Lipid Biosynthesis

CHAPTER 21 LIPID BIOSYNTHESIS Lipids are the principal form of stored energy in most higher organisms, as well as the major constituents of membranes. Anabolism is not simply the reverse of catabolism. Biosynthetic pathways typically diverge from breakdown pathways to overcome irreversible steps in catabolism. Like other anabolic pathways, the reaction sequences in lipid biosynthesis are endergonic and reductive. They use ATP as a source of metabolic energy and a reduced electron carrier (usually NADPH) as a reductant. Lipid biosynthesis, like other anabolic pathways, is subject to regulation to respond to cellular and organismal requirements. The places where catabolic and anabolic pathways diverge (Principle 2) provide opportunities to impose metabolic regulation to conserve resources and avoid futile cycles. Like other major classes of biological molecules, lipids have a plethora of cellular functions. Specialized lipids serve as pigments (retinal, carotene), cofactors (vitamin K), detergents (bile salts), transporters (dolichols), hormones (vitamin D derivatives, sex hormones), extracellular and intracellular messengers (eicosanoids, phosphatidylinositol derivatives), and anchors for membrane proteins (covalently attached fatty acids, prenyl groups, phosphatidylinositol). The ability to synthesize a variety of lipids is essential to all organisms. We will focus on eukaryotes, with occasional digressions to highlight important distinctions in bacteria and plants. 21.1 Biosynthesis of Fatty Acids and Eicosanoids Even when compared to other major classes of metabolites, the division between fatty acid biosynthesis and breakdown is particularly striking. The two processes occur by different pathways, catalyzed by different sets of enzymes, and, in eukaryotes, occur in different cellular compartments. Fatty acid breakdown occurs in the mitochondria, whereas biosynthesis occurs in the cytosol. Moreover, biosynthesis requires the participation of a three-carbon intermediate, malonyl-CoA, that does not appear in the path of fatty acid breakdown. The general pathway of fatty acid synthesis and its regulation now take center stage. We consider the biosynthesis of longer-chain fatty acids, unsaturated fatty acids, and their eicosanoid derivatives at the end of this section. Malonyl-CoA Is Formed from Acetyl- CoA and Bicarbonate The formation of malonyl-CoA from acetyl-CoA is an irreversible three-step process, catalyzed by acetyl-CoA carboxylase. In animal cells, all three steps are catalyzed in the cytoplasm by a single multifunctional polypeptide (Fig. 21-1). The enzyme contains a biotin prosthetic group covalently bound in amide linkage to the ε -amino group of a Lys residue in one of the domains of the enzyme molecule. The reaction catalyzed by this enzyme is very similar to other biotin-dependent carboxylation reactions, such as those catalyzed by pyruvate carboxylase (see Fig. 16-16) and propionyl-CoA carboxylase (see Fig. 17-12). A carboxyl group, derived from bicarbonate (HCO− 3), is first transferred to biotin in an ATP-dependent reaction. In a second step, the carboxyl group is carried by the biotin to a different active site, where the CO2 is transferred to acetyl-CoA in the third and final step to yield malonyl-CoA. As we will see, this carboxylation has the same function as the carboxylation of pyruvate by pyruvate carboxylase — it renders the next step in the reaction sequence much more favorable thermodynamically.

FIGURE 21-1 The acetyl-CoA carboxylase reaction. The mammalian acetyl- CoA carboxylase of the cytoplasm has three functional domains with distinct functions: biotin carrier protein; biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction; and transcarboxylase, which transfers activated CO2 (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA. Part of the biotin carrier protein domain and the long, flexible biotin arm rotate to carry the activated CO2 from the biotin carboxylase active site to the transcarboxylase active site. The active domain in each step is shaded in blue. The bacterial version of acetyl-CoA carboxylase is similar but has three separate polypeptide subunits (including a separate biotin carrier protein) that catalyze the three steps. Plant cells contain both types of acetyl-CoA carboxylase. Fatty Acid Synthesis Proceeds in a Repeating Reaction Sequence The long carbon chains of fatty acids are assembled in the cytosol in a repeating four-step sequence (Fig. 21-2), catalyzed by a system collectively referred to as fatty acid synthase. A saturated acyl group produced by each four-step series of reactions becomes the substrate for subsequent condensation with an activated malonyl group. With each passage through the cycle, the fatty acyl chain is extended by two carbons.

FIGURE 21-2 Addition of two carbons to a growing fatty acyl chain: a four- step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl- CoA, with elimination of CO2 from the malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β -keto product of the condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: the β -keto group is reduced to an alcohol, elimination of H2O creates a double bond, and the double bond is reduced to form the corresponding saturated fatty acyl group. In the four-step pathway, a condensation reaction is followed by a reduction-dehydration-reduction sequence to convert the C-3 carbonyl to a methylene. The latter three steps are the chemical reverse of the oxidation-hydration-oxidation sequence in the β oxidation of fatty acids (Fig. 17-8a). However, both the electron-carrying cofactor and the activating groups in the reductive anabolic sequence differ from those in the oxidative catabolic process. Recall that in β oxidation, NAD + and FAD serve as electron acceptors and the activating group is the thiol (— SH) group of coenzyme A. By contrast, the reducing agent in the synthetic sequence is NADPH and the activating groups are two different enzyme-bound — SH groups, as described below. In mammals, this synthetic system is called fatty acid synthase I (FAS I). There are seven active sites to catalyze the four-step cycle plus the charging steps described below. The active sites for different reactions lie in separate domains (Fig. 21-3a), all within a single multifunctional polypeptide chain (Mr240,000). Two of these multifunctional polypeptides function as a homodimer (Mr480,000; Fig. 21-3b). The two subunits seem to function independently. When all the active sites in one subunit are inactivated by mutation, fatty acid synthesis is only moderately reduced.

