Unesterified (Free) Fatty Acids
The nature and properties of individual fatty acids are discussed in our web pages dealing with Fatty Acids and Oxylipins, while those in esterified form are described in most of the remaining web pages in the Lipid Essentials pages. In brief, fatty acids are a major source of energy in cells, they are key components of membranes and they have vital signalling functions, often as precursors of oxylipins and related mediators; some essential fatty acids are required in the diet for animal life. However, in this web document, the properties of unesterified (free) fatty acids collectively as a single lipid class are described.
1. Occurrence and Uptake by Tissues
Free or unesterified fatty acids are ubiquitous if minor components of all living tissues. In animals, much of the dietary lipid is hydrolysed to free acids before it is absorbed and esterified as in lipid synthesis. Intact lipids in tissues can be hydrolysed to free acids by a variety of lipolytic enzymes (e.g. lipoprotein lipase, hormone-sensitive lipase, phospholipase A1, phospholipase A2), depending on the lipid class and tissue, before being metabolized in various ways including oxidation, desaturation, elongation or re-esterification. The various eicosanoids and other oxylipins are also fatty acids and exist mainly in unesterified form, but they have individual and distinctive properties that are dealt with in separate web pages on this site.
As free acids can interact with a wide range of enzyme systems in both specific and non-specific ways, they must be rapidly sequestered in tissues by various means to ensure that their activities are closely regulated. Monomeric fatty acids in the free state have very low solubilities in aqueous media, and normal concentrations in cells would be expected to be in the low nM range. In serum, they are transported between tissues bound to the protein albumin, which has up to six strong binding sites and a large number of weak binding sites where non-polar interactions are possible between the fatty acid hydrocarbon chains and uncharged amino acid side chains. In this way, the concentration of a long-chain fatty acid in serum can be increased by as much as 500 times above its normal maximum. However, the bound fatty acids can diffuse into the aqueous phase, where they might be rapidly taken up into the outer leaflet of the plasma membrane by a non-enzymatic mechanism.
There are three steps in the widely accepted model of the transport of unesterified fatty acids through cellular membranes that is believed to be the main route for entry of unesterified fatty acids into cells across the plasma membrane. These are adsorption of the fatty acid into the outer leaflet of the membrane, translocation across the membrane (“flip-flop”), and subsequent desorption into the cytosol; the last step is rate-limiting with model membranes in vitro. No receptor or transporter molecule is required. Control over this cross-membrane traffic is regulated by the balance between intracellular triacylglycerol synthesis from long-chain fatty acids in fat droplets, for example, and lipolysis in which the unesterified fatty acids are liberated from fat droplets.
Further mechanisms are believed to exist in specific tissues. While transport of unesterified fatty acids across membranes of all types of cell is important, the process of intestinal absorption has attracted intensive study because of the relatively high concentrations involved. Two main mechanisms are believed to operate. A fatty acid translocase (CD36 or scavenger receptor class B type 2 (SR-B2)) on its own or together with the peripheral membrane protein 'plasma membrane-associated fatty acid-binding protein' (FABPpm; 43 kDa) accepts medium-chain and long-chain fatty acids at the enterocyte surface to transport fatty acids across the apical membrane before these are bound by cytoplasmic FABP (FABPc); this enables them to enter the cellular metabolic pathways. Together, these proteins have a crucial role in chylomicron formation, assembly and trafficking from the endoplasmic reticulum to the Golgi, but CD36 has many additional functions as a multi-functional scavenger receptor with multiple ligands including cholesterol and phospholipids. For example in addition to its interaction with lipids, it mediates the secretion of intestinal peptides and it is important in the maintenance of intestinal homeostasis and the integrity of the epithelial barrier.
CD36 is a 75 to 88 kDa protein, depending on the extent of glycosylation, with two trans-membrane domains, and it has palmitoylation sites on both the N-terminal and C-terminal (cytoplasmic) tails of the protein, which anchor it in the membrane in order to function. It is recognized as a virtually ubiquitous constituent of the plasma membrane in all tissues and together with various fatty acid-binding proteins both facilitates and regulates the entry of fatty acids into cells. Indeed, it is considered to be a pivotal membrane protein that is involved in whole-body lipid homeostasis. As it has been implicated in lipid accumulation in heart and skeletal muscle induced by high fat diets with effects upon insulin resistance and type 2 diabetes, it is considered to be a promising therapeutic target for the metabolic syndrome. Similarly, by fueling tumor metastasis and therapy resistance by enhancing fatty acid uptake and oxidation, CD36 may be a factor in cancer. However, it should be recognized that efforts to inhibit its activity may limit the uptake of essential fatty acids.
