CHEMICAL BIOLOGY

Chemistry of Lipid Homeostasis

Paul N. Black, Whitney Sealls and Concetta C. DiRusso, Center for Metabolic Disease, Ordway Research Institute and Center for Cardiovascular Sciences, Albany Medical College, Albany, New York

doi: 10.1002/9780470048672.wecb284

Lipid metabolism encompasses a large part of intermediary metabolism and includes highly dynamic processes, which are subject to regulation at transcriptional and enzymatic levels. The maintenance of lipid metabolism or lipid homeostasis is multilayered and includes biosynthesis and degradation, the impact of external stimuli proceeding through signal transduction, the function of lipid-responsive transcription factors, and lipid trafficking. It also includes the production of bioactive lipids, which have regulatory roles in different lipid metabolic pathways, effect specific transcription factors, and influence a myriad of cellular processes. Lipid metabolism is responsive to external supplies of fatty acids and other precursors required for neutral lipid, glycerol phospholipid, and sphingolipids synthesis. In some cell types, fatty acid composition within membrane lipids must necessarily adapt to changes in temperature to maintain appropriate fluidity essential for biologic activity. Finally, lipids are diverse in both structure and abundance and differ among animals, plants, fungi, and bacteria. This article will focus on the fatty acid metabolism and, to some extent, its relationship to sterol metabolism, which underpins lipid homeostasis. Readers are referred to other articles in this series (see cross-references below), particularly on steroid and lipid synthesis, chemistry and chemical reactivity of lipids, and lipidomics.

Lipid homeostasis is maintained through a complex network of hormonal, neuronal, and environmental regulators. Inability to modulate lipid metabolism to maintain homeostasis is a hallmark of many disease states, including metabolic syndrome, obesity, diabetes, cardiovascular disease, and some cancers. Together, these diseases are the leading causes of morbidity and mortality in the United States and most other developed countries. It is hypothesized that high circulating levels of free fatty acids, occurring as a result of various metabolic disturbances, lead to excessive fatty acid internalization and the resultant lipotoxicity of normal cells and tissues. Furthermore, free fatty acids and activated fatty acids (fatty acyl-CoAs) are toxic to cells because of their hydrophobic properties, which dissolve membranes. These compounds interfere with enzyme function and, when in excess, serve to increase both oxidation rates and the synthesis of ceramide, which in turn leads to increases in cell death by apoptosis. The correlation between chronically increased plasma free fatty acids and triglycerides with the development of obesity, insulin resistance, and cardiovascular disease has led to the hypothesis that decreases in pancreatic insulin production, cardiac failure, and cardiac hypertrophy are from aberrant accumulation of lipids in these tissues (1).

Biologic Background

Lipids represent a diverse group of compounds, which are readily soluble in organic solvents such as chloroform or toluene and are essential for the structure and function of all living cells. Lipids include oils, fatty acids, waxes, steroids (e.g., cholesterol and steroid hormones), and other related compounds. Specific classes of lipids are the primary structural components of membranes, provide sources of metabolic fuel, and function as bioactive signaling molecules.