FIGURE 21-3 The structure of a fatty acid synthase type I system. Shown here is the structure of a single (monomeric) polypeptide chain of the mammalian (porcine) enzyme system. (a) All of the active sites in the mammalian system are located in different domains within a single large polypeptide chain. The different enzymatic activities are β -ketoacyl-ACP synthase (KS), malonyl/acetyl-CoA–ACP transferase (MAT), β -hydroxyacyl- ACP dehydratase (DH), enoyl-ACP reductase (ER), and β -ketoacyl-ACP reductase (KR). ACP is the acyl carrier protein. The seventh domain is a thioesterase (TE) that releases the palmitate product from ACP when synthesis is completed. The ACP and TE domains are disordered in the crystal and are therefore not shown in the structure. (b) The native dimeric structure is shown, with one polypeptide transparent to show how the two independently operating subunits come together. The linear arrangement of the domains in the polypeptide is shown below the structure. [Data from PDB ID 2CF2, T. Maier et al., Science 311:1258, 2006.] With FAS I systems, fatty acid synthesis leads to a single product. As it goes through the cycle, the acyl group is covalently linked to acyl carrier protein (ACP), which shuttles it from one active site to another in sequence. Acyl carrier protein is yet another contiguous part of the single FAS I polypeptide. No intermediates are released. When the chain length reaches 16 carbons, that product (palmitate, 16:0; see Table 10-1) leaves the cycle. Carbons C-16 and C-15 of the palmitate are derived from the methyl and carboxyl carbon atoms, respectively, of an acetyl-CoA used directly to prime the system at the outset (Fig. 21-4); the rest of the carbon atoms in the chain are derived from acetyl-CoA via malonyl-CoA. FIGURE 21-4 The overall process of palmitate synthesis. The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow; C-1 and C-2 of malonate, light red; and the carbon released as CO2, green. A er each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0). A somewhat different FAS I is found in yeast and other fungi, and is made up of two multifunctional polypeptides that form a complex with an architecture distinct from that of the vertebrate systems. Three of the seven required active sites are found on the α subunit and four on the β subunit. A different system, called FAS II, is found in plants and most bacteria. FAS II is a dissociated system; each step in the synthesis is catalyzed by a separate enzyme. Unlike FAS I, FAS II generates a variety of products, including saturated fatty acids of several lengths, as well as unsaturated, branched, and hydroxy fatty acids. An FAS II system is also found in vertebrate mitochondria, yet another indication of the bacterial origins of mitochondria in evolution. The Mammalian Fatty Acid Synthase Has Multiple Active Sites The multiple domains of mammalian FAS I function as distinct but linked enzymes. The active site for each enzyme is found in a separate domain within the larger polypeptide. Throughout the process of fatty acid synthesis, the intermediates remain covalently attached as thioesters to one of two thiol groups. One point of attachment is the — SH group of a Cys residue in one of the synthase domains (β -ketoacyl-ACP synthase; KS); the other is the — SH group of acyl carrier protein, a separate domain of the same polypeptide. Hydrolysis of thioesters is highly exergonic, and the energy released helps to make two steps ( and in Fig. 21-6) in fatty acid synthesis thermodynamically favorable. Acyl carrier protein is the shuttle that holds the system together, containing the prosthetic group 4′-phosphopantetheine, also found in coenzyme A (Fig. 21-5). The 4′-phosphopantetheine serves as a flexible arm, tethering the growing fatty acyl chain to the surface of the fatty acid synthase complex while carrying the reaction intermediates from one enzyme active site to the next. The same prosthetic group is used in FAS II systems. FIGURE 21-5 Acyl carrier protein (ACP). The prosthetic group is 4′- phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine, also found in the coenzyme A molecule, contains the B vitamin pantothenic acid. Its — SH group is the site of entry of malonyl groups during fatty acid synthesis. Coenzyme A, shown here for comparison, serves a similar chemical purpose in general metabolism. Fatty Acid Synthase Receives the Acetyl and Malonyl Groups Before the condensation reactions that build up the fatty acid chain can begin, the two thiol groups on the enzyme complex must be charged with the correct acyl groups ( Fig. 21-6a). First, the acetyl group of acetyl-CoA is transferred to ACP in a reaction catalyzed by the malonyl/acetyl-CoA–ACP transferase (MAT) domain of the multifunctional polypeptide. The acetyl group is then transferred to the Cys — SH group of the β -ketoacyl-ACP synthase (KS). The second reaction, transfer of the malonyl group from malonyl-CoA to the — SH group of ACP, is also catalyzed by malonyl/acetyl-CoA–ACP transferase. In the charged synthase complex, the acetyl and malonyl groups are activated for the chain-lengthening process. We now consider the first four steps of this process in some detail; all step numbers refer to Figure 21- 6. FIGURE 21-6 Sequence of events during synthesis of a fatty acid. (a) The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight S shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but it is attached to the KR domain. The phosphopantetheine arm of ACP ends in an — SH. (b) The enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow; C-1 and C-2 of malonate, light red; and the carbon released as CO2, green. Steps to are described in the text. Step Condensation The first reaction in the formation of a fatty acid chain is a formal Claisen condensation (see reaction class in Fig. 13-4) of the activated acetyl and malonyl groups to form acetoacetyl-ACP, an acetoacetyl group bound to ACP through the phosphopantetheine — SH group; simultaneously, a molecule of CO2 is produced. In this reaction, catalyzed by β -ketoacyl-ACP synthase, the acetyl group is transferred from the Cys — SH group of the enzyme to the malonyl group on the — SH of ACP, becoming the methyl- terminal two-carbon unit of the new acetoacetyl group. The carbon atom of the CO2 formed in this reaction is the same carbon originally introduced into malonyl-CoA from HCO− 3 in the acetyl-CoA carboxylase reaction (Fig. 21-1). Thus, CO2 is only transiently in covalent linkage during fatty acid biosynthesis; it is removed as each two-carbon unit is added. Why do cells go to the trouble of adding CO2 to make a malonyl group from an acetyl group, only to lose the CO2 during the formation of acetoacetate? The use of activated malonyl groups rather than acetyl groups makes the condensation reactions thermodynamically favorable. The methylene carbon (C-2) of the malonyl group, sandwiched between carbonyl and carboxyl carbons, forms a good nucleophile. In the condensation step, decarboxylation of the malonyl group facilitates nucleophilic attack of the methylene carbon on the thioester linking the acetyl group to β -ketoacyl-ACP synthase, displacing the enzyme’s — SH group. Coupling the condensation to the decarboxylation of the malonyl group renders the overall process highly exergonic. A similar carboxylation-decarboxylation sequence facilitates the formation of phosphoenolpyruvate from pyruvate in gluconeogenesis (see Figs. 14-17 and 14-18).

