Gene Transcription Factor Targets The key component of the inflammatory response of the innate immune system is the activation of NF-κB, which acts as master genetic switch to cause the expression of inflammatory proteins (such as the COX-2 enzyme and various inflammatory cytokines) that amplify the inflammatory response. Recent evidence has indicated that aspirin, salicyates and statins also inhibit the activation of NF-κB at high concentrations 17,18. It is now hypothesized that many of the cardiovascular benefits of statins may come from their anti-inflammatory actions as opposed to their cholesterol-lowering effects 101. If NF-κB can be considered to be an inflammatory gene transcription factor, then the PPAR family of gene transcription factors can be considered anti-inflammatory. There are a number of drugs that activate the PPAR systems. PPARγ is the gene transcription factor that induces the increased expression of fat oxidation enzymes. It is this transcription factor that is activated by drugs such as fibrates 102. These drugs are effective in lowering triglyceride levels and are widely used in cardiovascular treatment. Once activated, the PPARγ system has profound anti-inflammatory effects, including the generation of new healthy fat cells that help reduce lipotoxicity. The thiazolidinediones, a mainstay of diabetes treatment, are examples of such drugs that activate the PPARγ gene transcription factor 103. Regulatory Enzyme Targets Certain enzymes act as energy sensors and can regulate a great number of metabolic systems, especially dealing with glucose and lipid metabolism. AMP kinase occupies a key place in this metabolic control process. The diabetic drug metformin is one such drug that activates this particular enzyme 104,105. This enzyme not only generates adequate levels of ATP but also regulates a wide number of enzyme systems involved in glucose and lipid metabolism 106. It should be noted that the class of drugs that metformin belongs to were originally isolated from the French lilac 107. As a result, metformin is sometimes used as an off-label drug for weight loss 108 and cancer treatment 109,110. Satiety Hormones The primary problem in generating long-term weight loss is constant hunger. Another group of hormones derived from AA include endocannabinoids that are involved in generating hunger. The endocannabinoid receptor antagonist (rimonabant) was developed as an appetite-suppressant for the treatment of obesity and diabetes 111. This drug was never approved in the United States due to various neurological problems, including an increase in suicidal tendencies. Other hormones involved in the appetite control come from the gut, including the satiety hormones PYY and GLP-1. Currently the only long-term medical intervention to treat obesity and diabetes is gastric bypass surgery1 12,113. The most successful of these types of surgery is Roux-en-Y gastric bypass that reroutes much of the small intestine thus delivering most of the dietary nutrients to the distal part of the small intestine (i.e. ileum). Unlike other gastric bypass methods, this surgery also increases the release of gut hormones, such as PYY and GLP-1 from the L-cells in the ileum, to give a profound degree of satiety

9 Fatty acids and polyphenols in inflammatory gene transcription Neurotransmitter Targets Although we have many successful examples of drugs for the treatment of acute inflammation, cardiovascular, and diabetes, there are relatively few examples of drugs that are successful in the treatment of obesity. The most successful of these drugs are amphetamines that stimulate dopamine receptors in the brain leading to increased satiety. Unfortunately, they also lead to addiction 115. Fen-phen was a combination of a weaker amphetamine (phentermine) and removed from the market when it was discovered it increased the incidence of primary pulmonary hypertension and heart valve degeneration 116. A new combination of phentermine and the anti-epilepsy drug topiramate is under investigation, but has not yet been approved by the FDA. Phentermine and other ampthetamine derivatives are still in use today for the treatment of obesity but only for short-term use (approximately 12 weeks). In an effort to bypass these restrictions, various amphetamine-like drugs used to treat attention deficient conditions are often used as off-label weight-loss drugs. Anti-inflammatory Nutrition The goal of anti-inflammatory nutrition is to understand how pharmacological targets (especially those that activate inflammatory gene transcription factors) of inflammation can be impacted by dietary nutrients. This would include reduction of those dietary components (1) that directly activate the inflammatory responses of innate immune system, such as gene transcription factors, such as NF-κB, or (2) that indirectly activate NFκB by interacting with toll-like receptors. These dietary nutrients that induce an inflammatory response can disrupt hormonal signaling patterns between hormone receptors and their internal targets giving rise to insulin and leptin resistance. Dietary nutrients (AA, omega-6 fatty acids that can be converted to AA, and saturated fats) that induce inflammatory responses via NF-κB are ones that must be significantly reduced in the diet. Dietary