Classes of lipids

Fatty acids, the simplest class of lipids, are carboxylic acids containing a long aliphatic tail generally consisting of an even number of carbon atoms, which are either saturated or unsaturated (Table 1). Natural fats and oils generally have fatty acids with at least eight carbon atoms [e.g., caprylic acid (C8:0, n-octanoic acid)]. Saturated fatty acids (SFA) lack double bonds and, as a result, can form straight chains, which can be tightly packed. Common SFAs include butyric (C4:0, n-Butanoic acid), lauric (C12:0, n-Dodecanoic acid), myristic (C14:0, n-Tetradecanoic acid), palmitic (C16:0, n-Hexadecanoic acid), stearic (C18:0, n-Octadecanoic acid), and arachidic (C20:0, n-Eicosanoic acid). Monounsaturated fatty acids (MUFAs) contain one double bond along the chain, which generally occurs in a cis -configuration. Although some monounsaturated fatty acids have a trans-configuration, most found in nature are cis. This cis-configuration results in a fatty acid, which when part of a phospholipid molecule functions to increase membrane fluidity. Common MUFAs include palmitoleic acid (C16:1∆⁹, cis-9-Hexadecenoic acid) and oleic acid (C18:1∆⁹, cis-9-Octadecenoic acid). Polyunsaturated fatty acids (PUFAs) contain two or more double bonds, each in a cis-configuration. These fatty acids include the essential fatty acids in humans, linoleic acid (LA, C18:2 ω6; cis-,cis-9,12-Octadecadienoic acid), and α-linolenic acid (LN, C18:3 ω3; cis-,cis-,cis-9,12,15-Octadecatrienoic acid). Mammals can synthesize both SFAs and MUFAs but lack the enzymes required to introduce a double bond at the ω3 or ω6 position. The ω3 or ω6 fatty acids LA and LN are the precursors to the highly unsaturated fatty acids (HUFAs). The ω6 fatty acid LA is the precursor to the HUFA arachidonic acid (AA, C20:4, ω6; cis-,cis-,cis-,cis-5,8,11,14-Eicosatetraenoic acid), whereas the ω3 fatty acid LN is the precursor to the HUFAs eicosapentaenoic acid (EPA, C20:5 ω3; cis-,cis-,cis-,cis-,cis-5,8, 11,14,17-Eicosapentaenoic acid) and docosahexaenoic acid (DHA, C22:6 to3; cis-,cis-,cis-, cis-,cis-,cis-4,7,10,13,16,19- Docosahexaenoic acid). Essential fatty acids support the cardiovascular, reproductive, immune, and nervous systems, including the production of prostaglandins, which regulate body functions such as heart rate, blood pressure, blood clotting, fertility, and conception, and they are essential in the inflammatory response.

Table 1. Chemical names and descriptions of common fatty acids

Fatty acids C10:0 and longer have considerable detergent properties and are not found free within cells. Most cells contain small intracellular fatty acid-binding proteins (FABPs), which are thought to function in part to buffer the detergent effects of free fatty acids. In the commercial sense, soaps are the water-soluble sodium or potassium salts of fatty acids, which are made from fats and oils by treating them chemically with a strong base such as NaOH or KOH.

Fatty acids can be esterified to glycerol to form diacylglycerol, which can enter the phospholipid biosynthetic pathway or, with the addition of a third fatty acid, can be converted into triacylglycerol (TAG, triglyceride). TAGs represent the predominant storage lipid but are also present in blood plasma and, in association with cholesterol, are components of lipoprotein particles.

Fatty acids are major components of phospholipids, sphingolipids, and cholesterol essters. Phospholipids with a glycerol backbone are termed glycerophospholipids or phosphoglycerides. The major classes of glycerophospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and diphosphatidylglycerol (CL, cardiolipin). Sphingolipids differ from the glycerophospholipids in that they are derived from the aliphatic amino alcohol sphingosine. The sphingosine backbone can be O-linked to a charged head group such as choline. Fatty acids are attached through an amide linkage to sphingosine. Ceramides, sphingomyelins, and glycosphingolipids, which differ in their head groups, are the predominant forms of sphingolipids and function as structural components of membranes and in signal transmission and cell recognition. Finally, fatty acids are stored esterified to cholesterol in lipoprotein particles. Cholesterol is the major sterol found in plasma membranes and, in addition, provides essential functions in lipoprotein metabolism and lipid trafficking and bile acid metabolism. Readers are directed to Vance and Vance (2) and to the section entitled “Lipid Synthesis” in this series regarding the details of phospholipid, sphingolipids, and lipoprotein synthesis, including structures.

Fatty acid and fatty acid derivatives function as signaling molecules

Some of the earliest indications that fatty acids and fatty acid derivatives function as signaling molecules come from studies using the gram-negative bacterium Escherichia coli. The long chain acyl-CoA (LC-CoA) formed as a consequence of coupled transport-activation serves primarily as a substrate for P-oxidation, although it can be incorporated into membrane phospholipids. One particular feature of LC-CoA is that it functions as a bioactive lipid that governs the DNA-binding properties of a specific transcription factor (FadR), which negatively regulates genes required for fatty acid degradation and positively regulates key genes required for fatty acid biosynthesis. Studies summarized by DiRusso and Black (3), which culminated in three crystal structures of FadR, alone in the presence of DNA with the consensus-binding site and in the presence of myristoyl-CoA, clearly defined the bioactive role of LC-CoA as an important effector governing lipid metabolism in E. coli.