By using activated malonyl groups in the synthesis of fatty acids and activated acetate in their degradation, the cell makes both processes energetically favorable, although one is effectively the reversal of the other. The extra energy required to make fatty acid synthesis favorable is provided by the ATP used to synthesize malonyl-CoA from acetyl-CoA and HCO− 3 (Fig. 21-1). Step Reduction of the Carbonyl Group The acetoacetyl-ACP formed in the condensation step now undergoes reduction of the carbonyl group at C-3 to form D-β - hydroxybutyryl-ACP. This reaction is catalyzed by β -ketoacyl- ACP reductase (KR), and the electron donor is NADPH. Notice that the D-β -hydroxybutyryl group does not have the same stereoisomeric form as the L-β -hydroxyacyl intermediate in fatty acid oxidation (see Fig. 17-8). Step Dehydration The elements of water are now removed from C-2 and C-3 of D-β - hydroxybutyryl-ACP to yield a double bond in the product, trans-Δ2-butenoyl-ACP. The enzyme that catalyzes this dehydration is β -hydroxyacyl-ACP dehydratase (DH). Step Reduction of the Double Bond Finally, the double bond of trans-Δ2-butenoyl-ACP is reduced (saturated) to form butyryl-ACP by the action of enoyl-ACP reductase (ER); again, NADPH is the electron donor. The Fatty Acid Synthase Reactions Are Repeated to Form Palmitate Production of the four-carbon, saturated fatty acyl–ACP marks completion of one pass through the fatty acid synthase complex. In step , the butyryl group is transferred from the phosphopantetheine — SH group of ACP to the Cys — SH group of β -ketoacyl-ACP synthase, which initially bore the acetyl group (Fig. 21-6). To start the next cycle of four reactions that lengthens the chain by two more carbons (step ), another malonyl group is linked to the now unoccupied phosphopantetheine — SH group of ACP (Fig. 21-7). Condensation occurs as the butyryl group, acting like the acetyl group in the first cycle, is linked to two carbons of the malonyl-ACP group with concurrent loss of CO2. The product of this condensation is a six-carbon acyl group, covalently bound to the phosphopantetheine — SH group. Its β - keto group is reduced in the next three steps of the synthase cycle to yield the saturated acyl group, exactly as in the first round of reactions — in this case forming the six-carbon product.

FIGURE 21-7 Beginning of the second round of the fatty acid synthesis cycle. The butyryl group is on the Cys — SH group. The incoming malonyl group is first attached to the phosphopantetheine — SH group. Then, in the condensation step, the entire butyryl group on the Cys — SH is exchanged for the carboxyl group of the malonyl residue, which is lost as CO2 (green). This step is analogous to step in Figure 21-6. The product, a six-carbon β -ketoacyl group, now contains four carbons derived from malonyl-CoA and two derived from the acetyl-CoA that started the reaction. The β - ketoacyl group then undergoes steps through in Figure 21-6. Seven cycles of condensation and reduction produce the 16- carbon saturated palmitoyl group, still bound to ACP. For reasons not well understood, chain elongation by the synthase complex generally stops at this point, and free palmitate is released from the ACP by a hydrolytic activity (thioesterase; TE) in the multifunctional protein. We can consider the overall reaction for the synthesis of palmitate from acetyl-CoA in two parts. First, the formation of seven malonyl-CoA molecules: 7Acetyl-CoA + 7CO2+ 7ATP → 7malonyl-CoA + 7AD P + 7Pi (21-1) then seven cycles of condensation and reduction: Acetyl-CoA + 7malonyl-CoA + 14NAD PH + 14H+ → palmitate+ 7CO2+ 8CoA + 14NAD P+ + 6H2O (21-2) Notice that only six net water molecules are produced, because one is used to hydrolyze the thioester linking the palmitate product to the enzyme. The overall process (the sum of Eqns 21-1 and 21-2) is 8Acetyl-CoA + 7ATP + 14NAD PH + 14H+ → palmitate+ 8CoA + 7AD P + 7Pi+ 14NAD P+ + 6H2O (21-3) The biosynthesis of fatty acids such as palmitate thus requires acetyl-CoA and the input of chemical energy in two forms: the group transfer potential of ATP and the reducing power of NADPH. The ATP is required to attach CO2 to acetyl-CoA to make malonyl-CoA; the NADPH molecules are required to reduce the β -keto group and the double bond. In nonphotosynthetic eukaryotes there is an additional cost to fatty acid synthesis, because acetyl-CoA is generated in the mitochondria and must be transported to the cytosol. As we will see, this extra step consumes two ATP per molecule of acetyl-CoA transported, increasing the energetic cost of fatty acid synthesis to three ATP per two-carbon unit. Fatty Acid Synthesis Is a Cytosolic Process in Most Eukaryotes but Takes Place in the Chloroplasts in Plants In most eukaryotes, the fatty acid synthase complex (FAS I) is found in the cytosol, as are the biosynthetic enzymes for nucleotides, amino acids, and glucose. This location segregates synthetic processes from degradative reactions, many of which take place in the mitochondrial matrix. There is a corresponding segregation of the electron-carrying cofactors used in anabolism (generally a reductive process) and those used in catabolism (generally oxidative). Usually, NADPH is the electron carrier for anabolic reactions, and NAD + serves in catabolic reactions. The liver, the largest mammalian internal organ and a key metabolic center responding to large changes during feasting and fasting, provides a useful focus for this discussion. In hepatocytes, the [NAD PH]/[NAD P+] ratio is very high (~75) in the cytosol, furnishing a strongly reducing environment for the reductive synthesis of fatty acids and other biomolecules. The cytosolic [NAD H]/[NAD +] ratio is much lower (∼8× 10−4), so the NAD +- dependent oxidative catabolism of glucose can take place in the same compartment, and at the same time, as fatty acid synthesis. The [NAD H]/[NAD +] ratio in the mitochondrion is much higher than that in the cytosol, because of the flow of electrons to NAD + from the oxidation of fatty acids, amino acids, pyruvate, and acetyl-CoA. This high mitochondrial [NAD H]/[NAD +] ratio favors the reduction of oxygen via the respiratory chain. In hepatocytes and adipocytes, cytosolic NADPH is largely generated by the pentose phosphate pathway (Fig. 21-8a; also see