Dietary Activation of AMP-Activated Protein Kinase (AMPK) to Treat Insulin Resistance

Abstract

Lipodystrophy is a rare condition that generates lipotoxicity resulting in significant insulin resistance. However, lipodystrophy is only one of many chronic conditions associated with insulin resistance. Insulin resistance is defined as the impaired ability of insulin to activate GLUT4-mediated glucose transport into target cells. The molecular reason for the failure of glucose transport is less apparent. Considering the wide range of chronic conditions associated with insulin resistance, a new potential understanding of insulin resistance in terms of an accumulation of metabolic stressors resulting in the inhibition of 5′ adenosine monophosphate-activated protein kinase (AMPK) may be warranted. Since AMPK is under robust dietary control, nutrition, as opposed to pharmacological strategies, may be more appropriate to activate AMPK to treat insulin resistance in lipodystrophy and any condition associated with insulin resistance. The focus of this chapter is to outline an AMPK-centric theory of insulin resistance and the combination of defined dietary strategies likely to be necessary to activate AMPK to reduce insulin resistance.

1. Introduction

Although lipodystrophy syndromes are rare and heterogeneous disorders, they are characterized by a partial or complete loss of adipose tissue. This loss of adipose tissue results in lipotoxicity as body fat infiltrates into organs that are not designed to store fat [1]. These organs that are primarily affected are the liver and skeletal muscle. This lipotoxicity ultimately leads to insulin resistance that is strongly associated with many chronic diseases, including diabetes, cardiovascular disease and neurological disorders [2]. Furthermore, the loss of adipose tissue reduces leptin levels, thus inhibiting satiety signaling, resulting in increased appetite. This lack of satiety leads to greater calorie intake, increasing the metabolic stress in the organs already affected by lipotoxicity. This increased calorie intake leads to a vicious metabolic cycle of increasing diet-induced inflammation. This chapter discusses how dietary activation of AMP-activated protein kinase (AMPK) may attenuate insulin resistance and thus reduce the severity of lipodystrophy. The same dietary technology may also be applicable to treating a wide variety of other chronic conditions associated with insulin resistance.

2. Classification of lipodystrophy syndromes

The prevalence of various lipodystrophy syndromes is relatively rare and are comprised of a heterogeneous group of disorders. These various syndromes can be characterized as (congenital or acquired) generalized or partial lipodystrophies [3, 4, 5]. Generalized lipodystrophy is characterized by an almost complete loss of subcutaneous adipose tissue. This loss of adipose tissue results in low leptin levels leading to a significantly increased appetite [6]. Patients with partial lipodystrophy may exhibit excess adipose tissue accumulation in other areas of the body. Lipodystrophy syndromes usually manifest with several metabolic abnormalities associated with severe insulin resistance that include diabetes mellitus, hypertriglyceridemia, and hepatic steatosis which can progress to steatohepatitis.

Acquired lipodystrophy is a frequent side effect of HIV and antiretroviral treatments that can affect the adipose tissue by several mechanisms [7]. Another cause of acquired lipodystrophy syndrome results from cancer treatments using irradiation especially preceding bone marrow transplants [8].

Finally, localized lipodystrophy is often caused by repeated trauma in the same area, such as injections of insulin, corticosteroids, monoclonal antibodies, and antibiotics [9].

3. Lipotoxicity

Regardless of the cause of lipodystrophy, the immediate consequence is an increased deposition of fat in tissues that are not designed to store excess fat. These tissues include insulin-sensitive tissues such as the liver and skeletal muscle. This type of fat is classified as ectopic fat. The result of increased ectopic fat is the disruption of the normal metabolism in these tissues. The disturbance of normal metabolism by ectopic fat is known as lipotoxicity (or lipid-induced toxicity). When lipotoxicity occurs, the ability of these tissues to respond to insulin signaling is compromised, resulting in insulin resistance [10]. Insulin resistance can also occur in other organs such as the brain, where it inhibits insulin signaling in the hypothalamus, that has a crucial role in the regulation of satiety. Insulin resistance in the brain can also reduce the neuronal glucose uptake and use for energy production [11].

Lipotoxicity can also occur when the adipose tissue can no longer expand to store fat safely, leading to fat disposition in the liver and skeletal muscles [12].

Although obesity and type 2 diabetes mellitus are opposite clinical conditions compared to lipodystrophy, they also result in lipotoxicity leading to insulin resistance. In the case of obesity and type 2 diabetes mellitus, adipocytes become saturated with fat and any additional intake of dietary fat or lipids derived from de novo de novo lipogenesis are deposited as ectopic fat in non-adipose tissues. As shown in Figure 1, in lipodystrophy, the significant loss of adipose tissue drives any additional dietary fat or de novo synthesized fat to be deposited as ectopic fat in non-adipose tissues. More recently, compelling genetic studies have suggested that subtle partial lipodystrophy is likely to be a major factor in prevalent insulin-resistant type 2 diabetes mellitus [13].

Figure 1.

Lipodystrophy syndromes have been provided critical insights into adipocyte biology and the systemic consequences of impaired adipocyte function. In addition, compelling genetic studies have suggested that subtle partial lipodystrophy is likely a significant factor in prevalent insulin-resistant type 2 diabetes mellitus. In addition, compelling genetic studies have suggested that subtle partial lipodystrophy is likely a significant factor in prevalent insulin-resistant type 2 diabetes mellitus.