In a second transport mechanism, medium- and long-chain fatty acids are carried by fatty acid transport protein 4 (FATP4) across the apical membrane, where they are activated rapidly by plasma membrane acyl-CoA synthetase 1 (ACS1) to form acyl-CoA esters. Short-chain fatty acids simply diffuse across the membrane into the cell and eventually they reach the portal vein for distribution to other tissues.
Within cells, there is evidence to suggest that specific transporter proteins are involved in part to activate fatty acids by formation of acyl-coA prior to further esterification, but also to ensure vectorial transport so that specific fatty acids are directed towards particular purposes. Certainly within the cell, a family of fatty acid binding or transport proteins has essential functions in fatty acid trafficking pathways and in fatty acid activation, with many of these proteins being characteristic of particular tissues. These functions include uptake of dietary fatty acids in the intestine, targeting of fatty acids in the liver to either catabolic or anabolic pathways, regulation of storage in adipose tissue, targeting to β-oxidation pathways in muscle, and maintenance of phospholipid compositions in neural tissues. For example, Fatty Acid-Binding Protein 1 (FABP1) from liver is believed to be critical for fatty acid uptake and intracellular transport; it is also important in regulating lipid metabolism and cellular signalling pathways, as well as being protective against oxidative damage and other hepatic injuries. In addition, fatty acid binding proteins diminish the toxicity of unesterified fatty acids by reducing their effective concentrations within cells. It appears that cells have several overlapping mechanisms that ensure sufficient uptake and directed intracellular movement of the fatty acids required for their physiological functions.
In a single organ, many such factors can be involved and for example in the brain, it is essential that the levels of polyunsaturated fatty acids are maintained. This requires a complex interplay of fatty acids transport proteins that include fatty acid binding proteins, long chain acyl-coA synthases, fatty acid translocase (CD36) and a recently described major facilitator superfamily domain-containing protein (Mfsd2a), which is specific for docosahexaenoic acid (DHA).
In plants, fatty acids are synthesised in plastids, the photosynthetic organelle, and they must be exported for incorporation into lipids in the cytosol and endoplasmic reticulum for structural, signalling, storage and so forth in other tissues of the plant. So far, one protein has been identified in the chloroplast inner envelope with α-helical membrane-spanning domains and designated FAX1 (fatty acid export 1), which mediates this transport.
2. Biochemical Function of Unesterified Fatty Acids
Unesterified (free) fatty acids are released into plasma from the triacylglycerols of adipose tissue, and are thence transported to other tissues where they are utilized as a source of energy (see our web page on acylcarnitines, for example), for the synthesis of structural and storage lipids, or for the biosynthesis of lipid mediators. These processes are the subject of most of the web pages in the Lipid Essentials section of this website, and the following discussion is limited largely to unmodified fatty acids.
Signalling functions: Unesterified fatty acids can act as second messengers required for the translation of external signals, as they are produced rapidly as a consequence of the binding of specific agonists to membrane receptors in particular tissues and organelles, especially cytoplasmic droplets, which can then activate lipases selectively. In this way, they can substitute for the second messengers of some of the inositide pathways. Fatty acids are effective also in operating at specific intracellular locations reversibly to amplify or otherwise modify signals. For example, they influence the activities of protein kinases, phospholipases, G-proteins, adenylate and guanylate cyclases, and many other metabolic processes. Part of the action of fatty acids may occur indirectly via metabolism of arachidonic acid to eicosanoids. On the other hand, there is much evidence that fatty acids per se are messengers that mediate the responses of the cell to extracellular signals. Many of these reactions are specific to particular fatty acids. Thus, polyunsaturated fatty acids, including docosahexaenoic and arachidonic acids, activate retinoid X receptor (RXRs), which are obligatory heterodimeric receptors, and many other receptors including those discussed below.
In addition in animal tissues after release by lipases, long-chain polyunsaturated fatty acids are involved in regulating gene expression, mainly targeting genes that encode proteins with roles in fatty acid transport or metabolism. Fatty acid-binding proteins bind long-chain fatty acids with high affinity in the cytoplasm and transport them to nuclei, which they enter via the nuclear pores where they are able to form complexes with nuclear receptors to enable them to regulate receptor activation. The mechanisms by which modulation of gene transcription occurs are only partially resolved, and this is the subject of considerable research effort, especially with respect to the family of transcription factors, i.e. peroxisome proliferator-activated receptors (PPARs), in the nuclei of cells. The effects can be highly specific, different fatty acids binding to or activating different types of PPAR, although the PPARγ and hepatocyte nuclear factor 4α (HNF4α) are especially important.