In mammalian systems, the HUFAs derived from LA and LN yield an array of compounds with unique roles in a variety of cellular metabolic and signaling events (see “Prostaglandins” in this series). AA-and EPA-derived eicosanoids mediate plieotropic responses including, for example, pain, vasoconstriction, vasodilation, inflammation, and bronchoconstriction. Some HUFA and eicosanoids are ligands for transcription factors, particularly members of the peroxisomal proliferator activated receptor family (PPAR) involved in fatty acid and triacylglyceride metabolism and adipocyte differentiation. Certain classes of highly unsaturated fatty acids also decrease the activity of the Steroid Receptor Element-binding Protein (SREBP) family members, which activate genes required for LDL uptake, cholesterol, and fatty acid synthesis.

Dynamics of Fatty Acid Homeostasis

Lipid homeostasis is maintained through the multilayered regulatory networks of lipid metabolism, transport, and signal transduction. These processes are dynamic and respond to nutritional and environmental cues, which includes interplay between fatty acid synthesis, fatty acid degradation, and complex lipid and cholesterol synthesis, which are influenced by numerous cellular processes. Metabolic output to maintain homeostasis must be quickly adjusted to meet and adapt to the nutritional needs of the cell. In the context of lipid metabolism, it includes well-defined regulatory processes at both the enzyme and transcriptional levels. Inability for a cell or organism to maintain lipid homeostasis has been suggested to cause a number of diseases, including obesity, diabetes, atherosclerosis, and some cancers.

Fatty acid synthesis

Mammalian fatty acid synthesis is a complex process being partly governed by de novo pathways and also requiring essential dietary contributions (Fig. 1a). Quantitatively, de novo mammalian fatty acid synthesis produces primarily palmitate (C16:0) and oleate (C18:1), which constitute the bulk of the side chains of membrane lipids, storage TAGs, and sterol esters. Short and medium chain fatty acids are synthesized in lesser amounts but have some essential functions in cellular metabolism and growth. For example, myristate (C14:0) is added to proteins in posttranslational modification reactions and is often required for membrane association of proteins, many of which are involved in complex signal transduction events. Very long chain fatty acids (VLCFA; ≥C20) are essential components of sphingolipids, glycosyl phosphoinositides, and the lubricating waxes of skin and hair.

The essential dietary fatty acids include linolenic acid (LA, C18:2 ω6) and alpha-linolenic acid (LN, C18:3 ω3). Mammals are missing a fatty acid desaturase capable of introducing a double bond between C9 and the methylene end of the fatty acid. Hence, to synthesize essential HUFAs, LA and LN are both required in the diet (Fig. 1b). LA is the precursor to arachidonic acid (AA, C20:4 ω6) and derivative eicosanoids, whereas LN is the precursor to eicosapentaenoic acid (EPA, C20:5 ω3), docosahexaenoic acid (DHA,C22:6 ω3), and M3-derived eicosanoids, sometimes called resolvins (4). EPA and DHA are required in large quantities during embryonic and neonatal development, particularly for the formation of neuronal tissues (5). Some current evidence indicates the de novo synthetic pathway for DHA and EPA may be too limited to meet the growth and development requirements of infants and dietary supplementation is recommended for nursing mothers and synthetic formulas.

Four enzyme families are required for fatty acid synthesis. The first, acetyl-CoA carboxylase (Acac), synthesizes malonyl-CoA from acetyl-CoA and CO₂. Malonyl-CoA is the substrate for two other protein families, the fatty acid synthases (Fasn) and the fatty acid elongases (Elovl). The length of the acyl chain is determined by the specificity of the terminal reaction catalyzed by the thioesterase active site of Fasn. The mammalian liver Fasn synthesizes primarily palmitate. The palmitate produced is the substrate for two additional enzyme families, the A9-fatty acid desaturases and fatty acid elongases. There are at least three ∆9-fatty acid desaturase isozymes in mammals, Scd1, Scd2, and Scd3, which differ in their tissue distribution and activity (6). At least six members of the fatty acid elongase family have been identified in mammals. Although poorly defined at this time, current evidence suggests the Elos are condensing enzymes as opposed to a multienzyme complex, which carry out the steps to condense and reduce each 2-carbon unit added (7).

