Unesterified (Free) Fatty Acids

Fatty acids are a major source of energy in cells, they are structural components of membranes, and they are vital for signalling purpose, often as precursors of oxylipins and related mediators, while some essential fatty acids are required in the diet for animal life. In this web document, the properties of unesterified (free) fatty acids are considered collectively and are described in terms of their occurrence and biochemistry mainly as a single lipid class.

Many fatty acids have distinctive biological properties, and indeed the various eicosanoids and other oxylipins are fatty acids and most are only active in unesterified form, but these are so important that are dealt with in separate web pages on this site, i.e., Fatty Acids and Oxylipins, while those in esterified form are described in the remaining web pages in the Lipid Essentials section. Some fatty acids other than oxylipins affect cellular metabolism in unesterified form, and these are discussed briefly below.

1.   Occurrence and Uptake by Tissues

Structural formula of stearic acidFree 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, while 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 and re-esterification.

As free acids can affect 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 these interactions 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 many 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. The bound fatty acids can diffuse from this into the aqueous phase, where they might be rapidly taken up into the outer leaflet of the plasma membrane of epithelial cells before they are internalized.

There are three steps in the widely accepted model for the transport of unesterified fatty acids through cellular membranes that is believed to be the main route for entry of unesterified fatty acids into most epithelial 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, and 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 and lipolysis in which the unesterified fatty acids are liberated.

In further transport mechanisms, long-chain fatty acids are carried by fatty acid transport proteins (FATPs) across membranes, where they are activated rapidly by plasma membrane acyl-CoA synthetase 1 (ACS1) to form acyl-CoA esters. There are six isoforms of these with characteristic tissue distributions. Short-chain fatty acids simply diffuse across the membrane into the cell, and eventually they reach the portal vein for distribution to other tissues. The LDL receptor-related protein 5 (LRP5) transports unesterified polyunsaturated fatty acids selectively into a number of cell types and delivers them to intracellular compartments including lysosomes. In particular, this transport mechanism is required to enable fatty acids of the n‑3 series, i.e., α-linolenic, eicosapentaenoic and docosahexaenoic acids, to inhibit mTORC1, a protein complex that functions as a nutrient sensor to control protein synthesis.

While transport of unesterified fatty acids across membranes of all cell types is important, the process of intestinal absorption has attracted intensive study because of the relatively high concentrations involved. 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 acts in many more ways as a multi-functional scavenger receptor with multiple ligands including cholesterol and phospholipids. As well as its interaction with lipids, it mediates the secretion of intestinal peptides, and it is required for the maintenance of intestinal homeostasis and the integrity of the epithelial barrier.

Fatty acid uptake and metabolism in enterocytes

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 to enable it to work. It is recognized as a virtually ubiquitous constituent of the plasma membrane in all tissues that acts as a surface receptor together with various fatty acid-binding proteins to both facilitate and regulate the entry of fatty acids into cells. Indeed, it is a pivotal membrane protein that is crucial for whole-body lipid homeostasis. As it has been implicated in lipid accumulation in heart and skeletal muscle induced by high fat diets to impact upon insulin resistance and type 2 diabetes, it may be a therapeutic target for the metabolic syndrome.

CD36 is more than simply a fatty acid receptor, and it is widely expressed in many immune cells, both innate and adaptive but especially macrophages and T cells, where it is a signalling receptor that responds to danger-associated and pathogen-associated molecular patterns (DAMPs and PAMPs). As such it can integrate cell signalling and metabolic pathways and thereby influence immune cell differentiation and activation to affect the pathogenesis of diseases such as atherosclerosis and cancer.

Within animal cells, there is evidence to suggest that transporter/binding proteins assist activation of fatty acids to form acyl-coA prior to further esterification but also to ensure vectorial transport so that specific fatty acids are directed to where they are required. A family of fatty acid binding proteins (FABP) are utilized in fatty acid trafficking pathways and in fatty acid activation, with many of these proteins being characteristic of particular tissues and functions, which 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. They diminish the toxicity of unesterified fatty acids by reducing their effective concentrations within cells. In the liver, FABP1 is believed to be critical for fatty acid uptake and intracellular transport and for the regulation of lipid metabolism and cellular signalling pathways, as well as being protective against oxidative damage and other hepatic injuries.

