Role of Free Fatty Acids in Physiological Conditions and Mitochondrial Dysfunction

The role of free fatty acids (FFAs) as a source of energy and their functions in energy transport within the body are well established. Equally important is a role that FFAs play in oxidative stress following cell membrane depolarization. FFAs are physiologically active, not only as nutritional components, but also as molecules involved in cell signaling and stabilization of membranes via palmitoylation and myristoylation. Protein palmitoylation is involved in numerous cellular processes, including apoptosis, and neuronal transmission. Besides nuclear peroxisome proliferator-activated receptors that mediate the biological effects of FFAs, G protein-coupled receptors (GPCRs) that are activated by FFA, have been recently identified. Those multiple FFA receptors (FFARs), which function on the cell surface as activated FFAs, play significant roles in the regulation of energy metabolism and mediate a wide range of important metabolic processes. FFARs have been targeted in drug development for the treatment of type 2 diabetes and metabolic syndrome. FFAs upregulate transcription of uncoupling proteins, increasing their expression in brain, cardiac, and skeletal muscle that may be protective or cytotoxic, depending on the cellular energy state. Recently, FFA effects on the endothelial function and dysfunction are being recognized. FFAs play a key role in endothelium-dependent nitric oxide production. A disturbance of endothelial function, due to an imbalance in production and release of relaxing and constricting factors, has implications in the development of cardiovascular problems, such as hypertension, as well as neurotoxicity following loss of blood-brain barrier integrity. This review presents information on broad range of FFAs actions of prime importance for physiological processes. Understanding of FFA functions in the body is crucial for developing new therapeutic strategies against several metabolic disorders.


Introduction
In animals, many of the dietary lipids are hydrolysed to FFAs before being absorbed and utilized further for lipid synthesis.Lipids can be hydrolysed to FFAs in tissues by lipolytic enzymes, such as lipoprotein lipase, hormonesensitive lipase, and phospholipases A and C, before they are metabolised in many different pathways, including oxidation, desaturation, elongation and re-esterification [1].
FFAs play an important role as a source of energy for the body.Fat stored in adipose tissue is transported to other tissues in the form of FFAs hydrolyzed from triglycerides and combined with albumin [1].These satu-rated fatty acids (SFA), such as the 18-carbon stearic acid, the 16-carbon palmitic acid, and the 14-carbon myristic acid are also important for various biological functions such as the production of hormones, cellular membrane signaling, and the stabilization processes in the body [2,3].A biochemical process in which the body uses palmitic acid in stabilization processes by regulating G protein-coupled receptor (GPCR) signaling is called palmitoylation [4][5][6].
Protein transporters on the cell surface, including fatty acid translocase (FAT/CD36), tissue-specific fatty acid transport proteins (FATP), and plasma membrane bound fatty acid binding protein (FABP pm ), play an important role in transporting FFAs to the cell.Inside the cell, the thioester, coenzyme A (CoA) is added to the fatty acid by fatty acyl-CoA synthase (FACS), forming long-chain acyl-CoA.
The mitochondrial membrane is impermeable to acyl-CoA that enters mitochondria via the "carnitine shuttle".The first step of this biochemical process is mediated by carnitine palmitoyltransferase 1 (CPT1) that converts acyl-CoA into acylcarnitine.CPT1 binding of the long chain acyl-CoA to long chain acylcarnitine allows the fatty acid to be transported across the inner mitochondrial membrane via facilitated diffusion by carnitine translocase (CAT), which exchanges long chain acylcarnitines for carnitine.The long chain acylcarnitine is then converted back to long chain acyl-CoA by an inner mitochondrial membrane enzyme, carnitine palmitoyltransferase 2 (CPT2).The beta-oxidation pathway of fatty acid and long chain acyl-CoA yields acetyl-CoA that enters the mitochondrial tricarboxylic acid (TCA) cycle.Electron carriers, coenzymes NADH and FADH 2 , supplied by both fatty acid beta-oxidation and the TCA cycle, are used in the electron transport chain to produce energy stored in ATP molecules [1,7,8].