Fig. 14-30) and by malic enzyme (Fig. 21-8b). The pyruvate produced by the action of malic enzyme reenters the mitochondrion. FIGURE 21-8 Production of NADPH. Two routes to NADPH, catalyzed by (a) the pentose phosphate pathway and (b) malic enzyme. In the photosynthetic cells of plants, fatty acid synthesis occurs not in the cytosol but in the chloroplast stroma (Fig. 21-9). This makes sense, given that NADPH is produced in chloroplasts by the light-dependent reactions of photosynthesis: FIGURE 21-9 Subcellular localization of lipid metabolism. Yeast and animal cells differ from higher plant cells in the compartmentation of lipid metabolism. Fatty acid synthesis takes place in the compartment in which NADPH is available for reductive synthesis (i.e., where the [NAD PH]/[NAD P+] ratio is high); this is the cytosol in animals and yeast, and the chloroplast in plants. Processes in red type are addressed in this chapter. Acetate Is Shuttled out of Mitochondria as Citrate In nonphotosynthetic eukaryotes, nearly all the acetyl-CoA used in fatty acid synthesis is formed in mitochondria from pyruvate oxidation and from catabolism of the carbon skeletons of amino acids. Acetyl-CoA arising from the oxidation of fatty acids is not a significant source of acetyl-CoA for fatty acid biosynthesis in animals, because the two pathways are reciprocally regulated, as described below. The inner mitochondrial membrane is impermeable to acetyl- CoA, so an indirect shuttle transfers acetyl group equivalents across the membrane (Fig. 21-10). Intramitochondrial acetyl-CoA first reacts with oxaloacetate to form citrate, in the citric acid cycle reaction catalyzed by citrate synthase (see Fig. 16-7). Citrate then passes through the inner membrane on the citrate transporter. In the cytosol, citrate cleavage by citrate lyase regenerates acetyl-CoA and oxaloacetate in an ATP-dependent reaction. Oxaloacetate cannot return to the mitochondrial matrix directly, as there is no oxaloacetate transporter. Instead, cytosolic malate dehydrogenase reduces the oxaloacetate to malate, which can return to the mitochondrial matrix on the malate–α - ketoglutarate transporter, in exchange for citrate. In the matrix, malate is reoxidized to oxaloacetate to complete the shuttle. However, most of the malate produced in the cytosol is used to generate cytosolic NADPH through the activity of malic enzyme (Fig. 21-8b). The pyruvate produced is transported into the mitochondria by the pyruvate transporter (Fig. 21-10), then converted back into oxaloacetate by pyruvate carboxylase in the matrix. In the resulting cycle, two ATP molecules are consumed (by citrate lyase and pyruvate carboxylase) for every molecule of acetyl-CoA delivered to fatty acid synthesis. FIGURE 21-10 Shuttle for transfer of acetyl groups from mitochondria to the cytosol. The outer mitochondrial membrane is freely permeable to all these compounds. Pyruvate derived from amino acid catabolism in the mitochondrial matrix, or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the matrix. Acetyl groups pass out of the mitochondrion as citrate; in the cytosol they are delivered as acetyl-CoA for fatty acid synthesis. Oxaloacetate is reduced to malate, which can be returned to the mitochondrial matrix. The steps converting malate to oxaloacetate and oxaloacetate plus acetyl-CoA to citrate (indicated by blue arrows) are part of the citric acid cycle. The major fate of cytosolic malate, however, is oxidation by malic enzyme to generate cytosolic NADPH; the pyruvate produced returns to the mitochondrial matrix. Malate thus has two fates in metabolism. In the mitochondrial matrix, malate is part of the citric acid cycle. In the cytosol, malate degradation becomes a significant source of NADPH. Aer citrate cleavage to generate acetyl-CoA, conversion of the four remaining carbons to pyruvate and CO2 by malic enzyme generates about half the NADPH required for fatty acid synthesis. The pentose phosphate pathway contributes the rest of the needed NADPH. Fatty Acid Biosynthesis Is Tightly Regulated When a cell or an organism has more than enough metabolic fuel to meet its energy needs, the excess is generally converted to fatty acids and stored as lipids such as triacylglycerols. The reaction catalyzed by acetyl-CoA carboxylase is the rate-limiting step in the biosynthesis of fatty acids, and this enzyme is an important site of regulation. In vertebrates, palmitoyl-CoA, the principal product of fatty acid synthesis, is a feedback inhibitor of the enzyme; citrate is an allosteric activator (Fig. 21-11a), increasing Vmax. Citrate plays a central role in diverting cellular metabolism from the consumption (oxidation) of metabolic fuel to the storage of fuel as fatty acids. When the concentrations of mitochondrial acetyl-CoA and ATP increase, citrate is transported out of mitochondria; it then becomes both the precursor of cytosolic acetyl-CoA and an allosteric signal for the activation of acetyl-CoA carboxylase. At the same time, citrate inhibits the activity of phosphofructokinase-1 (see Fig. 14-23), reducing the flow of carbon through glycolysis. FIGURE 21-11 Regulation of fatty acid synthesis. (a) In the cells of vertebrates, both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA. In plants, acetyl-CoA carboxylase is activated by the changes in [M g2+] and pH that accompany illumination (not shown here). (b) Filaments of acetyl-CoA carboxylase from chicken hepatocytes (the active, dephosphorylated form), as seen with the electron microscope. Acetyl-CoA carboxylase is also regulated by covalent modification. Phosphorylation, triggered by the hormones glucagon and epinephrine or by high [AMP], inactivates the enzyme and reduces its sensitivity to activation by citrate, thereby slowing fatty acid synthesis. Phosphorylation occurs on at least three Ser residues and is catalyzed primarily by the AMP- activated protein kinase (AMPK). In its active (dephosphorylated) form, acetyl-CoA carboxylase polymerizes into long filaments (Fig. 21-11b); phosphorylation is accompanied by dissociation into monomeric subunits and loss of activity. The acetyl-CoA carboxylase of plants and bacteria is not regulated by citrate or by a phosphorylation-dephosphorylation cycle. Instead, the plant enzyme is activated by an increase in