The HUFAs (AA, EPA, and DHA), required by mammals, are synthesized by the microsomal elongases and A5 and A6 desaturases (8). The substrates for synthesis, LA and LN, are obtained primarily from vegetable oils in the diet. Elongation and desaturation of LA produces AA, whereas elongation and desaturation of LN produces EPA and DHA. Synthesis of DHA, which is concentrated in the brain, proceeds through the elongation and desaturation pathways to the product C24:6 ω3, which is then the substrate for one cycle of peroxisomal P-oxidation to yield C22:6 ω3 (8).

The liver is quantitatively the most important site of de novo fatty acid synthesis and processing and modification of dietary fatty acids. In liver, fatty acids are incorporated into phospholipids, triglycerides, and cholesterol esters, packaged in very low density lipoproteins, and exported to blood. Uptake of fatty acids in extrahepatic cells requires degradation of the complex lipid into component parts by lipases. Fatty acids are then imported by protein-mediated transport mechanisms involving fatty acid transport protein (FATP), fatty acid translocase (FAT)/CD36, and long chain acyl-CoA synthetase (ACSL) (9). This process is poorly understood, although efforts are underway to define the components and mechanisms mediating the selectivity and efficiency of uptake into cells and tissues (10).

Fatty acids are incorporated into complex lipids through de novo synthetic and remodeling pathways. As detailed below and shown in Fig. 2a, intracellular pools of acyl-CoA are involved in processes outside of lipid metabolism and, in many instances, function as important regulatory molecules. Figure 2b illustrates an overview of glycerol phospholipid synthesis and how fatty acids in the form of acyl-CoA enter these metabolic pathways. Readers are referred to the article entitled “Lipid Synthesis” in this series for specific details regarding these pathways.

Figure 1. Fatty acid synthesis in mammals. Gene encoding enzymes are shown in italics and are based on current evidence for substrate specificities. (A) De novo fatty acid synthesis. (B) Synthesis of the highly unsaturated fatty acids AA, EPA, and DHA from C18:2 ω3 and C18:3 ω3 obtained from the diet. Details are found within the text.

Figure 2. (A) Acyl-CoA metabolism within the cell. Fatty acids must be activated to acyl-CoA before metabolism. As shown, acyl-CoA pools derive from vectorial acylation [import/activation], lipolysis of triglyceride stores, chain turnover from complex lipids, and de novo biosynthesis. Acyl-CoAs are important signaling molecules, incorporated into complex lipids and triglycerides, are substrates for energy production through p-oxidation, and are substrates for protein N-myristoylation and palmitoylation. Acyl CoAs are also subject to chain modification, including desaturation and elongation. Acyl chains in complex lipids serve both structural and regulatory roles. (B) Overview of glycerol lipid metabolism in eukaryotes, with an emphasis on how fatty acids as acyl-CoAs enter these pathways. Glycerol-3-phosphate is converted into phosphatidic acid (PA) through a two-step process, with the intermediate monoacylglycerol phosphate (MAG-P). PA serves in the synthesis of diacylglycerol (DAG) or CDP-diacylglycerol (CDP-DAG). The former is required in the CDP-choline (CDP-C) and CDP-ethanolamine (CDP-E) pathways in the synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). DAG also serves in the synthesis of triacylglycerol (TAG). PE is converted into phosphatidylserine via phosphatidylethanolamine-serine transferase; the reverse reaction requires phosphatidylserine decarboxylase. CDP-DAG is involved in the biosynthesis of phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL). The asterisks show where acyl-CoA is a substrate in the reaction, including the conversion of lysoPC (LPC), lysoPE (LPE), and lysoCL to PC, PE, and CL, respectively. Also shown is the requirement of acyl-CoA for the generation of cholesterol esters (CHO-FA) from cholesterol (CHO).