components can also have an indirect influence on the inhibition of NF-κB. These indirect effects include: (1) the inhibition of the binding of saturated fats to the TLR-2 and TLR-4 receptors by omega-3 fatty acids as well as the activation of PPARγ and (2) polyphenol activation of anti-inflammatory gene transcription factors, such PPARγ and AMP kinase. A simple diagram of the impact of these dietary impacts on NF-κB activity is shown in Figure 3. Before detailing these molecular targets in the context of anti-inflammatory nutrition, it is best to start with our knowledge of the molecular targets in terms of current anti-inflammatory pharmacological approaches used today, especially in the treatment of obesity and diabetes. General Overview of Anti-Inflammatory Nutrition With the above as a short review of the molecular targets for reducing cellular inflammation, we can now turn to understanding how various dietary components can be successfully used to achieve this goal. Pro-Inflammatory Nutrients Anti-inflammatory drugs work by inhibiting the formation of downstream pro-inflammatory eicosanoids derived from AA. A more elegant approach is to go upstream and reduce the levels of AA in the cells thereby restricting the substrate required for the production of pro-inflammatory eicosanoids. There is no known drug that can reduce AA. Only the diet can 117,118. AA can directly activate NF-κB 90 or serve as substrate for the production of leukotrienes that activate NF-κB 42,43 ). Thus, the overall reduction of AA will not only reduce the production of pro-inflammatory eicosanoids, but also inhibit the release of gene products (COX-2 enzymes and inflammatory cytokines such as TNF-α, IL-1, and IL-6) that are expressed if NF-κB is activated. Omega-3 Fatty Acids and Polyphenols Omega-6 and Saturated Fatty Acids Figure 3. Dietary controls on the innate immune system. 1145

10 B. Sears, C. Ricordi The best approach to reduce AA is to directly limit its intake in the diet. Dietary sources richest in AA include organ meats and egg yolks. Since most red meat in America comes from cornbased feedlots providing both linoleic acid and high glycemic-index carbohydrates, this food should likewise be minimized. Epidemiological studies confirm the validity of that food ingredient limitation 119. However, even if an individual were following a strict vegetarian diet, they would be able to produce large amounts of AA if that vegetarian diet is rich in both omega-6 fatty acids and high glycemic-index carbohydrates that stimulate insulin secretion. As described earlier, the combination of these two dietary factors will increase the production of AA. Therefore, in addition to reducing the direct intake of AA, one should also have a dietary strategy for the simultaneous reduction of the dietary intake of omega-6 fatty acids, such as linoleic acid, and lowering of the levels of insulin generated by the diet. Reduction of dietary linoleic acid can be achieved by using fat sources low in omega-6 fatty acids, such as olive oil or nuts as well as using animal protein sources that are lower in saturated fat (such as chicken and lean fish) or rich in omega-3 fatty acids (such as fish like tuna or salmon). Reducing insulin levels would decrease the rate at which any linoleic acid remaining in the diet is converted into AA. This requires a reduction of the glycemic load of the diet by increasing the consumption of vegetables and fruits as the primary sources of carbohydrates and the simultaneous reduction of the consumption of high-glycemic carbohydrates, such as grains and starches. Another pro-inflammatory nutrient that should be reduced are saturated fatty acids as they can bind to TLR- 4, indirectly activating NF-κB Finally, what is often not appreciated is that the total calorie content of a meal can also raise insulin levels and increase inflammation. Therefore, maintaining a calorie-restricted diet is also essential for insulin control. It has been demonstrated that over-nutrition causes inflammation in the hypothalamus and disrupts the precise signaling balance of satiety and hunger hormones and thus increases appetite 120. Anti-Inflammatory Nutrients The omega-3 fatty acid eicosapentaenoic acid (EPA) is a weak inhibitor of the delta 5-desaturase enzyme. However, at high dietary intakes, the EPA can both decrease AA production as well as dilute out the concentration of AA in the cell membrane, thereby decreasing its potential of being converted into pro-inflammatory eicosanoids, such as leukotrienes 42,43. In this regard, EPA can be viewed to act as a weak corticosteroid. This is why the AA/EPA ratio is considered an excellent clinical marker for cellular inflammation. As stated earlier, high intakes of EPA can reduce inflammation in the adipose tissue By either directly inhibiting the formation of AA or diluting it out by the presence of high levels of EPA in target cells (especially in the adipose tissue), overall inflammation will be automatically reduced as long as there is constant supplementation with fish oils rich in EPA. Omega-3 fatty acids have been recently shown to activate the previous orphan binding receptor known as GPR-120. Upon binding to omega-3 fatty acids, the activated GPR-120 appears to reduce the pro-inflammatory activity