stromal pH and [M g2+], which occurs on illumination of the plant (see Fig. 20-35). Bacteria do not use triacylglycerols as energy stores. In Escherichia coli, the primary role of fatty acid synthesis is to provide precursors for membrane lipids; the regulation of this process is complex, employing guanine nucleotides (such as ppGpp; see Fig. 8-42) that coordinate cell growth with membrane formation. In addition to the moment-by-moment regulation of enzymatic activity, these pathways are regulated at the level of gene expression. For example, when animals ingest an excess of certain polyunsaturated fatty acids, the expression of genes encoding many lipogenic enzymes in the liver is suppressed. This gene regulation is mediated by a family of nuclear receptor proteins called PPARs, which we encountered in Section 17.2. If fatty acid synthesis and β oxidation were to proceed simultaneously, the two processes would constitute a futile cycle, wasting energy. We noted earlier (see Fig. 17-13) that β oxidation is blocked by malonyl-CoA, which inhibits carnitine acyltransferase I. Thus, during fatty acid synthesis, production of the first intermediate, malonyl-CoA, shuts down β oxidation at the level of a transport system in the inner mitochondrial membrane. This control mechanism illustrates another advantage of segregating synthetic and degradative pathways in different cellular compartments. Long-Chain Saturated Fatty Acids Are Synthesized from Palmitate Palmitate, the principal product of the fatty acid synthase system in animal cells, is the precursor of other long-chain fatty acids (Fig. 21-12). It may be lengthened to form stearate (18:0) or even longer saturated fatty acids by further additions of acetyl groups, through the action of fatty acid elongation systems present in the smooth endoplasmic reticulum (smooth ER) and in mitochondria. The more active elongation system of the ER extends the 16-carbon chain of palmitoyl-CoA by two carbons, forming stearoyl-CoA. Although different enzyme systems are used, and coenzyme A rather than ACP is the acyl carrier in the reaction, the mechanism of elongation in the ER is otherwise identical to that in palmitate synthesis: donation of two carbons by malonyl-CoA, followed by reduction, dehydration, and reduction to the saturated 18-carbon product, stearoyl-CoA.

FIGURE 21-12 Routes of synthesis of unsaturated fatty acids and their derivatives. Palmitate is the precursor of stearate and longer-chain saturated fatty acids, as well as the monounsaturated acids palmitoleate and oleate. Mammals cannot convert oleate to linoleate or α -linolenate (shaded), which are therefore required in the diet as essential fatty acids. Conversion of linoleate to other polyunsaturated fatty acids and eicosanoids is outlined. Unsaturated fatty acids are symbolized by indicating the number of carbons and the number and position of double bonds, as in Table 10-1. Linoleate and α -linolenate are important omega-6 and omega-3 fatty acids, respectively; they are also precursors for a wide range of unsaturated fatty acids that act as signaling molecules. Two-letter abbreviations specify the eicosanoid prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT). Particular classes of unsaturated fatty acids are further delineated by the number of double bonds, which defines subclasses referred to as series. For example, series 2 TX are thromboxanes with two double bonds in the hydrocarbon chain. Two key products of elongation pathways are linoleate, an omega- 6 fatty acid (see Chapter 10 for the alternative nomenclature), and α -linolenate, an omega-3 fatty acid. These are precursors for two extensive families of derivative unsaturated fatty acids, the omega-6 and omega-3 families. Humans cannot synthesize linoleate and α -linolenate and must obtain them in the diet. The ratio of omega-6 to omega-3 fatty acids in the diet, if too high, can lead to cardiovascular disease. The importance of this ratio may reflect the multitude of signaling molecules in the omega-6 and omega-3 families (Fig. 21-12), with their equally complex physiological effects. Several of these derivative unsaturated fatty acids are considered below. Desaturation of Fatty Acids Requires a Mixed-Function Oxidase Palmitate and stearate serve as precursors of the two most common monounsaturated fatty acids of animal tissues: palmitoleate, 16:1(Δ9), and oleate, 18:1(Δ9); both of these fatty acids have a single cis double bond between C-9 and C-10 (see Table 10-1). The double bond is introduced into the fatty acid chain by an oxidative reaction catalyzed by fatty acyl–CoA desaturase (Fig. 21-13), a mixed-function oxidase (Box 21-1). Two different substrates, the fatty acid and NADPH, simultaneously undergo two-electron oxidations. The path of electron flow includes a cytochrome (cytochrome b5) and a flavoprotein (cytochrome b5 reductase), both of which, like fatty acyl–CoA desaturase, are in the smooth ER. In plants, oleate is produced by a stearoyl-ACP desaturase (SCD) that uses reduced ferredoxin as the electron donor in the chloroplast stroma. FIGURE 21-13 Electron transfer in the desaturation of fatty acids in vertebrates. Blue arrows show the path of electrons as two substrates — a fatty acyl–CoA and NADPH — undergo oxidation by molecular oxygen. These reactions take place on the lumenal face of the smooth ER. A similar pathway, but with different electron carriers, occurs in plants. BOX 21-1 MEDICINE Oxidases, Oxygenases, Cytochrome P-450 Enzymes, and Drug Overdoses In this chapter we encounter several enzymes that carry out oxidation- reduction reactions in which molecular oxygen is a participant. The stearoyl- CoA desaturase (SCD) that introduces a double bond into a fatty acyl chain (see Fig. 21-13) is one such enzyme. The nomenclature for enzymes that catalyze reactions of this general type can be confusing. Oxidase is the general name for enzymes that catalyze oxidations in which molecular oxygen is the electron acceptor but oxygen atoms do not appear in the oxidized product. The enzyme that creates a double bond in fatty acyl–CoA during the oxidation of fatty acids in peroxisomes (see Fig. 17-14) is an oxidase of this type; a second example is the cytochrome oxidase of the mitochondrial respiratory chain (see Fig. 19-13). In the first