β-oxidation of fatty acids

Fatty acids represent an important source of metabolic fuel, especially for cardiac and skeletal muscle, and are degraded by β-oxidation. Before β-oxidation, fatty acids are first activated by acyl-CoA synthetase (ACSL). This activation step proceeds through an acyl adenylate intermediate and results in the formation of acyl-CoA. These molecules must first traverse the mitochondrial membrane before further metabolism. Acyl-CoA is converted to acylcarnitine by carnitine palmitoyl transferase 1 (CPT 1) on the inner surface of the outer mitochondrial membrane, which is then transported across the inner mitochondrial membrane by carnitine-acylcarnitine translocase in exchange for carnitine. Carnitine palmitoyl transferase 2 (CPT 2), located on the matrix side of the mitochondrial inner membrane, converts acylcarnitine back to acyl-CoA, which then enters the β-oxidation cycle. During each cycle of β-oxidation, the products are acetyl CoA, NADH, and FADH2. The first step in β-oxidation is an oxidation step, which is catalyzed by acyl-CoA dehyrogenase (ACDH) and yields FADH2. This step is followed by a hydration step catalyzed by enoyl-CoA hydratase and a subsequent oxidation step catalyzed by hydroxyl acyl-CoA dehydrogenase yielding NADH. Acetyl CoA is liberated by β-ketothiolase after cleavage of the bond between the β and γ carbons with the addition of CoA to the shortened fatty acid, which undergoes another round of β-oxidation. Regulation of β-oxidation is largely controlled by the concentration of free fatty acids available. Malonyl Co, a key intermediate in fatty acid biosynthesis, inhibits CPT 1, thereby inhibiting the transport of acyl-CoA into mitochondria, which represents an important metabolic regulatory switch and functions to modulate the fatty acid biosynthetic and fatty acid oxidative pathways in such a manner that these processes will not occur together.

Very long chain fatty acids are initially oxidized in the peroxisome where the initial oxidation step is catalyzed by acyl-CoA oxidase and the subsequent steps in β-oxidation are catalyzed by a multi-enzyme complex with hydratase, dehydogenase, and thiolase activities. Unsaturated fatty acids require additional enzymatic activities, including enoyl-CoA isomerase and dienoyl-CoA reductase. Readers are directed to Vance and Vance (2) for additional details regarding P-oxidation, including the details of the metabolic reactions.

Fatty acid trafficking

Fatty acids are very apolar compounds and readily partition into a lipid bilayer and biologic membrane. Given the expense to synthesize fatty acids, most cell types will take up exogenous fatty acids, which as acyl-CoAs, enter pathways of β-oxidation and complex lipid and TAG synthesis. The concentration of free fatty acids in the circulation, extracellular milieu, and within cells is extremely low as a consequence of their relative insolubility under aqueous conditions. Cells with high levels of fatty acid metabolism (either degradation or storage) transport exogenous fatty acids at higher rates when compared with those with low levels of lipid metabolism. In a number of cell types, fatty acid transport is inducible and commensurate with the expression of specific sets of proteins thought to participate in this process (11). Insulin regulates fatty acid uptake in several tissues, including adipose tissue and muscle (12). Fatty acid transport is protease-sensitive and can be blocked through protein modification and the use of specific antibodies. The blood-brain barrier efficiently accumulates polyunsaturated fatty acids over other fatty acid species (13).

Although several proteins have been designated as plasma membrane transport proteins in mammalian cells, most evidence supports the proposal that only three, FAT/CD36, FATP, and ACSL, have a significant impact on fatty acid uptake and trafficking in the major lipid metabolizing organs (intestine, liver, muscle, and adipose tissue), in the pancreas, and in vascular tissues.

FAT/CD36 is a glycoprotein found on the plasma membrane of a number of different cells types (e.g., skeletal muscle); the protein is also found in intracellular stores and, like Glut4, moves to the plasma membrane in response to insulin. There is some indication that FAT/CD36 deficiency contributes to the etiology of hereditary hypertrophic cardiomyopathy (14). Genetic linkage studies suggest a deficiency of FAT/CD36 is associated with hypertriglyceridemia and hyperinsulinemia in the spontaneously hypertensive rat. The most informative data on FAT/CD36 derives from studies of transgenic overexpressing and knockout mice. The over expression of FAT/CD36 in transgenic mice results in slightly lower body weight than control litter mates, reduced levels of triglycerides (LDL fraction), and elevated levels of circulating fatty acids. Mice with engineered deletions in the gene encoding FAT/CD36 are viable, yet have a significant decrease in binding and uptake of oxidized low density lipoprotein in peritoneal macrophages. These animals also have significant increases in fasting levels of cholesterol (HDL fraction), nonesterified free fatty acids, and triacylglycerol (LDL fraction). Each of these phenotypes is consistent with alteration in lipid trafficking pathways but do not necessarily indicate a deficiency in cellular uptake of free fatty acids.