It appears that cells have several overlapping mechanisms that ensure sufficient uptake and directed intracellular movement of the fatty acids required for distinct physiological purposes. In a single organ, there can be many such factors as in the brain, where required levels of polyunsaturated fatty acids must be 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 organelles, and they must be exported for incorporation into lipids in the cytosol and endoplasmic reticulum for structural, signalling and storage reasons 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 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 as far as possible to unmodified fatty acids in general. At high concentrations in vitro, the physical properties of free fatty acids are such that they can cause disruption of membrane structures, but usually, their cytotoxicity comes into play before this can become relevant in vivo.

Signalling: Unesterified fatty acids can act as second messengers required for the translation of external signals, as they are produced rapidly because of binding of certain agonists to membrane receptors in 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 in operating at particular intracellular locations reversibly to amplify or otherwise modify signals, and they are known to influence protein kinases, phospholipases, G-proteins, adenylate and guanylate cyclases, and many other enzymes and metabolic processes. Aside from the oxylipins, there is much evidence that many other fatty acids are messengers that mediate the responses of the cell to extracellular signals. Thus, polyunsaturated fatty acids, including docosahexaenoic and arachidonic acids, stimulate retinoid X receptor (RXRs), which are obligatory heterodimeric receptors, and further receptors as discussed below. Oleic acid is an endogenous ligand for the orphan nuclear receptor TLX/NR2E1, which governs neural stem and progenitor cell self-renewal and proliferation, and it is essential for the survival of neural stem cells; it can block the calcium-activated chloride channel TMEM16A/ANO1 in membranes with the potential for a beneficial impact on the cardiovascular system.

Scottish thistleIn animal tissues after release by lipases, long-chain polyunsaturated fatty acids regulate gene expression, mainly by 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 to form complexes with nuclear receptors for regulatory purposes. The mechanisms by which such 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, 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, of which the PPARγ and hepatocyte nuclear factor 4α (HNF4α) are of great importance.

Polyunsaturated fatty acids of both the (n-6) and (n-3) families may be beneficial by up-regulating the expression of genes encoding enzymes for oxidation of fatty acids, while at the same time down-regulating genes for enzymes for lipid synthesis and 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, are associated with the pathologies of these disease states, and they increase greatly the amounts of oxylipins in tissues such as the pancreas, skeletal muscle and adipose tissue. The interactions between oxylipins and PPARs are discussed in the various web pages dealing with these fatty acid metabolites, although some of the mediator effects appear to be independent of PPARs and are characterized by interactions 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 needed for brain function and development through their interactions with oxylipins. For example, FABP5 is a regulator of synaptic endocannabinoid signalling, while FABP3 and FABP5 interact with epoxyeicosatrienoic acids (EETs) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) through characteristic binding pockets. FABP4 is sometimes termed the adipocyte protein 2 (aP2) and is highly expressed in adipose tissue, where it is a lipid chaperone protein, although it does have a relatively wide-spread distribution among other tissues. In effect, it is 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 is proinflammatory towards 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 are located on the cell surface. Before their ligands were identified, they were first recognized as orphan receptors with a GPR nomenclature. 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 and intestinal cells, where it aids energy homeostasis by sensing circulating medium and long-chain free fatty acids produced by lipolysis of dietary and endogenously synthesised triacylglycerols. Two of the main natural ligands are the omega-6 fatty acid γ‑linolenic acid and the omega-3 fatty acid docosahexaenoic acid (DHA). 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 macrophages, it contributes to the innate immune response.

Scottish thistleShort-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), which stimulate the release of the hormone cholecystokinin, which causes the gallbladder to contract and release bile to aid digestion. In effect, free fatty acids act via these receptors as nutrient sensors to regulate energy homeostasis. FFAR3 and FFAR2 are expressed in many other cell types, including brain and some cancer cells, and other functions are now being revealed; in adipose tissue, activation of FFAR2 inhibits lipolysis and can stimulate adipogenesis (FFAR3 is not present).