case, the transfer of two electrons to H2O produces hydrogen peroxide, H2O2; in the second, two electrons reduce 1/2O2 to H2O. Many, but not all, oxidases are flavoproteins. Mixed-function oxidases oxidize two different substrates simultaneously; again, the molecular oxygen atoms do not appear in the oxidized products. Mixed-function oxidases act in fatty acid desaturation (fatty acyl–CoA desaturase; see Fig. 21-13) and in the last step of plasmalogen synthesis (see Fig. 21-30). Oxygenases catalyze oxidative reactions in which oxygen atoms are directly incorporated into the product molecule, forming a new hydroxyl or carboxyl group, for example. Dioxygenases catalyze reactions in which both oxygen atoms of O2 are incorporated into the organic product. An example of a dioxygenase is tryptophan 2,3-dioxygenase, which catalyzes the opening of the five-membered ring of tryptophan in the catabolism of this amino acid. When the reaction takes place in the presence of 18O2, the isotopic oxygen atoms are found in the two carbonyl groups of the product (shown in red):

Monooxygenases, more common and more complex in their action, catalyze reactions in which only one of the two oxygen atoms of O2 is incorporated into the organic product, the other being reduced to H2O; an example is squalene monooxygenase (see Fig. 21-37). Monooxygenases require two substrates to serve as reductants of the two oxygen atoms of O2. The main substrate accepts one of the two oxygen atoms, and a cosubstrate furnishes hydrogen atoms to reduce the other oxygen atom to H2O. The general reaction equation for monooxygenases is AH + BH2+ O— O → A— OH + B + H2O where AH is the main substrate and BH2 is the cosubstrate. Because most monooxygenases catalyze reactions in which the main substrate becomes hydroxylated, they are also called hydroxylases. They are also sometimes called mixed-function oxygenases to indicate that they oxidize two different substrates simultaneously. Monooxygenases are divided into several classes, depending on the nature of the cosubstrate. Some use reduced flavin nucleotides (FM NH2 or FAD H2), others use NADH or NADPH, and still others use α -ketoglutarate as cosubstrate. The enzyme that hydroxylates the phenyl ring of phenylalanine to form tyrosine is a monooxygenase that uses tetrahydrobiopterin as cosubstrate (see Fig. 18-23). (This is the enzyme that is defective in the human genetic disease phenylketonuria.) The most numerous and most complex monooxygenation reactions are those employing a type of heme protein called cytochrome P-450. Like mitochondrial cytochrome oxidase, enzymes containing a cytochrome P-450 domain can react with O2 and bind carbon monoxide, but they can be differentiated from cytochrome oxidase because the carbon monoxide complex of their reduced form absorbs light strongly at 450 nm — thus the name P-450. Cytochrome P-450 enzymes catalyze hydroxylation reactions in which an organic substrate, RH, is hydroxylated to R— OH, incorporating one oxygen atom of O2; the other oxygen atom is reduced to H2O by reducing equivalents that are furnished by NADH or NADPH but are usually passed to cytochrome P- 450 by an iron-sulfur protein. Figure 1 shows a simplified outline of the action of cytochrome P-450. FIGURE 1 Simplified cytochrome P-450 reaction cycle. One large family of P-450–containing proteins consists of two general types: those highly specific for a single substrate (like typical enzymes) and those with more promiscuous binding sites that accept a variety of substrates, generally similar in being hydrophobic. In the adrenal cortex, for example, a specific cytochrome P-450 participates in the hydroxylation of steroids to yield the adrenocortical hormones (see Fig. 21-49). There are dozens of P-450 enzymes that act on specific substrates in the biosynthetic pathways to steroid hormones and eicosanoids (Fig. 2). Cytochrome P-450 enzymes with broader specificity are important in the hydroxylation of many different drugs, such as barbiturates and other xenobiotics (substances foreign to the organism), particularly if they are hydrophobic and relatively insoluble. The environmental carcinogen benzo[a]pyrene, found in cigarette smoke, undergoes cytochrome P-450–dependent hydroxylation during detoxification. Hydroxylation of xenobiotics, sometimes combined with the attachment of a polar compound such as glucuronic acid to the hydroxyl group, makes them more soluble in water and allows their excretion in urine. Hydroxylation (and glucuronidation) inactivates most drugs, and the rate at which it occurs can determine how long a given dose of a medication remains in the blood at therapeutic levels. FIGURE 2 Pathways of sterol biosynthesis, showing the steps requiring cytochrome P-450 enzymes. Humans differ in their levels of drug-metabolizing enzymes, both because of their genetics and because past exposure to substrates can induce the synthesis of higher levels of P-450 enzymes. Ethanol and barbiturate drugs share a P-450 enzyme. Long-term heavy drinking induces synthesis of this enzyme. Then, because the barbiturate is inactivated and cleared faster, larger doses are required to get the same therapeutic effect. If an individual takes this larger-than-usual dose of barbiturate and then also drinks alcohol, competition between the alcohol and the barbiturate for the limited amount of enzyme means that both alcohol and barbiturate are cleared more slowly. The resulting high levels of these two central nervous system depressants can be lethal. Similar complications arise when an individual takes two drugs that happen to be inactivated by the same P-450 enzyme; each drug increases the effective dose of the other by slowing its inactivation. It is therefore essential for physicians and pharmacists to know about all of a patient’s prescribed and over-the-counter drugs and supplements, as well as a history of heavy drinking, or smoking, or exposure to environmental toxins. The SCD of animals (as studied in mice) has an important role in the development of obesity and the insulin resistance that oen accompanies obesity and precedes development of type 2 diabetes mellitus. Mice have four isozymes, SCD1 through SCD4, of which SCD1 is the best understood. Its synthesis is induced by dietary saturated fatty acids, and also