4. Insulin resistance

The concept of insulin resistance was first discussed more than 80 years ago [14]. Insulin resistance can be viewed as the inability of insulin to promote the uptake of glucose into its target tissues. However, its relationship to a larger group of chronic conditions began to be more recognized by the work of Dr. Gerald Reaven [15]. Figure 2 shows some of the chronic diseases associated with insulin resistance.

Figure 2.

Chronic diseases that are associated with insulin resistance.

Nonetheless, it is still unclear exactly what causes insulin resistance [16, 17]. However, it is known that insulin resistance is also associated with chronic low-level inflammation [18, 19, 20, 21].

4.1 Putative mechanisms of insulin resistance

The most precise measurement of insulin resistance is represented by the hyperinsulinemic euglycemic glucose clamp [22]. Less precise, but more easily conducted is the use of the HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) test requiring the measurement of both fasting insulin and glucose levels. Other methods of determining insulin resistance would include elevated fasting glucose or elevation in the long-term marker of blood glucose glycated hemoglobin (HbA1c).

On the other hand, determining the intracellular mechanisms that are the underlying cause of insulin resistance has proven more challenging. Randle et al. first postulated that insulin resistance might be caused by fatty acids competing with glucose for substrate oxidation [23]. Through a series of complicated metabolic steps, the result would potentially lead to an increase in circulating glucose levels and decreased glucose uptake by the target cells. Another variation of this mechanism of insulin resistance was the proposed inhibition of glycogen synthase by free fatty acids [24]. In this proposal, insulin resistance occurred primarily in the liver, not in the skeletal muscle.

However, ¹³C nuclear magnetic resonance spectroscopy experiments seemed to refute this hypothesis, suggesting that much of the glucose uptake occurred in the skeletal muscle [25]. This result shifted thinking on the mechanism of insulin resistance from a glucose-centric theory to a more lipid-centric view. Another potential lipo-centric mechanism has been hypothesized to be the increased production of ceramides in the cell. This proposed mechanism would potentially disrupt the AKT signaling pathway [26]. This would prevent the translocation of the glucose transporter type 4 (GLUT4) to the cell surface needed to transport extracellular glucose into the cell. However, new information on the role of 5′ adenosine monophosphate-activated protein kinase (AMPK) in insulin resistance represented a swing back to a glucose-centric approach [27].

Insulin resistance is also strongly associated with low-level chronic systemic inflammation [28]. One clue of the cause of that inflammation emerged when it was determined that the tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, was associated with insulin resistance [29, 30]. One potential suggested pathway was that TNF-α might inhibit insulin receptor substrate 1 (IRS-1) by phosphorylation [31]. Another proposed variation is that IRS-1 inhibition might be induced by free fatty acids activating the Toll-like receptor 4 (TLR4) on the cell surface [32]. This activation of TLR-4 by free fatty acids would activate NF-κB (nuclear factor kappa B), the master gene transcription factor for inflammation. One of the cytokines produced by an activated NF-κB is TNF-α. This inflammatory hypothesis was reinforced by clinical studies using high-dose aspirin or salsalate to treat type 2 diabetes mellitus [33, 34, 35] and c-Jun N-terminal Kinase (JNK) inhibitors to treat insulin resistance [36].

Although there is no current consensus on the molecular cause of insulin resistance, it appears to involve a mixture of lipid, glycemic, and inflammatory stressors in the cells of insulin-sensitive target tissues. The result is making the cell less efficient in taking up glucose from the circulation.

4.2 An alternative mechanism of insulin resistance

As shown in Figure 2, many chronic diseases associated with insulin resistance seem to have little direct connection to impairment of the classical insulin signaling system mediated by phosphatidylinositol 3-kinase (PI3K). Therefore, an alternative hypothesis explaining insulin resistance may be via insulin-independent mechanisms that inhibit AMPK activity. AMPK is the master regulator of metabolism in every cell. Therefore, any inhibition of AMPK activity will have significant implications in intracellular metabolism, especially in the reduced ability of the GLUT-4 to translocate to the cell surface and to mediate the glucose transport into the cell for its metabolic needs. It is also known that activation of AMPK increases the phosphorylation of phosphodiesterase 4 (PDE4) [37]. The phosphorylated PDE4 would then inhibit lipolysis in the adipose tissue, thereby decreasing the levels of free fatty acids that would induce greater hepatic glucose production [38]. This would provide another insulin-independent pathway for the reduction of blood glucose levels.

However, controlling the activities of GLUT-4 and PDE4 represents only a limited number of intracellular metabolic processes regulated by AMPK, as shown in Figure 3.

Figure 3.

Metabolic effects of AMPK activation. Green arrows on the “spokes” indicate activation, red lines with a bar at the end indicate inhibition. Abbreviations: ACC1 and ACC2, acetyl-CoA carboxylase 1 and 2; AMPK, 5′ adenosine monophosphate-activated protein kinase (AMPK); APC, antigen presenting cells; Glut4, glucose transporter protein4; GS, glycogen synthase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa-B; NOS, nitric oxide synthase; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1-alpha; SREBP-1c, sterol regulatory element-binding protein 1c.