Fatty acid transport protein 1 (FATP1) and long chain acyl-CoA synthetase 1 (ASCL1) were identified as components of a fatty acid transport system in expression cloning experiments, which specifically targeted cellular fatty acid uptake (15). As both FATP and ACSL1 were selected in these experiments, it was suggested that fatty acid transport occurred by a coupled transport-activation (15). This mechanism was first shown to occur in gram-negative bacteria and is referred to as vectorial acylation (3). Six different isoforms of FATP have subsequently been identified experimentally in mice, rats, and humans (e.g., in mice, mmFATP1-6).

The mouse FATP1 structural gene contains a peroxisome proliferator-activated receptor response element (PPRE) from- 458 to-474. FATP1 expression is induced fourfold and 5.5-fold, respectively, by PPARa and PPARy in the presence of their respective activators in a PPRE-dependent manner (16). FATP1 expression is down regulated by insulin in cultured 3 T3-L1 adipocytes and up regulated by nutrient depletion in murine adipose tissue (17). An insulin response sequence has been identified in the mouse FATP1 structural gene, which is also found in the regulatory region of other genes negatively regulated by insulin, including those encoding phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, and insulin-like growth factor-binding protein 1 (16).

Knockout mice have been generated for FATP1 (18), FATP2 (19), FATP4 (20), and FATP5 (21), which have provided some insights into the roles of these proteins in fatty acid trafficking. Deletion of FATP1 protects animals from developing insulin resistance on a high fat diet and during chronic lipid infusion (18). Animals with a deletion in the gene encoding FATP2 were only evaluated for changes in very long chain acyl-CoA synthetase activity, which was partially diminished, and for accumulation of very long chain fatty acids, which were unchanged. The animals have not been evaluated for fatty acid uptake and no other phenotypes were noted (19). Deletion of FATP4 results in neonatal lethality (20), which is believed to be due to a restrictive dermopathy that prevents expansion of the diaphragm upon birth. Deletion of FATP5 in the mouse results in aberrant bile acid conjugation, which in some manner is linked to the regulation of body weight (21).

Bioactive fatty acids and cellular signaling

Work describing the transcriptional control of fatty acid metabolism in E. coli set the stage showing LC-CoA is an important regulatory molecule. The signaling proceeding through long chain LC-CoA is of particular significance as the transcription factor FadR controls the expression of a significant number of genes involved in fatty acid degradation, fatty acid biosynthesis, acetate metabolism, and the stress response (3). Acyl-CoA is now recognized as an important regulatory molecule that influences a number of different metabolic processes (Fig. 2a). As noted above, fatty acids also serve as important bioactive fatty acids, which impact on a number of cellular processes related to and distinct from lipid homeostasis (22). In mammalian systems, the highly unsaturated fatty acids derived from LA and LN yield a range of compounds with unique roles in a variety of cellular metabolic and signaling events. AA-and EPA-derived eicosanoids mediate plieotropic responses including, for example, pain, vasoconstriction, vasodilation, inflammation, and bronchoconstriction. Some HUFA and eicosanoids are ligands for transcription factors, particularly members of the peroxisomal proliferator activated receptor family (PPAR) involved in fatty acid and triacylglyceride metabolism and adipocyte differentiation. Certain classes of HUFA also decrease the activity of the SREBP family members, which activate genes required for LDL uptake, cholesterol, and fatty acid synthesis (23). Numerous studies have implicated HUFA in the prevention and management of diabetes, cardiovascular disease, and metabolic syndrome, although the mechanisms are largely undefined (24). Current evidence indicates eicosanoids derived from EPA may counter some effects of eicosanoids derived from AA (25). Hence, there is much interest in establishing a balanced ratio of M6:M3 fatty acids, generally recommended to be 2:1 (26, 27).