by the action of SREBP and LXR, two protein regulators of lipid metabolism that activate transcription of lipid-synthesizing enzymes (described in Section 21.4). Mice with mutant forms of SCD1 are resistant to diet- induced obesity and do not develop diabetes under conditions that cause both obesity and diabetes in mice with normal SCD1. Mammalian hepatocytes can readily introduce double bonds at the Δ9 position of fatty acids but cannot introduce additional double bonds between C-10 and the methyl-terminal end. Thus, as noted above, mammals cannot synthesize the omega-6 family precursor linoleate, 18:2(Δ9,12), or the omega-3 family precursor α -linolenate, 18:3(Δ9,12,15). Plants, however, can synthesize both; the desaturases that introduce double bonds at the Δ12 and Δ15 positions are located in the ER and in chloroplasts. The ER enzymes act not on free fatty acids but on a phospholipid, phosphatidylcholine, that contains at least one oleate linked to the glycerol (Fig. 21-14). Both plants and bacteria must synthesize polyunsaturated fatty acids to ensure membrane fluidity at reduced temperatures. FIGURE 21-14 Action of plant desaturases. Desaturases in plants oxidize phosphatidylcholine-bound oleate to polyunsaturated fatty acids. Some of the products are released from the phosphatidylcholine by hydrolysis. Because they are necessary precursors for the synthesis of other products, linoleate and α -linolenate are essential fatty acids for mammals; they must be obtained from dietary plant material. Once ingested, linoleate may be converted to certain other polyunsaturated acids, particularly γ -linolenate, eicosatrienoate, and arachidonate (eicosatetraenoate), all of which can be made only from linoleate (Fig. 21-12). Similarly, α -linolenate is converted to two important derivatives, eicosapentaenoic acid (EPA) and docosa-hexaenoic acid (DHA). Arachidonate, 20:4(Δ5,8,11,14), EPA, 20:5(Δ5,8,11,14,17), and DHA, 22:6(Δ4,7,10,13,16,19) , are essential precursors of distinct classes of eicosanoids, lipids with important regulatory functions. The 20- and 22-carbon fatty acids are synthesized from linoleate and -linolenate by fatty acid elongation reactions analogous to those described on page 753. Eicosanoids Are Formed from 20- and 22-Carbon Polyunsaturated Fatty Acids Eicosanoids are a family of very potent biological signaling molecules that act as short-range messengers, affecting tissues near the cells that produce them. In response to hormonal or other stimuli, phospholipase A2, present in most types of mammalian cells, attacks membrane phospholipids, releasing arachidonate from the middle carbon of glycerol. Enzymes of the smooth ER then convert arachidonate to prostaglandins, beginning with the formation of prostaglandin H2(PGH2), the immediate precursor of many other prostaglandins and thromboxanes (Fig. 21-15a). The two reactions that lead to PGH2 are catalyzed by a bifunctional enzyme, cyclooxygenase (COX), also called H2 prostaglandin synthase. In the first step, the cyclooxygenase activity introduces molecular oxygen to convert arachidonate to PGG2. The second step, catalyzed by the peroxidase activity of COX, converts PGG2 to PGH2. FIGURE 21-15 The “cyclic” pathway from arachidonate to prostaglandins and thromboxanes. (a) A er arachidonate is released from phospholipids by the action of phospholipase A2, the cyclooxygenase and peroxidase activities of COX (also called prostaglandin H2 synthase) catalyze the production of PGH2, the precursor of other prostaglandins and of thromboxanes. (b) Aspirin inhibits the first reaction by acetylating an essential Ser residue on the enzyme. Ibuprofen and naproxen inhibit the same step, probably by mimicking the structure of the substrate or an intermediate in the reaction. KEY CONVENTION Prostaglandins with different functional groups on the ring are given different letter designations: A, B, C, D, E, F, G, H, and R. The subscript number following the letter, as in PGH2 and PGG2, indicates the number of double bonds. Prostaglandins with two double bonds, all of which are derived from arachidonate, are referred to as series 2 prostaglandins; those with three double bonds, derived from EPA, as series 3 (Fig. 21-12). Similar naming patterns are used for other classes of eicosanoids described below. Series 2 prostaglandins have important roles in the immediate response to stress or injury, including inflammation, pain, swelling, and dilation of blood vessels. Series 3 prostaglandins, in general, act more slowly and usually moderate the responses associated with series 2 prostaglandins. Mammals have two isozymes of prostaglandin H2 synthase, COX-1 and COX-2. These have different functions but closely similar amino acid sequences (60% to 65% sequence identity) and similar reaction mechanisms at both of their catalytic centers. COX-1 is responsible for synthesis of the prostaglandins that regulate the secretion of gastric mucin, and COX-2 is responsible for synthesis of the prostaglandins that mediate inflammation, pain, and fever. Pain can be relieved by inhibiting COX-2. The first drug widely marketed for this purpose was aspirin (acetylsalicylate; Fig. 21-15b). The name “aspirin” (from a for acetyl and spir for Spirsaüre, the German word for the salicylates prepared from the plant Spiraea ulmaria) appeared in 1899 when the drug was introduced by the Bayer company. Aspirin irreversibly inactivates the cyclooxygenase activity of both COX isozymes, by acetylating a Ser residue and blocking each enzyme’s active site. The synthesis of prostaglandins and thromboxanes is thereby inhibited. Additional widely used nonsteroidal anti-inflammatory drugs (NSAIDs; Fig. 21-15b), ibuprofen and naproxen, inhibit the same pair of enzymes. However, the inhibition of COX-1 can result in undesired side effects, including stomach irritation and more serious conditions. In the 1990s, NSAID compounds that had a greater specificity for COX-2 were developed as advanced therapies for severe pain. Three of these drugs were approved for use worldwide: rofecoxib (Vioxx), valdecoxib (Bextra), and celecoxib (Celebrex). Though initially considered a success, Vioxx and Bextra were withdrawn as field reports and clinical studies connected the drugs with an increased risk of heart attack and stroke. Celebrex