These other metabolic actions of AMPK include stimulation of fatty acid oxidation to reduce lipotoxicity and inhibition of NF-κB, which would decrease the production of inflammatory cytokines such as TNF-α. Thus, inhibition of AMPK activity can be related to many of the glycemic, lipid, and inflammatory stressors associated with insulin resistance.

This alternative theory of insulin resistance is reinforced by studies suggesting that the mechanism of PPAR-γ agonists such as the thiazolidinediones (TZDs) - that are used to treat insulin resistance in patients with type 2 diabetes mellitus - may be more due to the activation of AMPK as opposed to the TZDs being ligands of the Peroxisome proliferator-activated receptor gamma (PPAR-γ) [39, 40, 41, 42].

It is also known that insulin resistance is strongly associated with reduced AMPK activity [43, 44, 45, 46]. Consequently, metabolic disorders such as obesity, metabolic syndrome, type 2 diabetes mellitus, and non-alcoholic fatty liver disease (NAFLD) that are strongly associated with insulin resistance, have been directly linked to decreased AMPK activity [47, 48, 49]. Furthermore, other chronic conditions related to increased insulin resistance include hypertension [50], cardiovascular disease [51], polycystic ovary syndrome [52], chronic kidney disease [53], various types of cancer [54], depression [55], and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [56]. Each of these conditions can be linked to a decrease in the activity of AMPK.

Since AMPK activity is under robust dietary control, a promising treatment of insulin resistance may be through dietary activation of AMPK [57, 58]. If feasible, such dietary activation of AMPK would increase the transport of GLUT-4 to the cell surface to bring glucose into the cell independent of insulin signaling. In addition, activating AMPK would reduce the levels of accumulated lipotoxic fat by increasing fatty acid beta-oxidation and reducing the production of pro-inflammatory cytokines such as TNF-α via its inhibition of NF-κB activity [59, 60].

Thus, inhibition of AMPK activity could explain many of the clinical manifestations of insulin resistance without invoking the events at the insulin receptor and its downstream signaling pathways as the focal point of the phenomenon of insulin resistance. It would also explain why calorie restriction and exercise are so helpful in reducing insulin resistance because of their direct effects on AMPK activation.

4.3 Drug treatments of insulin resistance

While lipodystrophy is rare, two other metabolic conditions (obesity and type 2 diabetes mellitus) associated with insulin resistance are far more common.

Obesity and type 2 diabetes mellitus initially appear to present a clinical paradox in that they are characterized by excess accumulation of stored fat in the adipose tissue. In contrast, lipodystrophy is a consequence of the loss of adipose tissue [61].

However, all three conditions are characterized by ectopic fat. In the case of lipodystrophy, the accumulation of ectopic fat is caused by a lack of adipose tissue. In contrast, in obesity and type 2 diabetes mellitus, the proliferation of ectopic fat is caused by the inability of the existing adipocytes to store additional fat. Thus, reduction of excess body fat for the treatment of obesity and type 2 diabetes mellitus should be expected with increased AMPK activity. Biopsies of adipose tissue before and after gastric bypass surgery or significant weight loss confirm that the levels of AMPK in the adipose tissue rise significantly [62, 63].

The role of AMPK in reducing insulin resistance in type 2 diabetes mellitus is further suggested because most of the drugs used to treat diabetes operate through activation of the AMPK pathway. These drugs include metformin[64, 65], sodium-glucose cotransporter-2 (SGLT2) inhibitors [66], dipeptidyl peptidase-4 (DPP-4) inhibitors [67] and glucagon-like peptide-1 (GLP-1) receptor agonists [68].

Therefore, a highly structured dietary program that can significantly activate AMPK should be useful in treating insulin resistance, whether it is associated with loss of adipose tissue (i.e., lipodystrophy) or with a reduced capacity of storing excess fat in the adipose tissue (i.e., obesity and type 2 diabetes mellitus).

5. Dietary activation of AMPK

Dietary activation of intracellular signaling pathways is more complex than pharmacology, but it has the advantage of having a far greater therapeutic index. It is more complicated because it usually requires multiple points of dietary intervention to sufficiently activate AMPK and thus greater patient compliance [57, 58]. In essence, there is no single ideal nutrient to optimally activate AMPK to the extent necessary to reduce severe insulin resistance. On the other hand, when using multiple dietary interventions, there is no need to use excessive levels of any single dietary intervention, thereby ensuring an exceptionally high therapeutic index for a dietary program that is likely to be required for a lifetime.

Three specific dietary interventions have the most significant impact on the activation of AMPK: (a) an anti-inflammatory calorie-restricted diet, (b) omega-3 polyunsaturated fatty acids (omega-3 PUFAs), and (c) polyphenols. Although each dietary intervention operates through different mechanisms to enhance AMPK activity, they have significant synergistic interactions [57, 58].

5.1 Anti-inflammatory, calorie-restricted diets

The description of an anti-inflammatory calorie-restricted diet must be highly defined, making it possible to be replicated in controlled clinical studies. This definition includes the total reduction of calories and the macronutrient composition that will increase satiety.

As confirmed by tissue biopsies, calorie restriction remains the most validated approach to activate AMPK [69]. On the other hand, it is usually the most difficult dietary intervention to enhance AMPK activity because it requires dietary compliance. Calorie restriction ideally aims to reduce total calorie intake by 25% of the recommended daily calories needed for current weight maintenance. However, the CALERIE (Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy) study demonstrated that trying to achieve a 25% reduction by simply reducing calories in young non-obese subjects without adjusting macronutrient ratios of the diet resulted in less than half that calorie restriction guideline being achieved. Nonetheless, significant clinical benefits were observed in the subjects during a 2-year study [70].