In particular, polyunsaturated fatty acids of both the (n-6) and (n-3) families may exert beneficial effects by up-regulating the expression of genes encoding enzymes involved in oxidation of fatty acids, while at the same time down-regulating genes for enzymes involved in lipid synthesis. They also influence glucose metabolism. As a result, unesterified fatty acids may mitigate the undesirable symptoms of the metabolic syndrome and may even reduce the risk of heart disease. In contrast, abnormal PPAR activation can be a factor in the lipotoxicity observed with obesity, insulin resistance, type 2 diabetes and hyperlipidemia. Abnormally elevated levels of non-esterified fatty acids in plasma, for example, are associated with the pathologies of these disease states. Also, they increase greatly the amounts of key bioactive lipids in tissues such as the pancreas, skeletal muscle and adipose tissue. Eicosanoids interact with PPARs strongly also, but this is discussed in the various web pages dealing with these fatty acid metabolites.
However, some of the mediator effects appear to be independent of PPARs and are characterized by involvement with cell surface receptors instead. In adult brains, the fatty acid-binding protein-3 (FABP3) may assist in consolidating and maintaining the differentiated status of neurons through selective use of polyunsaturated fatty acids, while FABP5, FABP7 and FABP8 are similarly involved in brain function and development. FABP4 is also known as adipocyte protein 2 (aP2) and is highly expressed in adipose tissue, where it functions as a lipid chaperone protein, although it does have a relatively wide-spread distribution among other tissues. It is also considered to be a secreted hormone that is transported in serum and has roles in maintaining glucose homeostasis by facilitating communication between adipocytes and more distant organs. The levels of FABP4 in the circulation have been correlated with the incidence of metabolic disease, and reduced concentrations are associated with improved metabolic health.
Palmitic acid has been shown to modulate autophagy via a secondary signalling pathway, for example in hepatic steatosis, a condition caused by high amounts of fat in the liver that result in lipotoxicity. In general, signalling by exogenous saturated fatty acids exerts proinflammatory effects on dendritic cells and macrophages.
G protein-coupled receptors for free fatty acids (FFAR): Four main receptors of this type (FFAR1 to FFAR4) have been identified that function on the cell surface and have important roles in the regulation of metabolism and immunity in many different ways, for example for the regulation of inflammation and the secretion of peptide hormones. Before their ligands were identified, they were first recognized as orphan receptors ('GPR'). Some of these are activated by short-chain and others by medium- and long-chain free fatty acids, and the presence of the unesterified carboxyl group is essential. FFAR1 (FFA1 or GPR40) is strongly expressed in pancreatic β-cells, where it is activated by a broad range of medium- to long-chain fatty acids, but especially by long-chain polyunsaturated acids and not by those with trans-double bonds. In the pancreas, FFAR1 enhances glucose-dependent insulin secretion on exposure to these fatty acids. Although FFAR1 is expressed to a lesser extent in brain, it is activated by a wide range of polyunsaturated fatty acids and is involved in neurodevelopment and neurogenesis and in some neuropathological conditions, including inflammatory pain, Alzheimer's disease and Parkinson's disease. In intestinal cells, FFAR1 functions as a nutrient-sensing receptor detecting dietary fats as they are converted to the free acids and are absorbed into the tissue.
Short-chain fatty acids, such as acetate, propionate, butyrate and valerate produced by the gut microbiota, are ligands for FFAR3 (FFA3 or GPR41) and FFAR2 (FFA2 or GPR43). When activated, these stimulate the release of the hormone cholecystokinin, which in turn causes the gall-bladder to contract and release bile to aid digestion. In effect, free fatty acids act via these receptors as nutrient sensors to regulate energy homeostasis. However, FFAR3 and FFAR2 are expressed in many other cell types, including brain and some cancer cells, and other functions are now being revealed. For example in adipose tissue, activation of FFAR2 (FFAR3 is not present) inhibits lipolysis and can stimulate adipogenesis.
FFAR4 (FFA4 or GPR120) has little sequence identity with the other family members. It is attracting particular interest as it is expressed in many different tissues, but especially the lower intestine, lung, spleen, and adipose tissue, and is activated most strongly by polyunsaturated fatty acids of the (n-3) family, i.e. α-linolenic, eicosapentaenoic and docosahexaenoic (DHA) acids together with palmitoleic acid, although many other fatty acids are partial agonists at least. It is possible that different types of fatty acids elicit differing responses, although how this is accomplished at a molecular level is not yet known. For example, the platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to chemotherapeutics via this receptor, while effects on inflammatory responses have been noted that are unique to DHA. More generally, by interacting with the anti-inflammatory M2 phenotypes of macrophages, FFAR4 influences appetite control and such conditions as obesity, type 2 diabetes, cancer and inflammatory diseases. It has also been linked to increasing Ca2+ concentrations in cells.