of macrophages in the adipose tissue 92. This may be the primary mechanism for the previously observed reduction in macrophage levels in adipose tissue upon supplementation with omega-3 fatty acids Inhibition of the NF-κB is another key target for anti-inflammatory nutrition. Both omega-3 fatty acids and polyphenols can inhibit the activation of NF-κB 121,122. However, one requires a therapeutic level of these nutrients to have any significant inhibition of NF-κB. PPARγ is an anti-inflammatory gene transcription factor. Its activation creates the stimulus for the production of new healthy fat cells that enhance the capacity of the adipose tissue for re-sequestering accumulated fat in other tissues thus reversing lipotoxicity. This will reverse much of the metabolic damage caused by the lipotoxicity, but it will make the individual fatter in the process. PPARγ is stimulated by adiponectin whose release from fat cells can be enhanced by the increased consumption of omega-3 fatty acids In addition, EPA and DHA can also directly activate PPARγ 123. Satiety The primary treatment for obesity, metabolic syndrome, and diabetes is weight loss. Successful weight loss means a long-term control of the balance between hunger and satiety. Many of the hormones involved in the modulation of hunger and satiety are directly generated by diet. Insulin is a key hormone in the development of obesity. 1146

11 Fatty acids and polyphenols in inflammatory gene transcription If insulin levels remain elevated, the stored fat in the adipose tissue will remain sequestered due to its inhibition of the hormone-sensitive lipase in healthy fat cells. In addition, insulin in the blood is a hunger hormone because of its ability to lower blood glucose levels. A diet rich in highglycemic carbohydrates will increase the postprandial levels of insulin, thus rapidly decreasing blood glucose levels leading to hunger. This sets up a cycle of increased calorie (primarily refined carbohydrate) consumption generating increased inflammation in the hypothalamus that further dissociates hunger and satiety signals 120. Ironically, insulin can also function as a satiety hormone if it can reach the hypothalamus But if one has insulin resistance (induced by cellular inflammation), then the high levels of insulin in the blood are unable to relay their message to the key cells in the hypothalamus, and potential satiety effects of insulin are blunted. Leptin is a hormone released from the fat cells that is also involved in satiety. Like insulin, it must also reach the hypothalamus to exert its satiety actions. Obese individuals often display both insulin and leptin resistance , possibly because both hormones use similar intercellular signaling pathways so that hyperinsulinemia could lead to reduction of the efficiency of the leptin pathway. Inhibition of endocannabinoid binding to its receptors in the brain is the mechanism of action for rimonabant, a drug developed for the treatment of obesity. Since endocannabinoids are derived from AA, reduction of its levels in the brain should reduce hunger. Unfortunately, the half-life of AA in the brain is long in humans 124. However, increasing the levels of EPA in the brain can inhibit the binding of endocannabinoids 130. Since the halflife of EPA in the brain is very short 131, this requires maintaining a therapeutic level of EPA in the blood to create a constant gradient necessary for the constant flow of the EPA into the brain. This gradient can only be maintained by a diet either very high in fatty fish consumption or supplemented by fish oil rich in EPA. As mentioned earlier, much of the success of gastric bypass surgery is related to the increase in satiety hormones (PYY and GLP-1) generated from the ileum 132. The release of these hormones can be enhanced by slowing down the rate of digestion and absorption of protein and carbohydrate in a meal so that greater amounts of these nutrients can reach the L-cells in the ileum. Lowering the glycemic load of the diet by the inclusion of fiber-rich carbohydrate sources (especially those rich in soluble fiber) can slow the digestion and absorption process of both protein and carbohydrate. At the same time increasing the protein content of that meal will also increase PYY levels 133. The slower rates of digestion and absorption mean more of these hormone agonists will appear in the ileum and thus generate higher levels of these satiety hormones. Neurotransmitters Amphetamines increase dopamine levels, which is instrumental in decreasing hunger 115. EPA and DHA can also increase dopamine levels in animal models 134. It has been demonstrated that dietary supplementation of high-dose EPA and DHA can further reduce the symptoms of attention deficit hyperactivity disorder (ADHD) in children already on their optimal level of amphetamine derivatives used to treat this condition 135,136. The ability of EPA and DHA to increase dopamine levels in animal studies and also to treat ADHD is suggestive that they may be useful in the reduction of hunger. AMP Kinase AMP kinase is the primary regulator of metabolic activity. This particular enzyme is stimulated by metformin 105,106. High levels of polyphenols can activate AMP kinase It s activation is key for not only increasing ATP levels, but