Simply restricting total calorie intake ignores the importance of the macronutrient balance of the remaining calories to prevent hormonal disturbances that can lead to increased hunger, loss of lean body mass, and increased inflammation. Protein is the primary macronutrient that needs to be maintained in any type of calorie restriction diet. This macronutrient is essential for improved hunger control and maintenance of muscle mass during calorie restriction. Thus, a calorie-restricted diet should provide about 90 grams of protein per day for an average woman and about 110 grams of protein per day for an average male. Furthermore, this daily protein intake should be evenly spread uniformly throughout the day. This requirement is because the hormonal changes induced by any meal will last only 5 hours before returning to baseline [71]. It has been demonstrated that increased protein intake is also essential to maintain glucagon levels between meals that will help stabilize blood glucose levels, thereby reducing hunger [71]. Another reason is that adequate protein intake is critical for dietary compliance because it enhances the release of gut satiety hormones such as peptide YY (PYY) and GLP-1, further reducing appetite [72]. Thus, consuming adequate protein content at every meal will significantly reduce hunger making long-term calorie restriction more feasible, especially in those individuals with existing insulin resistance [73]. This concept is known as “protein leveraging” [74].

Reducing inflammation requires relatively consistent control of the protein-to-carbohydrate ratio. Regardless of the amount of protein at a meal, it must be balanced by an appropriate level of low-glycemic load carbohydrates to maintain a balance of insulin-to-glucagon in the blood between meals [71, 75]. This balance of insulin to glucagon is also critical in controlling the desaturase enzymes that convert omega-6 fatty acids into arachidonic acid (AA) [76]. Insulin increases the activity of these enzymes, whereas glucagon decreases their activity. Reducing the formation of AA diminishes the potential generation of pro-inflammatory eicosanoids, as AA is the precursor of these eicosanoids. Furthermore, a lower glycemic index of the carbohydrates used at a meal is associated with a lower level of insulin secretion and with reduced AA formation. In addition, low-glycemic index carbohydrates tend to be rich in fermentable fiber that increases the signaling intensity of PYY and GLP-1 from the gut to the appetite control centers in the hypothalamus [77].

Finally, one must consider the total fat content of an anti-inflammatory calorie-restricted diet. First, the fat content should be low because the goal is to activate AMPK to increase the oxidation of existing ectopic fat. Therefore, any excess intake of dietary fat would slow the process. Furthermore, both omega-6 fatty acids and saturated fats can be regarded as pro-inflammatory fats; hence their level should be significantly reduced. The molecular reason is that the omega-6 fatty acids are building blocks necessary for generating eicosanoids, and saturated fats (primarily palmitic acid) can interact with the Toll-like receptor 4 (TLR-4) to activate NF-κB, which will increase the synthesis of cytokines and pro-inflammatory eicosanoids via up-regulation of cyclooxygenase-2 (COX-2) [78]. Therefore, most of the limited fat content of an anti-inflammatory diet should come from monounsaturated fatty acids.

Thus, the definition of an anti-inflammatory calorie-restricted diet becomes decreasing calorie intake by least 500 calories per day below the level estimated to maintain current body weight yet providing adequate protein to maintain lean muscle mass and glucagon levels. That amount of protein is balanced with moderate levels of low-glycemic-load carbohydrates (primarily non-starchy vegetables and limited amounts of fruits). A good starting point for developing the appropriate micronutrient balance of a calorie-restricted anti-inflammatory diet would be about one-third more low-glycemic index carbohydrates to low-fat protein at each meal. Finally, such a diet is low in total fat (especially omega-6 fatty acids and saturated fats) but high in fermentable fiber. Such a diet was first proposed in 1995 [75]. Numerous clinical trials over the years have supported the use of such a defined diet, especially in the treatment of type 2 diabetes mellitus [79, 80, 81, 82, 83, 84, 85, 86, 87, 88].

The question is, how long can such a calorie-restricted anti-inflammatory diet be maintained? The answer is potentially indefinitely. The resulting lack of hunger is due to a combination of increased satiety, better control of blood glucose levels to prevent hypoglycemia between meals, and reduced inflammation in the satiety control centers in the hypothalamus. Finally, much of the success in maintaining satiety is based on maintaining a balance of protein-to-glycemic load ratio at each meal [71].

Because a calorie-restricted anti-inflammatory diet is based on protein needs, determining the daily protein requirements for an individual is critical for success. Since the goal is to lose ectopic body fat, but not muscle mass, the subject must consume adequate protein to maintain their existing lean body mass. However, daily protein requirements are not determined by weight, but by lean body mass since total fat mass requires little incoming dietary protein to maintain its biological functions. To determine lean body mass requires determining the total body fat content and then subtracting it from the current body weight. Techniques such bioelectrical impedance analysis (BIA) or dual X-ray absorptiometry (DXA) can accurately determine both compartments [89]. Slightly less accurate measurements can be done by measurement at various body positions. Individuals with a body mass index (BMI) of about 25 Kg/m² have nearly one-third of their total weight as body fat [90]. Since females will have a higher percentage of body fat than males at the equivalent BMI, they have a lower lean body mass and a lower dietary protein requirement to maintain their lean body mass. Although an individual with lipodystrophy will have much lower total body fat levels, the calculation is still the same. Therefore, a good starting point for daily protein requirements for males and females is approximately 1.2 grams of protein per Kg of lean body mass.