Lipotoxicity: Excess accumulation of free fatty acid in non-adipose tissues leads to cell dysfunction and cell death, a process known as lipotoxicity, and this has been linked to the pathogenesis of various human diseases. In particular, plasma levels of free fatty acids are usually elevated in obesity because the increased mass of adipose tissue can release more while their clearance may be reduced. Elevated plasma free fatty acid levels in turn inhibit the anti-lipolytic action of insulin, further increasing the rate of release into the circulation. Triacylglycerols that might otherwise be produced when esterification of fatty acids is disrupted are considered to be inert and non-toxic. Similarly, if excess fatty acids cannot be oxidized in mitochondria, remodelling of membranes leads to organelle dysfunction including stress in the endoplasmic reticulum, while signalling cascades are activated, cellular homeostasis in general is disrupted, and excessive cycles of oxidative phosphorylation occur downstream of β-oxidation, resulting in generation of reactive oxygen species and oxidative stress. The physical properties of unesterified fatty acids are such that they cause disturbance to membrane structures.
As a simplistic generalization, saturated and unsaturated fatty acids have opposing actions; saturated fatty acids are associated mainly with adverse effects on cell metabolism, while oleic acid and polyunsaturated fatty acids tend to have beneficial effects. Free saturated fatty acids, especially palmitic acid, influence metabolic pathways that promote steatosis and affect cholesterol metabolism. In contrast, polyunsaturated fatty acids of the (n-3) family, which are more potent than those of the (n-6) family in this respect, function as activators of fatty acid oxidation and as feedback inhibitors that limit the synthesis of new fatty acids. Surprisingly, polymethyl-branched fatty acids, such as phytanic and pristanic, may also have a function of this kind. In some circumstances, both the free acids per se and their coenzyme A esters may be involved, and the balance between the two may be regulated by the activity of thioesterases.
In relation to cancer, fatty acid and lipid biosynthesis is extensively reprogrammed. Fatty acids provide substrates for energy production, and this is of great importance to meet the energy demands of these cells, which proliferate with great rapidity; they also require a considerable supply of fatty acids for structural purposes. Fatty acids are involved also in gene transcription as they trigger signals necessary for tumorigenesis, and enable cancer cells to migrate and generate distant metastasis.
Biocidal properties: Free fatty acids have potent antimicrobial, antiviral and antifungal properties, and they exert such effects in some living systems, especially the skin and mucosa of the mouth and lung. Unsaturated fatty acids seem to have the greatest effects, but this may be in part because they can insert more readily into membranes. Although they are powerful detergents and will inhibit very many enzymes systems in a non-specific manner, it is apparent the biocidal properties are often dependent on specific fatty acids in particular tissues. For example, sapienic (cis‑6‑hexadecenoic) acid, a distinctive constituent of the lipids of human sebaceous glands, is active against Gram-positive and Gram-negative organisms by disrupting the bacterial plasma membrane while upregulating biosynthesis of proteins involved in the stress response. At high concentrations in vitro, free fatty acids are known to perturb membrane structures, but cytotoxic effects come into play before this can become relevant.
Plants: Some related processes appear to occur in plants. When infected by bacteria and fungi, plants recognize molecular signatures including particular fatty acids, which can trigger a form of immunity that assists in resisting the infection. For example, arachidonic and eicosapentaenoic acids, which do not occur in higher plants and are released during fungal infection, engage plant signalling networks to induce resistance to the pathogens. They elicit a cascade of responses, including an oxidative burst and the transcriptional activation of genes involved in the hypersensitive response. Similarly, in bacteria, it has been demonstrated that the bacterial fatty acid transport and trafficking system leads to fatty acid-responsive regulation of gene expression.
Accurate measurement of free fatty acids concentrations in plasma and tissues can be a useful measure of metabolic status. Unfortunately, it is very easy to generate free acids artefactually by faulty storage or extraction. Lipases can continue to function slowly in some tissues, even at -20°C, and the process will accelerate if tissues are allowed to thaw prior to extraction. In one important series of experiments, it was shown that if animal tissues were pulverized and extracted at -70°C, very low levels only of free fatty acids were detected in comparison to more conventional procedures [Kramer, J.K.G. and Hulan, H.W. J. Lipid Res., 19, 103-106 (1978)]. High concentrations reported occasionally in the literature are obviously impossible in living tissues and are due to inappropriate sample handling. Following extraction of lipids from tissues by a suitable procedure, a free fatty acid fraction can be isolated by thin-layer chromatography or by solid-phase extraction chromatography on a bonded amine phase. This can be methylated for analysis by gas chromatography with an internal standard for quantification purposes.
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