Cardiovascular Diabetology BioMed Central Hypothesis

Despite the marked advances in research on insulin resistance (IR) in humans and animal models of insulin resistance, the mechanisms underlying high salt-induced insulin resistance remain unclear. Insulin resistance is a multifactorial disease with both genetic and environmental factors (such as high salt) involved in its pathogenesis. High salt triggers insulin resistance in genetically susceptible patients and animal models of insulin resistance. One of the mechanisms by which high salt might precipitate insulin resistance is through its ability to enhance an oxidative stress-induced inflammatory response that disrupts the insulin signaling pathway. The aim of this hypothesis is to discuss two complementary approaches to find out how high salt might interact with genetic defects along the insulin signaling and inflammatory pathways to predispose to insulin resistance in a genetically susceptible model of insulin resistance. The first approach will consist of examining variations in genes involved in the insulin signaling pathway in the Dahl S rat (an animal model of insulin resistance and salt-sensitivity) and the Dahl R rat (an animal model of insulin sensitivity and salt-resistance), and the putative cellular mechanisms responsible for the development of insulin resistance. The second approach will consist of studying the over-expressed genes along the inflammatory pathway whose respective activation might be predictive of high salt-induced insulin resistance in Dahl S rats. Variations in genes encoding the insulin receptor substrates -1 and/or -2 (IRS-1, -2) and/or genes encoding the glucose transporter (GLUTs) proteins have been found in patients with insulin resistance. To better understand the combined contribution of excessive salt and genetic defects to the etiology of the disease, it is essential to investigate the following question: Question 1: Do variations in genes encoding the IRS -1 and -2 and/or genes encoding the GLUTs proteins predict high salt-induced insulin resistance in Dahl S rats? A significant amount of evidence suggested that salt-induced oxidative stress might predict an inflammatory response that upregulates mediators of inflammation such as the nuclear factor- kappa B (NF-kappa B), the tumor necrosis factor-alpha (TNF-α) and the c-Jun Terminal Kinase (JNK). These inflammatory mediators disrupt the insulin signaling pathway and predispose to insulin resistance. Therefore, the following question will be thoroughly investigated: Question 2: Do variations in genes encoding the NF-kappa B, the TNF-α and the JNK, independently or in synergy, predict an enhanced inflammatory response and subsequent insulin resistance in Dahl S rats in excessive salt environment? Finally, to better understand the combined role of these variations on glucose metabolism, the following question will be addressed: Question 3: What are the functional consequences of gene variations on the rate of glucose delivery, the rate of glucose transport and the rate of glucose phosphorylation in Dahl S rats? The general hypothesis is that "high-salt diet in combination with defects in candidate genes along the insulin signaling and inflammatory pathways predicts susceptibility to high salt-induced insulin resistance in Dahl S rats".

Antidiabetic glitazones exhibit side effects such as weight gain, edema, and increased risk of myocardial infarctions [6,7], which have limited the use of these drugs in diabetic patients with high lipid levels. The PPARα/γ dual agonists, which reached clinical trials, have been suspended for safety issues [8]. Although much information is not available, these dual agonists are believed to exhibit similar side effects as with glitazones [2,9]. Bezafibrate, a traditional PPARα agonist, has recently been identified as a safe and synthetic pan agonist for all PPAR isotypes [10,11], although with relatively low potency and low affinity (Kd ~5 μM for PPARδ [5]). Development of drugs from natural origin against chronic diseases, such as metabolic syndrome and diabetes, has gain focus recently. Because metabolic system can easily excrete biomolecules and their derivatives, thereby avoid undesirable effects. Fish oil, which contains docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), is traditionally used as functional food against metabolic diseases. These beneficial health effects of DHA and EPA are thought to arise from their binding and activating PPARs [12,13]. Phytanic acid, a natural PPAR agonist from human diet, has also been shown to enhance glucose uptake and thereby increases insulin sensitivity, however with less capacity to differentiate adipocytes [14]. A recent study has reported an extract of Fuligo candida, Fuligocandin B, to induce 15-deoxy-Δ 12,14 prostaglandin J 2 (15d-PGJ 2 ), which is the most potent endogenous PPARγ ligand [15,16].
The hypothesis of this article is drawn on the assumption of PPARα/γ dual agonists from naturally originated molecules, that the derivatives of DHA and EPA would show stronger affinities for PPARs than the parent compounds. Therefore, these compounds may be used as putative drugs against diabetes and metabolic syndrome.

Presentation of hypothesis
The hypothesis is based on (a) functional roles of PPARs -PPAR agonists reduce blood glucose and lipid levels, and (b) structural details of ligand binding cavity of PPARs, which suggest how a ligand fits into the cavity.

Functional roles of PPARs
PPARs perform their activities by endogenous ligands produced by metabolism of fatty acids. Unmetabolized fatty acids can also act as PPAR ligands. Activities of these ligands vary according to their binding specificities for different PPARs, and on distributions of these ligands in different organs [17]. Because of this diversity, not all endogenous PPAR ligands are characterized yet. However, PPAR activities can be summarized as lipid sensors by their complementary actions. PPARα is expressed mainly in the tissues with high capacity for fatty acid oxidations, e.g. liver, heart, skeletal muscle etc. On the other hand, PPARγ is expressed predominantly in the adipose tissue but is PPAR agonists Figure 1 PPAR agonists. also expressed in immune and inflammatory cells, mucosa of the colon and placenta. If the fatty acid concentrations are increased, PPARα uptakes and oxidizes fatty acids and their metabolites [18], and PPARγ enhances storage of fatty acids in the adipose tissue [19]. These combined activities of PPARα and PPARγ cause increased utilization of glucose than fatty acids in the skeletal muscles, and cause enhanced insulin sensitivity (Fig. 2). Although PPARδ's role is not well defined, it is also implicated for fatty acid oxidation [20].