Once the protein levels are established based on lean body mass, the levels of carbohydrates and fat needed to fulfill the requirements of the anti-inflammatory component of the calorie-restricted diet are automatically determined. Typically, this diet would be approximately 40 percent of the total carbohydrates coming from low-glycemic index carbohydrates (primarily non-starchy vegetables and limited fruits), 30 percent of the total protein consisting of low-fat protein, and 30 percent of total fat mainly composed of monounsaturated fat sources such as nuts, olive oil, or avocados. This percentage of macronutrients would provide 90 grams of protein, 120 grams of low-glycemic index carbohydrates, and 40 grams of fat for an average female consuming 1200 calories per day or 110 grams of low-fat protein, 150 grams of low-glycemic index carbohydrates, and 50 grams of fat for an average male consuming 1500 calories per day. In addition, since most dietary carbohydrates come from low-glycemic sources, this diet would also be rich in fermentable fiber. Thus, an anti-inflammatory calorie-restricted diet is adequate in protein, moderate in carbohydrates, and low in fat but high in fermentable fiber. Although the levels of carbohydrates are considered moderate in terms of grams, one would need to consume approximately eight servings of non-starchy vegetables and two servings of fruit per day to reach those carbohydrate levels.

However, there are also dietary inhibitors of AMPK. The first of these dietary inhibitors is excess calories. Any calorie-restricted diet makes up for the decreased intake of incoming calories for metabolic needs by the increased oxidation of stored fat. The efficacy of the oxidation of stored fat is ultimately controlled by AMPK activity [91]. Therefore, as one takes in more calories than described above, AMPK activity will decrease, and the oxidation of stored body fat will slow down.

Eventually, there may be a point in time at which excess body fat has been sufficiently reduced. This state is usually indicated by the beginning of the physical appearance of the abdominal muscles that are generally covered by a layer of excess fat. The beginning of the physical appearance of one’s abdominal muscles roughly corresponds to a body fat percentage of approximately 22 percent for females and 15 percent for males. These levels of body fat are consistent with being metabolically fit, as stated by the American Council on Exercise. Therefore, if any further body fat loss occurs, one simply adds more non-inflammatory monounsaturated fat to the base calorie-restricted diet until the abdominal muscles are barely perceptible, indicating that the body has an adequate level of stored body fat.

The second dietary inhibitor of AMPK activity would be excess glucose intake. AMPK is also a glucose sensor [92]. Thus, as glucose intake increases, AMPK activity will slow down.

Finally, it has been demonstrated that excess leucine can inhibit AMPK activity in isolated rat skeletal muscle [93]. This inhibition of AMPK by leucine appears to be associated with an increase in lactate/pyruvate ratio and can be overcome by using the pharmacological AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR).

5.2 Omega-3 fatty acids

The use of an anti-inflammatory calorie-restricted diet is only one of several specific dietary interventions to enhance AMPK activity. Although helpful in reducing inflammation, the described anti-inflammatory calorie-restricted diet is not very useful for the complete resolution of residual inflammation. The primary dietary component to reach this goal is adequate intake of long-chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA and DHA are the building blocks for a powerful group of lipid mediators known as specialized pro-resolving mediators (SPMs). These lipid mediators are critical for the resolution of inflammation and stimulating the activity of AMPK by their interaction with specific receptors [94, 95, 96]. Therefore, combining adequate omega-3 fatty acids with a calorie-restricted anti-inflammatory diet is synergistic for AMPK activation. A typical starting dosage for omega-3 fatty acids would be 2–3 grams of EPA and DHA per day to make adequate levels of SPMs. For comparison, the average American consumes approximately 100 mg of EPA and DHA per day [57]. Therefore, it is likely that supplementation of omega-3 fatty acids would be needed to activate AMPK.

5.3 Polyphenols

Polyphenols are a class of compounds found in many plant foods that includes flavonoids, phenolic acids, lignans, and stilbenes. Regularly consuming polyphenols is thought to boost digestion and brain health, as well as protect against heart disease, type 2 diabetes mellitus, and even certain cancers [97].

Polyphenols provide a distinct third dietary pathway to activate AMPK [98]. Whereas calorie restriction activates AMPK by energy restriction and omega-3 fatty acids activate AMPK by producing SPMs, polyphenols indirectly activate AMPK by interacting with various sirtuin (SIRT) enzymes. SIRTs are deacetylating enzymes that are activated by polyphenols. Once activated by polyphenols, SIRT1 can facilitate the deacetylation of an upstream kinase, liver kinase B1 (LKBT1), promoting the activation of AMPK that increases fatty acid oxidation [99]. An additional benefit is that once AMPK is activated, it will also increase the synthesis of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) to accelerate the salvage pathway to replenish nicotinamide adenine dinucleotide (NAD⁺), which is required for continued deacetylation mediated by the SIRT enzymes [100]. This cycle is shown in Figure 4.

Figure 4.