Degradation of Cellular Membranes in Energy Insults
Although FFAs serve in the first instance as an important energy substrate, high concentrations of FFAs are associated with insulin resistance, fatty liver disease, atherosclerosis and myocardial dysfunction.Increased concentrations of FFAs are risk factors for the development of type 2 diabetes [9][10][11].The increased metabolism of FFAs leads via an inhibition of insulin receptor substrate-1 (IRS1) and phosphoinositol 3-kinase to reduced the intracellular effects of insulin [9].Moreover, FFAs lead directly to reduction in the uptake and phosphorylation of glucose and to a reduced glycogen synthesis.FFAs also impair the insulin signal transduction in the liver [9].FFAs play a role in the development of fatty liver disease because 60% of the triglycerides stored in the liver originate from circulating FFAs [12].FFAs are also independent risk factors for the development of arterial hypertension [13].It seems likely that an alpha a 1adrenergic activation may partially be responsible for the link between FFAs and arterial hypertension [13].In addition, FFAs raise the oxidative stress, lead to a reduced production of vasodilatory nitric oxide (NO) and reduce the insulin mediated vasodilation [13].FFAs also stimulate the proliferation of smooth muscle cells of the vessel walls.This causes vascular hypertrophy and vessel-wall rigidity and is associated with the development of arterial hypertension [13].A raised concentration of FFAs with subsequent increased beta-oxidation in the cardiomyocytes can be harmful for the heart in various ways.Comparatively, more oxygen is needed for producing energy through beta-oxidation than through glycolysis.This "oxygen wastage" through beta-oxidation has an extremely negative effect, particularly in diseases that are associated with increased energy requirements by the cardiomyocytes, such as heart failure or acute coronary syndromes [14,15].In addition, FFAs contribute to the development of atherosclerosis and have a proatherosclerotic effect onendothelial cells, macrophages and smooth muscle cells of the vascular walls [16][17][18].

Oxidative Stress in Mitochondrial Dysfunction and FFAs
Reduction in cellular energy production can lead to a significant loss of mitochondrial membrane potential in all cells, including neurons.Energy depletion and cellular membrane depolarization are associated with mitochondrial dysfunction due to ischemia/hypoxia or exposure to toxicants lead to accumulation of intracellular Ca 2+ and excitotoxicity as well as oxidative stress.Glutamate excitotoxicity contributes to oxidative stress through a disruption of reduced glutathione (GSH) synthesis.GSH acts as an endogenous scavenger of reactive oxygen species (ROS).But far more important for ROS generation is the effect of Ca 2+ activation of phospholipases, liberation of FFA and their metabolism to prostaglandins or eicosanoids (Figure 1) [19][20][21][22][23][24][25][26][27].
Mitochondrial dysfunction due to irreversible inactivation of mitochondrial complex II by the mitochondrial inhibitor, 3-nitropropionic acid (3-NPA), results in a decreased ATP/ADP ratio and an increased lactate/pyruvate ratio [20].The concentration of FFAs in all brain regions increases significantly as well [22].An increase in FFAs is followed by production of ROS and activation of endogenous radical oxygen scavengers [28].Increases in FFA levels may be correlated with uncoupling protein concentrations in heart and skeletal muscle tissue [27].
Other studies suggest that FFAs and oxidative stress lead to activation of several transcription factors including activator protein-1 (AP-1), comprised of the proteins c-Fos and c-Jun, and nuclear peroxisome proliferatoractivated receptor-alpha (PPARα) that have a bipotential role in mediating cell regeneration or death depending on conditions of cell plasticity and repair [29,30].
In vitro preliminary studies with SHSY-5Y neuroblastoma cells exposed to 10 and 0 nm ferric oxide (Fe 2 O 3 ) 3 nanoparticles (NPs) showed depletion of dopamine and conditions for oxidative stress [31].Therefore, we examined the response of brain and liver FFAs in adult male Sprague-Dawley rats treated systemically with Fe 2 O 3 -NPs at 25, 50 and 100 mg/kg.Rats were sacrificed 72 h after injection.Concentrations of saturated FFAs (palmitic and stearic acids) in the liver and brain did not change following exposure to iron NPs.However, unsaturated brain FFAs (oleic and linoleic acids) decreased in a dose-dependent fashion in the caudate nucleus (p < 0.05).In the liver, the concentration of unsaturated FFAs increased significantly at 25 and 50 mg/kg (p < 0.05).A link between a long-term regulatory involvement of FFAs and early activation of the inducible transcription factors such as c-Fos and c-Jun proteins (AP1) in a differential response to metabolic stress in brain and liver was suggested [31].
(PPARs) act as sensors of FFAs [28].These receptors maintain homeostasis under physiological and pathophysiological conditions by coordinating the expression of proteins that are involved in the uptake, synthesis, transport, storage, degradation and elimination of lipids.However, some of the biological effects of FFAs may be mediated by alternative mechanisms and may be mediated independently of PPARs.Recently, G protein-coupled receptors (GPCRs) activated by FFAs have been identified as FFAs receptors (FFARs) that function on the cell surface and play significant roles in nutritional regulation [32,33].The FFARs not only act as nutrientsensing receptors, independent of PPARs, but also are known to mediate a wide range of important metabolic processes.These receptors have the ability to monitor FFA levels in the intestine, pancreas, macrophages, and adipose tissue, and are considered to be a major drug target for the treatment of type 2 diabetes due to their role in energy homeostasis [34,35].Relatively small hydrophobic molecules, such as FFAs, serve this signaling purpose.The two most common modifications, myristoylation and palmitoylation, differ with respect to the type and chemical nature of fatty acid attachment to the polypeptide receptor backbone.Myristic acid, a 14-carbon saturated fatty acid, is a source of myristoyl groups utilized within the body to stabilize many different proteins, including proteins in the immune system [36].Myristoyl group is commonly added cotransitionally to the amino terminus of glycine in receptor-associated kinases to confer the membrane localization of the enzyme.Myristoylation occurs in signal transduction via G protein when myristoyl group links the G protein to the inner surface of the plasma membrane allowing the G protein to interact with its receptor [37][38][39][40].Myristoleic acid is biosynthesized from myristic acid by the enzyme delta-9-desaturase.This unsaturated fatty acid acylates an NH 2 terminus of a protein related to signal transduction in photoreceptors in the retina [41].Furthermore, the presence of myristoleic acid is of diagnostic value in patients with defects of longchain fatty acid oxidation [42], and it is known to be cytotoxic to tumor cells.Myristoleic acid induces apoptosis and necrosis in human LNCaP prostate cancer cells [43].