Lipid-responsive transcription factors and fatty acid homeostasis

The peroxisomal proliferator activated receptors (PPAR) are considered to be internal fatty acid sensors. To date, three distinct PPARs have been discovered, α, γ, and δ, each encoded by a separate gene with distinct tissue specific distributions. PPARs are ligand-activated nuclear transcription factors that heterodimerize with retinoic acid receptor X (RXR). On activation, the PPAR/RXR heterodimer enters the nucleus to elicit transcription of a set of target genes that contain a PPAR response element termed PPRE in their promoter (28). PPARα is primarily found in the liver and is involved in regulating the genes involved in fatty acid catabolism whereas PPARγ is primarily involved in the adipocyte differentiation and fatty acid storage. PPARδ has a wide range of tissue distribution, but its specific role in the regulation of fatty acid metabolism in the liver has not been well elucidated. The natural ligands for the PPARs are ω3 and ω6 PUFAs, HUFAs, and fatty acid-derived metabolites (e.g., leukotriene B₄ and 8(S)-hydroxy-eicosatetranoic acid) (29, 30). Recent work has shown that very long chain fatty acids, including C20:4 ω-6 and C20:5 ω-3, C22:6 ω-3, and branched-chain fatty acids are potent activators of PPARa (31). Although there is some disagreement whether PUFAs or HUFAs are more potent PPAR agonists, HUFAs are more potent toward PPARa (32). PPARa regulates genes involved in mitochondrial β-oxidation, including those encoding straight-chain acyl-CoA dehydrogenase (ACDH) and carnitine palmitoyl transferase (CPT1 and 2). PPARa also regulates key peroxisomal genes, including those encoding acyl-CoA oxidase (AOX), L-bifunctional protein, and 3-ketoacyl-CoA thiolase (33), involved in the β-oxidation of very long chain fatty acids (VLCFAs), which is required in the synthesis of DHA.

Sterol regulatory element-binding proteins (SREBP1a, SREBP1c, and SREBP2) are transcription factors that control both cholesterol and fatty acid biosynthesis. SREBP-1c regulates genes involved in fatty acid synthesis and storage, whereas SREBP-2 regulates genes involved in cholesterol and bile acid synthesis. SREBP-1a is thought to regulate both fatty acid and cholesterol biosynthesis. SREBPs are regulated by intracellular oxysterol levels such that when elevated, their proteolytic cleavage and subsequent activation is prevented. SREBPs serve to balance PPAR-regulated fatty acid oxidation. PUFAs and HUFAs are believed to suppress fatty acid synthesis by decreasing the activities of SREBP1c and LXR (34). The active form of SREBP is negatively regulated by PPAR, PUFAs, and HUFAs (22, 35, 36). In addition, SREBP transcription is controlled by LXR demonstrating crosstalk between PPARs and LXR (35). More specifically, PPAR competes with LXR for their shared heterodimer partner, RXR, thereby decreasing LXRs activity. The balance between fatty acid biosynthesis and breakdown is important in many disease states and all of the above-mentioned transcription factors play a role in maintaining this balance.

Chemical Tools and Techniques For Studying Lipid Metabolism

Extraction of lipids and the foundations for current analytical studies

The fundamental step in all analytical studies on lipids and lipid metabolites begins with extraction methods. Two papers, Folch et al. (37) and Bligh and Dyer (38), are routinely cited methods describing lipid extraction and should be consulted as general guides. Details of these methods can be found in the Lipid Library (http://www.lipidlibrary.co.uk/topics/extract/index.htm) and in Cyberlipids (http://www.cyberlipid.org/extract/extr0002.htm).

Thin layer and high performance liquid chromatography

Early studies employed thin layer chromatography (TLC) in the separation, identification, and quantification of individual classes of lipids. These techniques have been optimized for different classes of glycerol phospholipids, neutral lipids, sphingolipids, and fatty acids. Methods and general references to TLC can be found in the Lipid Library (http://www.lipidlibrary.co.uk/topics/tlc/index.htm) and in Cyberlipids (http://www.cyberlipid.org/fraction/frac0005.htm#top). High performance liquid chromatography (HPLC) has now become routine in the separation and quantification of lipids. Given the optical properties of lipids, the use of evaporative light-scattering detectors is most optimal for detection and quantification. The most comprehensive information on the use of HPLC in analytical lipid studies can be found at Cyberlipids (http://www.cyberlipid.org/index.htm).