Crosstalk between SIRT and AMPK. Any decrease in the cell’s energy state measured by an increased AMP/ATP ratio will activate AMPK. This activation of AMPK increases NAMPT activity that produces NAD⁺ required for SIRT deacetylation activity. SIRT then deacetylates LKB1, which activates AMPK. Abbreviations: AMP, 5′-adenosine monophosphate; AMPK, 5′ adenosine monophosphate-activated protein kinase; ATP, adenosine-5′-triphosphate; LKB1, liver kinase B1; NAMPT, nicotinamide phosphoribosyltransferase; SIRT, Sirtuins.

By increasing NAD⁺ levels, the SIRT enzymes can maintain the deacetylation of the p65 Lys310 protein that maintains NF-κB in an inactive state in the cytoplasm, preventing its entry into the nucleus and reducing the synthesis of cytokines and COX-2 enzymes as inflammatory mediators [101].

Another benefit of polyphenol-induced AMPK activation is the increase in the activity of the gene transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) that will increase the synthesis of various antioxidant enzymes such glutathione reductase and superoxide dismutase [102]. The increase in these antioxidant enzymes will reduce oxidative stress within the cell.

The primary source of dietary polyphenols are fruits and vegetables. Unfortunately, most polyphenols in these food sources are usually either water-insoluble or have polymeric structures. In either case, they will have low bioavailability. However, such polyphenols may be further metabolized into less complex phenolic compounds by the microbiome in the gut. These less structurally complex phenolic compounds may have greater bioavailability but less potential for stimulating the SIRT enzymes. However, some polyphenols (such as monomeric anthocyanins) are directly absorbed and have a more significant impact on activating the SIRT enzymes [103]. If one is supplementing with a water-soluble extract rich in monomeric anthocyanins, a good starting dose would be 450 mg per day. Significant reductions in oxidative stress and oxidation of low-density lipoproteins (LDLs) are observed at this dose [104].

Thus, there are three separate dietary pathways for AMPK activation, as shown in Figure 5.

Figure 5.

A graphical description of the dietary interventions that can activate AMPK to reduce insulin resistance. Abbreviations: AMPK, 5′ adenosine monophosphate-activated protein kinase; SIRT, Sirtuins; SPMs, specialized pro-resolving mediators.

Any one of the three dietary pathways is beneficial, but when all three are being used simultaneously, the diet provides a robust but non-toxic methodology to overcome AMPK inhibition. Of these three dietary pathways to activate AMPK, calorie restriction requires the most dietary discipline, whereas adequate intake of omega-3 fatty acids and polyphenols can be easily achieved by supplementation. However, the potential benefits of omega-3 fatty acid and polyphenol supplementation will be significantly attenuated without adequate calorie restriction since excess calories and glucose are dietary inhibitors of AMPK activity. Thus, calorie and glucose restriction become key to ensuring that all three dietary interventions can activate AMPK by working synergistically. Furthermore, using such a synergistic dietary strategy does not require the potential excess intake of omega-3 fatty acids or water-soluble polyphenols to overcome the inhibitory actions of increased calorie or glucose intake that would inhibit AMPK activity.

6. Clinical markers of AMPK activation

Treating insulin resistance using dietary inventions to activate AMPK can be achieved by reducing the intracellular stress on lipid, glycemic, and inflammatory, metabolic responses within the cell. Therefore, the more these three metabolic responses are maintained within appropriate ranges, the greater the degree to which the AMPK becomes activated.

Since AMPK is constrained to remain in the cell, measuring AMPK activity directly without a tissue biopsy is impossible. However, since AMPK is the master regulator of metabolism, several surrogate blood markers can be used to determine whether the dietary interventions described above are achieving their goals of increased intracellular AMPK activity. Such blood markers must be easy to obtain and readily interpreted to make any necessary dietary adjustments to fine-tune them to the individual. The three clinical markers that meet these criteria are the following:

• Reducing lipid stress: a primary factor in causing insulin resistance is lipid stress. The triglyceride to high-density lipoprotein cholesterol (TG/HDL-C) ratio is one marker of insulin resistance, especially hepatic insulin resistance [105, 106, 107, 108, 109, 110, 111, 112]. Ideally, the TG/HDL-C ratio (measured in mg/dL) should be less than 1.

• Reducing inflammatory stress: inflammatory stress is caused by an imbalance in the production of eicosanoids and SPMs. Thus, to maintain an optimal balance of eicosanoids and SPMs, the AA/EPA ratio should be between 1.5 and 3. Within this target AA/EPA range, a significant reduction of various cytokines can be observed relatively quickly [113, 114, 115]. Unfortunately, Americans’ average AA/EPA ratio is greater than 20 [116, 117], indicating an unfavorable balance of eicosanoids to SPMs. This imbalance fuels the inflammatory component of insulin resistance.

• Reducing glucotoxicity: the HbA1c level can be used as a long-term marker of glucose control and glucotoxicity. The HbA1c levels should be maintained between 4.9% and 5.1%, indicating the lack of glucose inhibition of AMPK activity [118].

Only when all three of these three clinical markers are in their proper ranges can AMPK activity be considered optimized for an individual.

It should be noted that optimized AMPK activity differs from continuous AMPK activity. Metabolism is highly dynamic, allowing the cell to adapt to changing conditions rapidly. This optimization using the above-described blood markers enables AMPK to respond to rapidly changing cellular needs for more efficient metabolic activity. In other words, optimization of AMPK activity provides metabolic resilience. If AMPK is constantly active, it can become desensitized to changes in cellular energy status [119]. Maintaining the surrogate blood markers of AMPK activity within these operating limits allows AMPK to retain the necessary metabolic resilience required for maintaining cellular homeostasis.