also to regulate lipid and carbohydrate metabolism. As with EPA, therapeutic levels of polyphenols are required due to their low bioavailability probably due to their rapid degradation in the small intestine. Increased Thermogenesis The higher the protein content of the diet, the more thermogenesis is increased 141,142. The underlying cause may be activation of protein synthesis. To activate such protein synthesis requires a combination of adequate protein (in particular the amino acid leucine) and a consistent level of insulin in the blood to activate mtor to stimulate protein synthesis during the post-prandial period 143. Data suggest that it takes g of high-quality protein at a meal to provide adequate levels of leucine to stimulate this protein synthesis 144. Developing an Anti-Inflammatory Diet Anti-inflammatory nutrition is understanding how individual nutrients affect the same molecular targets affected by pharmacological drugs. This is 1147

12 B. Sears, C. Ricordi only the first step in developing an anti-inflammatory diet. Such a diet should contain all nutrient considerations described above, as well as being a diet that can promote compliance for a lifetime. The first question concerns the protein, carbohydrate, and fat composition for such an anti-inflammatory diet. Virtually every nutritional authority would recommend no more than 30% of the total calories should come from fat. Lower-fat diets are simply too difficult to maintain for a sustained basis as demonstrated in long-term studies However, the fat composition must also be low in both omega-6 and saturated fats because of their ability to increase cellular inflammation by their interaction with various components of the innate immune system. The most common dietary fats that are low in both omega-6 and saturated fats are olive oil and most nuts. Thus these food ingredients should constitute the bulk of the dietary fat in any anti-inflammatory diet. The next question is the amount of protein. Most dietitians would recommend consumption of no more low-fat protein than would fit on the palm of your hand. This translates into about 3 oz of a low-fat protein source (this would contain about 20 g of protein) for a typical female and approximately 4 oz of low-fat protein (this would contain about 30 g of protein) for a typical male. However, the protein has to be at this level at every meal to supply adequate levels of leucine to activate protein synthesis to increase thermogenesis 144. If one takes into account the protein content of carbohydrate sources and potential snacks, then the average female should be consuming about g of low-fat protein per day and the average male about g of low-fat protein per day. This amount of protein would have to be equally spaced at each meal to provide the necessary levels of protein for the enhanced release of PYY from the L-cells in the ileum after each meal thereby controlling satiety. The carbohydrate content should be able to maintain a stable level of insulin between meals. This can be achieved with about 40 g of low glycemic-load carbohydrates at each meal. The vast bulk of the carbohydrates should come from sources with the highest content of polyphenols with the least amount of carbohydrates, meaning the consumption of primarily colorful nonstarchy vegetables, moderate amounts of fruits, limited amounts of whole-grains and a radical reduction of the dietary intake of refined carbohydrates or any carbohydrate that has a high glycemic index (white rice, white bread, white potatoes, etc). Although there is some controversy about whether a low glycemic- load diet leads to improved weight loss 148,149, there is no question that a low glycemic-load diet will generate a lower inflammatory burden This may be the result of a high glycemic-load diet increasing the activity of NF-κB 153. Finally, there is the question of meal timing. The hormonal control benefits of any meal will last only about five hours 154. To achieve this hormonal control requires three low-calorie meals and two even lower-calorie snacks spaced throughout the day so that five hours never pass between a meal or snack. Such an anti-inflammatory diet would consist of about 1,500 calories per day (about 50 g of monounsaturated fat, 100 g of low-fat protein, and 150 g of low glycemic-load carbohydrates per day). On a calorie basis, that is about 30% of the calories as fat, 30% as protein, and 40% as carbohydrates. These are the dietary recommendations made by one of the authors in Similar dietary recommendations were made by the Joslin Diabetes Research Center at Harvard Medical School for the treatment of obesity, metabolic syndrome and diabetes in , and confirmed by their own pilot studies with type 2 diabetic patients 156. To this anti-inflammatory diet foundation supplemental omega-3 fatty acids at the level of 2-3 g of EPA and DHA should be added per day, either through a high fish consumption or supplementation with fish oil supplements rich in EPA. Finally, a diet rich in colorful, non-starchy vegetables also contributes adequate amounts of polyphenols to help inhibit NF-κB as well as activate AMP kinase. In many respects, this proposed anti-inflammatory diet has similarities to a Mediterranean diet. Both are rich in vegetables and fruits. Both emphasize the moderate intake of low-fat protein sources, such as chicken and fish. Both recommend the use of monounsaturated fats, like olive oil and nuts. The differences are in the carbohydrate composition. An anti-inflammatory diet radically restricts the use of bread and grains (especially refined grain products), and makes up for it with increased consumption of more colorful (i.e. rich in polyphenols) vegetables and fruits. That one seemingly small difference will have tremendous hormonal and genetic consequences leading to a lowered inflammatory burden. The goal of an anti-inflammatory diet is the reduction of cellular inflammation. Of course, this same reduction of cellular inflammation should 1148