Gas chromatography and mass spectrometry

The identification of individual classes of fatty acids has relied on the use of gas chromatography (GC), equipped with a flame ionization detector. Lipids are saponified after extraction and the fatty acids converted to methyl esters. The fatty acid methyl esters (FAME) are separated using GC. The use of standards allowed for the identification of individual species of fatty acids based on retention time. The method is quite sensitive and permits the quantification of fatty acid species. The mode of detection has been enhanced with the use of mass spectrometry (MS), which allows for the detection and quantification of unknowns, thus increasing the utility of these methods. The Lipid Library section on the gas chromatography of lipids (http://www.lipidlibrary.co.uk/GC_lipid/01_intro/index.htm) provides a comprehensive description of these methods.

Fluorescent fatty acids using fatty acid trafficking and signaling

A number of different fluorescent fatty acids are in use to investigate various aspects of fatty acid homeostasis. Among these are the C18, C16, and C12 fatty acid-5-aminofluorescein conjugates octadecoylaminofluorescein (OAF), hexadecoylaminofluorescein (HAF), and dodecoylaminofluorescein (DAF), which have been used to study membrane dynamics in gram-negative bacteria. Studies on the intracellular fatty acid-binding proteins have employed anthroloxy-labeled fatty acids, whereas biophysical studies addressing fatty acid transport have used the naturally occurring cis-paranaric acid. Many of the current studies addressing fatty acid trafficking use BODIPY (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene)-labeled fatty acids, which has been adapted for high throughput technologies to select for small molecules that inhibit one or more steps in fatty acid trafficking (39). The BODIPY-derived fatty acid molecules can be visualized using fluorescence microscopy, which provides a tool to monitor intracellular trafficking.

Research Directions—2007

High resolution studies using LC-MS/MS

The identification and quantification of lipids, particularly minor species that are likely to have bioactive roles, has lagged behind their hydrophilic counterparts. Understanding these minor lipid compounds is essential to have a complete picture of all of the factors involved in governing lipid homeostasis. With the advent of developing analytical tools that combine HPLC with tandem mass spectrometry, we are now in a position to acquire a significant and novel body of information on complex lipids and lipid metabolites, and to determine how these compounds fit into larger metabolic schemes essential for addressing issues of health and disease.

Lipid maps

The emerging field of metabolomics is defined as the study of metabolites and metabolic interrelationships and encompasses the global analysis of the plethora of small molecules generated in the process of metabolism. To effectively use this information for understanding both normal physiology and pathophysiology, we must first have a firm grasp on the interrelationships between metabolic enzymes and the pathways in which they participate. The study of the metabolic intermediates of lipid metabolism and the enzymes participating in these metabolic pathways comprise the field of lipidomics (see “Lipidomics” in this series). A new resource in lipidomics is the Lipid MAPS (Metabolites And Pathways Strategy) consortium (http://www.lipidmaps.org/), which has been established to aid investigators. Lipid MAPS serves 1) to separate and detect all of the lipids in a specific cell and discover and characterize any novel lipids that may be present, 2) to quantify each of the lipid metabolites present and the changes in their levels and location during cellular function, and 3) to define the biochemical pathways for the synthesis of each lipid and develop lipid maps that define the interaction networks (40). This consortium is directed to finding and cataloging the myriad of lipid compounds, including those that are less abundant, those that are transient intermediates in the metabolic pathways, and those with pronounced biologic activity. The consortium is establishing a comprehensive database available to researchers addressing questions within the lipidomics field (e.g., see Reference 41). Independent researchers and smaller research consortia of investigators with common interests in lipidomics, such as those described within this proposal, can access these databases and, as importantly, can provide information to be cataloged within Lipid MAPS.

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Further Reading

Vance DE, Vance JE. Biochemistry of Lipids, Lipoproteins and Membranes, 4th edition. 2002 Elsevier, New York.

See Also

Lipidomics

Lipid Bilayers, Properties of

Lipid Synthesis

Lipid Signals

Chemistry and Chemical Reactivity of Lipids

Lipoproteins, Chemistry of

Mass Spectrometry: Small Molecules

Metabolism, Cellular Organization of