Structure of PPARs
Like other nuclear receptors, 3D structure PPARs consists of a DNA binding domain in the N-terminus and a ligand binding domain (LBD) in the C-terminus [21]. In canonical mechanism, ligand binding to PPARs causes conformational changes in the receptor, which release corepressor and recruit coactivator. Then the receptors form compulsory heterodimers with another nuclear receptor, named retinoid X-receptor (RXR), and the resulting complex finally bind to DNA of target genes [22] (Fig.  3).

Complementary actions of PPARα and PPARγ
To date, 37 X-ray crystallographically determined structures of PPAR-LBDs are deposited in the Protein Data Bank (PDB) in different apo-and holo forms. These structures shows that the LBD is composed of 13 helices and a small four-stranded β-sheet (Fig. 3B). The lower part of LBD contains a Y-shaped cavity with the volume of 1300-1400 Å 3 for ligand binding. This cavity includes 34 amino acids; each arm of the cavity is ~12 Å in length. One of the arms is hydrophilic but the other two are hydrophobic. While the amino acids in the polar arm form H-bonds with the ligand polar atoms, those in the hydrophobic arms form non-specific interactions with hydrophobic part of the ligands. Across PPARs, ~80% amino acids in the cavity are conserved, and the overall size of the cavity is also similar [23]. However, the topology is different inside the cavities of different PPARs. These differences influence ligand specificities. Ligand binding stabilizes the LBD in the active conformation; as such coactivator and RXR can bind to the activated LBD [24].

Fatty acids and their derivatives as PPAR agonists
For centuries, consumption of fatty fish is considered to protect against metabolic diseases. During early 1970s, Danish physicians discovered that Greenland Eskimos consuming fatty fishes exhibited low incidence of heart diseases and arthritis despite high-fat diet. This finding suggested beneficial effects of DHA and EPA. Fish oil supplementation successfully passed clinical trials and is now effectively used for treating metabolic syndrome [25].
Usually, PPAR ligands have three essential parts for optimal binding: (a) polar head group, (b) linker region, and (c) hydrophobic tail. DHA and EPA have a carboxylic group which serves as the polar head group, and their long chains form the required linker and hydrophobic regions. In addition, X-ray structures of PPARs show some free spaces proximal to the polar arm of binding cavity [26], and substitutions at α or β positions on DHA and EPA would fit into these spaces. Specifically, hydrophobic sub- stituents that complement the size of free spaces would be a choice. However, substituents may not fit into PPARδ because of its narrow polar regions [27]. Size and hydrophobic/hydrophilic ratio of substituents may influence the ratio of binding between PPARα and PPARγ [28]. A high PPARα/PPARγ ratio of affinities of putative ligands would be safer dual agonists, because the side effects are thought to arise from high PPARγ affinity [2].

Structure and functions of PPARs
Limitation of the hypothesis EPA and DHA have many beneficial health effects which are not typical for PPAR ligands. For example, their capacity to reduce coronary heart disease, blood pressure, primary heart attack and rheumatoid arthritis [29][30][31] are not observed with typical PPAR ligands. Also, various developmental problems including attention-deficit/hyperactivity disorder have been linked to biological deficiencies in polyunsaturated fatty acids [32]. But these fatty acids do not show many PPAR effects such as reversing insulin resistance [33,34], which may occur due to dissociation between n-3 polyunsaturated fatty acid and lipid metabolism and insulin action in insulin resistant state [35]. Therefore, an interesting observation will be whether substitutions at α or β positions on these fatty acids impart such typical PPAR effects.

Testing of hypothesis
Ligand binding assays and gene transactivation assays would confirm the hypothesis. Ligand binding assays quantify the binding affinities such as Kd or IC 50 values. Previously published experiments with fatty acids showed inconsistent binding affinities. Krey et al. [5] reported binding affinities in 5-10 μM range, while Lin et al [36] reported this range of 5-17 nM. The latter range corresponds to the intracellular free fatty acid concentrations which are in the range of 7-50 nM [37]. Until now, no data are available about the affinities of DHA, DHA derivatives, and EPA derivatives. Therefore, fluorescence-based methods as used by Lin et al. may provide correct binding affinities. In transactivation assays of PPARα/γ genes, precautions should be taken to remove unwanted fatty acids in the recombinant proteins [38]. In both ligand binding and transactivation assays, ligands with high ratio of PPARα/γ affinity and activity should be chosen, which mean that compounds showing less PPARγ affinities and activities would be ideal.

Implication of the hypothesis
The ligand binding cavity of PPARs is 3-4 times larger than the other nuclear receptors, indicating their capability to accommodate and bind variety of natural and synthetic lipophilic acids. Many previous studies have revealed the roles of natural PPAR agonists against specific diseases. For example, natural PPAR agonists such as 15d-PGJ 2 are emerging as important regulators of immunity and inflammation [39][40][41]. 15d-PGJ 2 has also been implicated for antitumor activities [42]. PPARα mediates the anti-inflammatory actions of palmitoylethanolamide, the naturally occurring amide of palmitic acid and ethanolamine [43]. Synthetic PPARγ agonists glitazones have been reported and used in phase I-II human clinical trials as anticancer agents [44].
Present hypothesis is framed on the basis of using natural PPAR agonists that small structural changes in the molecular structure of fatty acids have a great influence on activating different PPARs [45]. Therefore, this hypothesis bridges the concept of natural PPAR agonists and the use of structural information in designing new drugs against diabetes and metabolic syndrome. The derivatives may also be used as anti-inflammatory and anticancer agents.

Competing interests
The author declares no competing interests.
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