"Active" (Regulatory) Role of FFAs
In contrast, palmitoylated proteins contain the 16carbon saturated fatty acid palmitate, attached via a thioester linkage to one or more cysteine residues.Palmitoylation is a posttranslational reaction that appears to be mediated by a membrane-bound palmitoyl acyl transferase.Unlike myristoylation, which is generally a relatively stable lipid modification, palmitoylation is reversible through the thioester (S-acylation).Since linkage between the palmitate and protein is readily cleaved, cycles of palmitoylation and depalmitoylation occur in a regulated manner for many proteins [44].
The palmitoylation process is involved in multiple diseases, such as degenerative Huntington's disease, various cardiovascular and T-cell mediated immune disorders, and cancer [57].

Effects Induced by Fatty Acids in the Cardiovascular System
Cardiovascular disease (CVD) remains the leading cause of death worldwide, possibly as a result of population growth, increased longevity and exposure to risk factors associated with high blood pressure, obesity, hyperglycemia and hyperlipidemia [58].The severity of cardiovascular problems has led to global concern and consequently to study of the relationship between CVD and dietary fat.Over the years, much research has been focused on the effects and imbalance between SFA, monounsaturated fatty acids, polyunsaturated fatty acids (PUFA), trans-fatty acids (TFA), and most recently, conjugated linoleic acid (CLA).It has become clear that the relationship between total fat intake and CVD is related more to the quality than to the quantity of fat consumed [59].

Endothelium
Endothelium, or intima, of the inner wall of blood vessels is a selectively permeable barrier between the blood and the vascular tissue, whose function is to mediate the integrity and metabolism of vascular wall (i.e., vascular tone and inflammatory response) through synthesis and release of vascular modulators [60].Therefore, disturbance of endothelial function due to an imbalance in production and release of relaxing and constricting factors has implications for the development of CVD [61].
One of the most potent vasodilator agents released from the endothelium is NO, a free radical with an ultrashort half-life and highly versatile functions.NO is synthesized by endothelial nitric oxide synthase (eNOS), an endothelial Ca 2+ -dependent enzyme modulated by palmitoylation and myristoylation processes [62].NO diffuses into smooth muscle cells (SMC) of the blood vessels and promotes vasodilation [63].Some studies have reported a modulating effect of FFAs on endothelium-dependent NO production.Chronic exposure to a 2:1 (w/w) mixture of oleate/palmitate significantly reduces NO production induced by phospholipase C-activating receptor agonists, such as bradykinin and ATP-dependent vasodilators, in bovine aortic endothelial cells [64].In rat aortic endothelial cells, palmitic acid (PA) has been reported to decrease NO bioavailability via the pAKT/PeNOS/NO pathway [65].However, the steroidogenic acute regulatory protein inhibits PAinduced-inflammation and attenuates impairment of NO bioavilability via pAKT/PeNOS/NO pathway.It also regulates the lipid metabolism, decreasing intracellular lipid levels in endothelial cells.Those actions may offer protection against endothelial dysfunction.
Polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) omega-3 fatty acids, are recognized to have beneficial effects against atherosclerosis [66].The incorporation of PUFA in the endothelial membrane phospholipids promotes synthesis of series 3 prostaglandins and series 5 leukotrienes, favoring vasodilation and anti-aggregation [67].It has been recently shown that EPA has cardiovascular-protective effects via activation of protein kinase and up-regulation of the mitochondrial uncoupling protein-2 (UCP-2), leading to an increase in NO production [68].The molecular mechanism underlying UCP-2 upregulation is not known.The nuclear PPAR-gamma (PPARγ) receptors could be involved, since an agonist of PPARγ promoted a significant increase in UCP-2 expression [68].
More cellular and molecular studies at the level of palmitoylation and myristoylation should be conducted as these reactions play a key role in the regulation of the eNOS activity.The identification of those signaling pathways induced by EPA in endothelial cells opens a new avenue for potential drug development in the treatment of atherosclerosis or endothelial dysfunction in human hyperlipidemia.
Endothelial dysfunction and associated inflammatory events are critical for the initiation of cardiovascular diseases such as atherosclerosis.The mechanisms by which selected fatty acids induce endothelial cell activation, oxidative stress, and inflammation are not fully understood.