13 Fatty acids and polyphenols in inflammatory gene transcription also result in consistent fat loss and resolution of metabolic syndrome and type 2 diabetes if our working hypothesis is correct that cellular inflammation is a driving force for the accumulation of body fat. This is accomplished by affecting the same molecular targets that have been elucidated by pharmacological agents. The success of this anti-inflammatory diet can be measured clinically by various markers of cellular inflammation as mentioned earlier as well as improvement of metabolic conditions (i.e. metabolic syndrome, diabetes, cardiovascular disease, etc) that are associated with obesity. At a the molecular level, the ultimate marker of success of an anti-inflammatory diet is the maintenance of the AA/EPA ratio in the plasma between 1.5 and 3 as found in the Japanese population 157. Conclusions The ultimate treatment of obesity, metabolic syndrome and type 2 diabetes lies in re-establishing hormonal and genetic balance that generates satiety instead of constant hunger as well as balancing pro- and anti-inflammatory gene transcription factors. This can be achieved by reducing cellular inflammation induced by the diet. Pharmacological agents can pinpoint those previously unknown molecular targets that induce cellular inflammation. The purpose of anti-inflammatory nutrition is to determine which food ingredients can affect the same molecular targets as drugs and determine what the therapeutic concentrations are for those nutrients required to affect the same molecular targets. Only then can you develop an anti-inflammatory diet that has to be used like a drug at the right time and right level to keep cellular inflammation under control by silencing inflammatory genes. Obesity, metabolic syndrome and type 2 diabetes are chronic conditions that are ultimately caused by increased diet-induced cellular inflammation. Cellular inflammation is generated by the mismatch of our current diet and our genes. Therefore, anti-inflammatory nutrition should be considered a form of gene-silencing technology, in particular the silencing of the genes involved in the generation of cellular inflammation. Pharmacological agents often work downstream from the true primary molecular target of cellular inflammation (NF-κB), whereas anti-inflammation nutrition works upstream to reduce the dietary factors that activate NF-κB to generate cellular inflammation. Not only is upstream targeting using an anti-inflammatory diet a more elegant way to treat obesity and associated co-morbid conditions, but it has an almost infinite therapeutic index compared to pharmacological agents. This dietary approach provides a new avenue to treat many other chronic diseases ultimately driven by cellular inflammation. References 1) VACHHARAJANI V, GRANGER DN. Adipose tissue: a motor for the inflammation associated with obesity. IUBMB Life 2009; 61: ) OLEFSKY JM. IKKepsilon: a bridge between obesity and inflammation. Cell 2009; 138: ) WELLEN KE, HOTAMISLIGIL GS. Obesity-induced inflammatory changes in adipose tissue. J Clin Invest 2003; 112: ) COGGINS M, ROSENZWEIG A. The fire within: cardiac inflammatory signaling in health and disease. Circ Res 2012; 110: ) KOLB H, MANDRUP-POULSEN T. The global diabetes epidemic as a consequence of lifestyle-induced low-grade inflammation. Diabetologia 2010; 53: ) GONDA TA, TU S, WANG TC. Chronic inflammation, the tumor microenvironment and carcinogenesis. Cell Cycle 2009; 8: ) GLASS CK, SAIJO K, WINNER B, MARCHETTO MC, GAGE FH. Mechanisms underlying inflammation in neurodegeneration. Cell 2010; 140: ) FLEGAL KM, CARROLL MD, OGDEN CL, CURTIN LR. Prevalence and trends in obesity among US adults, JAMA 2010; 303: ) AMERICAN DIABETES ASSOCIATION Fact Sheet. 10) PRADHAN A. Obesity, metabolic syndrome, and type 2 diabetes: inflammatory basis of glucose metabolic disorders. Nutr Rev 2007; 65: S ) SERHAN CN, BRAIN SD, BUCKLEY CD, GILROY DW, HASLETT C, O'NEILL LA, PERRETTI M, ROSSI AG, WAL- LACE JL. Resolution of inflammation: state of the art, definitions and terms. FASEB J 2007; 21: ) SERHAN CN. Resolution phase of inflammation: novel endogenous anti-inflammatory and pro-resolving lipid mediators and pathways. Annu Rev Immunol 2007; 25: ) SEARS B. OMEGARX ZONE, REGAN BOOKS. New York, NY, ) SEARS B. The Anti-inflammation Zone. Regan Books, New York, NY, ) Sears B. Toxic Fat. Regan Books, New York, NY, ) HOTAMISLIGIL GS, ERBAY E. Nutrient sensing and inflammation in metabolic diseases. Nat Rev Immunol 2008; 8:

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