FFAR4 (FFA4 or GPR120) has little sequence identity with the other family members, and it is attracting particular interest because of how it affects immune regulation. It is expressed in many different tissues, such as the lower intestine, lung, spleen and adipose tissue, and it is activated most strongly by polyunsaturated fatty acids of the (n-3) family, together with palmitoleic acid, although many other fatty acids are partial agonists at least. It is believed that different types of fatty acids elicit differing responses at a molecular level by changes to effector coupling of FFAR4 to Gi or Giq trimers or to other proteins or fatty acid hormones; aromatic residues within the FFAR4 ligand pocket are then able to recognize the positions of the double bonds. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) causes systemic resistance to chemotherapeutics via this receptor, while effects on inflammatory responses have been noted that are unique to DHA. FFAR4 has been linked to increasing Ca2+ concentrations in cells, but more generally, by interacting with the anti-inflammatory M2 phenotypes of macrophages and with neutrophils, it influences appetite control and such conditions as obesity, type 2 diabetes, cancer and inflammatory diseases.

Lipotoxicity: Excess accumulation of free fatty acids in non-adipose tissues leads to cell disruption and death, a process known as lipotoxicity, and this has been linked to the pathogenesis of various human diseases. 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, inhibiting the anti-lipolytic action of insulin and further increasing the rate of release into the circulation; triacylglycerols that might otherwise be produced when esterification of fatty acids is disrupted are 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 triggered, cellular homeostasis in general is disrupted, and excessive cycles of oxidative phosphorylation occur downstream of β-oxidation to result in generation of reactive oxygen species and oxidative stress.

As a simplistic generalization, saturated and unsaturated fatty acids have opposing actions; saturated fatty acids tend to affect cell metabolism adversely in the main, while oleic acid and polyunsaturated fatty acids are often beneficial. Free saturated fatty acids such as 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, serve to induce fatty acid oxidation and act as feedback inhibitors to limit the synthesis of new fatty acids. Surprisingly, polymethyl-branched fatty acids, such as phytanic and pristanic, can act in this way also. 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 thioesterases.

Scottish thistle Cancer: In most cancers, fatty acid and lipid biosynthesis is extensively reprogrammed with increased expression of fatty acid synthases, FATPs and FABPs. Fatty acids provide substrates for structural purposes and energy production to meet the growth and energy demands of these cells, which proliferate with great rapidity. Free fatty acids affect gene transcription by triggering signals necessary for tumorigenesis, and they enable cancer cells to migrate and generate distant metastasis; high expression of FABP5 in tumours is associated with poor prognosis. By fuelling tumour metastasis and therapy resistance by enhancing fatty acid uptake and accumulation, the transport protein CD36 may be a factor in cancer cells where it can be highly expressed. Similarly, immune cells in the tumour microenvironment overexpress CD36 and undergo metabolic reprogramming with immunosuppressive effects.

Biocidal properties: Free fatty acids have potent antimicrobial, antiviral and antifungal properties, and these have been observed in some living systems, such as the skin and mucosa of the mouth and lung, most often with unsaturated fatty acids, which can insert more readily into membranes. Although they are powerful detergents and will inhibit many enzymes systems in a random manner, it is apparent that the biocidal properties are often dependent on specific fatty acids in particular tissues. For example, sapienic acid (cis‑6‑hexadecenoic), a distinctive constituent of the lipids of human sebaceous glands, disrupts the bacterial plasma membrane while upregulating biosynthesis of proteins involved in the stress response in Gram-positive and Gram-negative organisms.

Plants: Some processes related to those in animals 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. Arachidonic and eicosapentaenoic acids, which do not occur in higher plants, are released from fungi during infection to engage plant signalling networks and induce resistance to the pathogens by eliciting a cascade of responses, including an oxidative burst and the transcription of genes for 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.

3.   Analysis

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 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 identification and quantification purposes.

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