Specific dietary interventions can modulate each surrogate blood marker of intracellular AMPK activity. For example, the TG/HDL-C ratio can be significantly reduced by following a calorie-restricted diet for 6 weeks [120]. In addition, the dietary intake of omega-3 fatty acids strongly influences the AA/EPA ratio in relatively short periods of time [113, 114]. Finally, the dietary intake of polyphenols to activate AMPK will significantly reduce HbA1c levels within 3 months in pre-diabetic subjects [121]. Furthermore, there is significant crosstalk of the various dietary components as they become synergistic in their cumulative actions [58].

7. Clinical examples of dietary modulation of AMPK activity

Numerous clinical examples demonstrate that the individual dietary interventions described above are effective in chronic conditions characterized by insulin resistance.

7.1 Anti-inflammatory calorie-restricted diets

Numerous examples of treating type 2 diabetes mellitus with an anti-inflammatory calorie-restricted diet have been described [80, 83, 85, 86, 87, 88]. In addition, some of the studies range up to 5 years in duration, indicating long-term compliance using an anti-inflammatory calorie-restricted diet is possible [84].

7.2 Omega-3 fatty acids

High-dose omega-3 fatty acids have successfully treated NAFLD, which is a common complication of severe insulin resistance [122]. It has also been demonstrated that an adequate intake of omega-3 fatty acids can cause a statistically significant reduction in pro-inflammatory cytokines [113, 114]. The decrease in pro-inflammatory cytokine levels is likely the result of increased AMPK activity that would inhibit NF-κB activity.

7.3 Polyphenols

Supplementation with monomeric delphinidins (one class of anthocyanins) has been shown to reduce HbA1c in pre-diabetic individuals [121]. This clinical result suggests an increase in AMPK activity that would be consistent with the increase in GLUT-4 transport to the cell surface. In addition, higher doses of monomeric delphinidins also reduce oxidative stress in smokers [104]. This decrease in oxidative stress is most likely mediated by increased AMPK activity that would increase Nrf2-mediated up-regulation of antioxidant enzymes [102].

These clinical examples used only a single dietary intervention to activate AMPK. Thus, it might be reasonably expected that combinations of any two or ideally all three dietary interventions described above would have synergistic effects on reducing insulin resistance.

8. Treating insulin resistance using systems-based biology

Systems-based biology is based on the interconnected signaling pathways within the cell that are required to maintain homeostasis. Furthermore, homeostasis requires rapidly switching from anabolic to catabolic states for cell maintenance. Because of these complex intracellular signaling relationships, a pharmaceutical intervention focused on one pathway may adversely affect other intracellular signaling pathways in the same cell, thereby generating significant side effects.

Our working hypothesis is that dietary activation of AMPK can effectively coordinate these intracellular pathways to decrease various metabolic stresses within the cell that drive insulin resistance. How AMPK activity is intimately connected to many of these diverse signaling pathways is shown in Figure 6.

Figure 6.

Effect of dietary activation of AMPK on various regulatory proteins and gene transcription factors. Abbreviations: AMPK, 5′ adenosine monophosphate-activated protein kinase; FOXO, Forkhead box transcription factors class O; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa-B; Omega-3 PUFAs, Omega-3 polyunsaturated fatty acids; PI3K/AKT, phosphatidylinositol 3-kinase/AKT (protein kinase B); SIRT, Sirtuins. Green lines = activation; red lines = inhibition; black lines = mutual activation or mutual inhibition.

This figure shows significant cross-signaling between these various metabolic systems within the cell and potential inhibition or activation of one system by another. In some cases, there is mutual activation, such as between AMPK and SIRT or between PI3K/AKT (Phosphatidylinositol 3-kinase/protein kinase B), mTOR (mammalian target of rapamycin) and NF-κB. In other cases, there can be reciprocal inhibition between systems, such as between PI3K/AKT and AMPK. Finally, there can also be unidirectional inhibition or activation between various signaling pathways.

AMPK may represent the molecular link between these diverse signaling systems and the diet. This control is possible since AMPK is an evolutionarily conserved energy sensor that controls metabolism. In essence, AMPK becomes the checkpoint for metabolic control that links diet to other intracellular signaling systems.

However, one can only routinely monitor the blood, not the cell’s interior. This inaccessibility of direct measurement of AMPK is why constant monitoring of the blood markers described above provides an easily obtained insight into AMPK activity. In doing so, it may be possible to maintain these other internal cellular signaling pathways within their optimal operating parameters that are personalized to the subject.

The goal is to have AMPK able to respond dynamically to changing conditions in the cell. For example, there will be times when inflammation must be up-regulated but then returned to a quiescent state to maintain homeostasis. This molecular resilience requires keeping AMPK activity within discrete operating boundaries determined from surrogate markers in the blood. As pointed out above, each of these surrogate markers is under robust dietary control.

On the other hand, if AMPK activity is significantly inhibited, this leads to the over-expression of pro-inflammatory signaling systems shown in Figure 6. One of the linked systems that would increase with a decrease in AMPK activity is represented by the activation of NF-κB. Increased NF-κB activity is associated with increased inflammatory state related to cardiovascular disease [123] and cancer [124]. Likewise, reduced activity of AMPK would lead to potentially excessive activity of mTOR and PI3K/AKT signaling pathways associated with cancer [125, 126].