Vascular Smooth Muscle and Heart
The Ca 2+ channels and large-conductance calcium-activated potassium (BK) channels operate as a functional unit in the regulation of smooth muscle tone in the blood vessels [69].A recent study has shown that DHA metabolites (16, produced in the cytochrome P450 (CYP450) pathway induce vasodilatation through activation of BK channels in vascular smooth muscle cells [70,71].Like DHA, EPA is metabolized by CYP.However, an EPA metabolite, 17,18-epoxyeicosatetranoic acid, activates the vascular BK channels independent of intracellular Ca 2+ concentration or local sarcoplasmic reticulum Ca 2+ release [72].
As in the endothelium, CLA has protective effects on SMC by lowering the eicosanoid release from resting SMC; probably as a result of a reduced cellular pool of amino acids.CLA may compete with LA for the incorporation into the membrane phospholipids, interfering with the production of amino acids, and in consequence with the production of its derivatives [73,74].In activated SMC, the reduction of cytokine-stimulated prostanoid release by cis-9, trans-11-CLA and trans-10, cis-12-CLA is mediated by the PPARγ-dependent inhibition of the NF-κB pathway, which lowers the expression of genes involved in prostanoid synthesis.This action could contribute to the anti-inflammatory and antiatherogenic effects induced by CLA [73,75].
In cardiomyocytes, eNOS is linked with the plasma membrane by the saturated fatty acids, myristic and palmitic acids, and subsequent formation of a heteromeric complex of eNOS with the caveolin-3 protein into the caveolae, which are small indentations in plasma membranes.The co-localization of caveolin-3 and eNOS may facilitate both eNOS activation by cell surface receptors and NO release at the cell surface for intercellular signaling [76].Palmitic acid induces deregulation of cardiovascular homeostasis by a decrease in the caveolin-3 level, and consequently the translocation of eNOS from the plasma membrane to a perinuclear location in the cardiomyocytes, which is related to a decrease of the phosphorylation of eNOS at its Thr495 inhibitory site and decrease of NO production [77].
Apoptosis may play an important role in the pathogenesis of several cardiovascular diseases, such as ischemia, infarction and heart failure [78].Autophagy has been recognized recently as an important modulator of apoptotic cell death [79].Palmitic acid and TFA, such as vaccenic acid and elaidic acid induce apoptosis regulated by autophagy in cardiomyocytes and cardiomyoblasts, whereas EPA protects against palmitate-induced autophagy and apoptosis via the regulation of cardiolipin loss, translocation of Bcl-2-associated X protein (Bax) to the mitochondria, release of cytochrome c, and caspase-3 activation [79][80][81][82].
The mechanisms by which selected fatty acids induce endothelial cell activation, oxidative stress, and inflammation are not fully understood.Further studies will help to identify specific and selective endogenous and exogenous molecules capable of preventing the detrimental effects induced by SFA.

Conclusions
The "passive" role of FFAs as a source of energy, mainly in peripheral tissues but also in brain, has been well recognized.FFAs are established biomarkers of neuronal membrane degradation and have directly toxic effect in the pathogenesis of ischemic-hypoxic brain damage.
The "active" roles of unesterified fatty acids, such as the effects of polyunsaturated fatty acids on up-and down-regulation of gene expression or on FFAs cell surface G coupled receptor myristoylation and palmitoylation, where they help to mediate and modify receptor signaling, are still poorly understood.
The same poor understanding pertains to the active role of FFAs in endothelial function, where they may modulate endothelium-dependent nitric oxide production or mediate inflammatory reactions in endothelial dysfunction.
Understanding mechanisms of FFA actions in the body is crucial for prevention and new drug development for multiple diseases, such as metabolic and degenerative disorders, cardiovascular and T-cell mediated immune disorders, and various forms of cancer.