How the activity of AMPK acts as the intracellular central hub linking various other cellular signaling systems is described in more detail in the following paragraphs.

8.1 NF-κB signaling pathway

One of the primary benefits of activating AMPK is the inhibition of NF-κB, thus reducing pro-inflammatory cytokine and eicosanoid formation. The lowering of inflammation is achieved through several routes orchestrated by AMPK [60, 127]. One pathway is inhibiting NF-κB by the direct activation of AMPK [127]. Another pathway is activating sirtuin 1 (SIRT1) by increasing NAD⁺ levels [128]. AMPK activates the rate-limiting enzyme in the NAD⁺ salvage pathway that provides the necessary NAD⁺ to enable SIRT1 to deacetylate the p65 Lys310 component of NF-κB to prevent its binding to the cell’s DNA that is required to express inflammatory mediators [129].

Additional AMPK-mediated pathways that inhibit NF-κB activity include the activation of the Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) [130] and the phosphorylation of Forkhead box transcription factors class O (FOXO) [127].

8.2 mTOR signaling pathway

Activation of AMPK is the primary inhibitor of mTOR. At the molecular level, mTOR inhibition is due to phosphorylation of the raptor component of mTORC1 and TSC2 [131]. In addition, the association of increased SIRT1 activity with mTOR inhibition [132] can be induced by the AMPK’s activation of the rate-eliminating enzyme of the NAD+ salvage pathway [128]. On the other hand, any increase in AKT activity will up-regulate mTOR, which activates NF-κB [133].

8.3 PI3K/AKT signaling pathway

The PI3K/AKT signaling pathway is activated by insulin and results in cellular growth activation [134]. If the PI3K/AKT signaling pathway is overactive, this will inhibit the activity of AMPK [135, 136]. On the other hand, any increase in AMPK activation will inhibit AKT activity [135, 136].

Therefore, the inhibition of AMPK and FOXO activity by AKT can be reduced by dietary activation of AMPK. PI3K activity is reduced at the most basic level by lowering blood insulin levels following an anti-inflammatory calorie-restricted diet. The reduction of PI3K stimulation by insulin leads to decreased activation of AKT. Long-term studies using the previously described anti-inflammatory calorie-restricted diet have demonstrated success in the long-term management of type 2 diabetes mellitus [81, 84].

8.4 FOXO signaling pathway

The FOXO family of gene transcription factors consist of FOXO1, FOXO3, FOXO4, and FOXO6. The FOXO family is vital in controlling cellular senescence, stem cell maintenance, and lifespan in animal models [137]. FOXO upregulation can be achieved either by phosphorylation via AMPK or deacetylation by SIRT2 [138, 139]. In addition to the direct effect of AMPK activation on FOXO, any increase in AMPK activity will increase the activity of the rate-limiting enzyme (NAMPT) in the synthesis of NAD⁺, thereby activating SIRT, which also increases FOXO activity [140].

An indirect route to activate FOXO is via the AMPK-induced inhibition of AKT [138]. On the other hand, any upregulation of AKT by a deficit in AMPK activity will reduce FOXO activity [141, 142, 143]. This central role of AMPK in FOXO activation may explain why activation of AMPK has been hypothesized to control the aging process [144].

Another inflammatory pathway that can be modulated by AMPK is JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway, which mediates cytokine signaling [145].

Considering the complexity of these interactions with cellular signaling mechanisms in the cell, optimizing AMPK activity may have a far greater potential to bring a cell back to homeostasis than any current or proposed potential drug therapies. Furthermore, the dietary interventions described above have a potential therapeutic index that is significantly higher in reducing insulin resistance compared to any drug therapy.

9. Conclusions

We hypothesize that the ability to reduce severe insulin resistance caused by lipodystrophy and other conditions associated with insulin resistance may be achieved by increasing AMPK activity. Our hypothesis is based on the ability of AMPK to modulate internal cellular signaling through systems-based biology. While there is no single specific nutrient to optimize the body’s internal capacity to alleviate insulin resistance, an appropriate combination of dietary interventions can alter signaling pathways that can lead to the molecular goal of increasing AMPK activity.

This concept of requiring a defined variety of multiple dietary interventions to achieve the appropriate activation of AMPK is no different from using various combinations of chemotherapeutic drugs to treat cancer. However, unlike numerous combination drug therapies used for cancer treatment, each dietary intervention described earlier can be easily modulated on a personalized basis using the clinical markers described earlier.

In conclusion, understanding the complex interaction of highly defined dietary interventions that result in the activation of AMPK may provide a new comprehensive nutritional strategy to treat insulin resistance induced by lipodystrophy. The same dietary technology is also applicable to many other chronic conditions associated with insulin resistance. Furthermore, the dietary approach we have outlined can be optimized individually using validated blood markers to orchestrate various internal cellular signaling systems. Using such blood markers to titrate each dietary component that impacts activation of AMPK to their appropriate ranges moves precision nutrition into the realm of personalized medicine.

Acknowledgments

This study was supported by the Inflammation Research Foundation, Peabody, MA, USA.

Conflicts of interest

BS and AKS are employed by Zone Labs, Inc., a medical foods company.

Author contributions

BS and AKS wrote the paper.