is still on the market but is being used with increased caution. The detailed reasons for the problems with these drugs are still not clear but serve as a cautionary note. We are increasingly aware of the complexity of the web of these signaling interactions, and predicting the consequences of targeting specific components with pharmaceutical agents remains an imperfect process. Thromboxane synthase, present in blood platelets (thrombocytes), converts PGH2 to thromboxane A2, from which other series 2 thromboxanes are derived (Fig. 21-15a). The series 2 thromboxanes induce constriction of blood vessels and platelet aggregation, early steps in blood clotting. Low doses of aspirin, taken regularly, reduce the probability of heart attacks and strokes by reducing thromboxane production. Thromboxanes, like prostaglandins, contain a ring of five or six atoms; the pathway from arachidonate to the series 2 prostaglandins and thromboxanes is sometimes called the “cyclic” pathway, to distinguish it from the “linear” pathway that leads from arachidonate to the leukotrienes, which are linear compounds (Fig. 21-16). Leukotriene synthesis begins with the action of several lipoxygenases that catalyze the incorporation of molecular oxygen into arachidonate. These enzymes, found in leukocytes and in heart, brain, lung, and spleen, are mixed- function oxidases of the cytochrome P-450 family (see Box 21-1). The various leukotrienes differ in the position of the peroxide group introduced by the lipoxygenases. The linear pathway from arachidonate, unlike the cyclic pathway, is not inhibited by aspirin or other NSAIDs. FIGURE 21-16 The “linear” pathway from arachidonate to leukotrienes. Pathogenic organisms, as well as irritants such as air pollution and tobacco smoke, trigger an inflammatory response in the affected tissue, which consists of two phases: initiation and resolution. Eicosanoids of the omega-6 family are critical to initiation — playing key roles in recruiting leukocytes, making blood vessels more permeable, and stimulating chemotaxis and migration of immune system cells. As the source of tissue damage is brought under control, the inflammation must be resolved and the tissue brought back to its normal state. Resolution of inflammation is called catabasis, and it is promoted by several classes of signaling molecules; prominent among these are several leukotrienes and prostaglandins. Many eicosanoids of the omega-3 family (including series 3 prostaglandins and thromboxanes) are anti-inflammatory, although the classification is not absolute; individual eicosanoids can be inflammatory in one tissue and anti-inflammatory in another. Catabasis is also promoted by a set of eicosanoids termed specialized pro-resolving mediators (SPMs). The first family of SPMs to be discovered was the lipoxins, followed more recently by resolvins, protectins, and maresins. All SPMs are derived from essential fatty acids (Fig. 21-12). They affect different target cells and tissues in different ways. The sum of their action is to promote removal of debris, microbes, and dead cells, to restore blood vessel integrity, and to regenerate tissue. Particular SPMs also reduce pain and fever, and play roles in resolving the tissue inflammation leading to diabetes, obesity, and asthma. Further research on SPMs thus has potential for the development of new pharmaceutical targets. Plants also derive important signaling molecules from fatty acids. As in animals, a key step in the initiation of signaling is activation of a specific phospholipase. In plants, the fatty acid substrate released by phospholipase action is α -linolenate. A lipoxygenase then catalyzes the first step in a pathway that converts α - linolenate to jasmonate, a substance known to have signaling roles in defense against insects, resistance to fungal pathogens, and maturation of pollen. Jasmonate also affects seed germination, root growth, and fruit and seed development. SUMMARY 21.1 Biosynthesis of Fatty Acids and Eicosanoids Malonyl-CoA, a key precursor of fatty acids, is synthesized by the action of acetyl-CoA carboxylase. Beginning with malonyl-CoA and acetyl-CoA, fatty acids are synthesized in a repeating cycle of four steps. Long-chain saturated fatty acids are synthesized from acetyl- CoA by a cytosolic system of six enzymatic activities plus acyl carrier protein (ACP). There are two types of fatty acid synthase. FAS I, found in vertebrates and fungi, consists of multifunctional polypeptides. FAS II is a dissociated system found in bacteria and plants. Both contain two types of OSH groups (one furnished by the phosphopantetheine of ACP, the other by a Cys residue of β - ketoacyl-ACP synthase) that function as carriers of the fatty acyl intermediates. Malonyl-ACP, formed from acetyl-CoA (shuttled out of mitochondria) and CO2, condenses with an acetyl bound to the Cys OSH to yield acetoacetyl-ACP, with release of CO2. This is followed by reduction to the D-β -hydroxy derivative, dehydration to the trans-Δ2-unsaturated acyl-ACP, and reduction to butyryl- ACP. NADPH is the electron donor for both reductions. Fatty acid synthesis is regulated at the level of malonyl-CoA formation. Six more molecules of malonyl-ACP react successively at the carboxyl end of the growing fatty acid chain to form palmitoyl- ACP — the end product of the fatty acid synthase reaction. Free palmitate is released by hydrolysis. Fatty acid synthesis occurs in the cytosol of animal cells, and in chloroplasts in plants. Acetate is exported from the mitochondria as citrate. Fatty acid synthesis is tightly regulated, principally by regulation of acetyl-CoA carboxylase. Palmitate may be elongated to the 18-carbon stearate. Palmitate and stearate can be desaturated to yield palmitoleate and oleate, respectively, by the action of mixed-function oxidases. Mammals cannot make linoleate and must obtain it from plant sources; they convert exogenous linoleate to arachidonate, the parent compound of eicosanoids (prostaglandins, thromboxanes, leukotrienes, and specialized pro-resolving mediators), a family of very potent signaling molecules. The synthesis of prostaglandins and thromboxanes is inhibited by NSAIDs that act on the cyclooxygenase activity of prostaglandin H2 synthase. 21.2 Biosynthesis of Triacylglycerols Most of the fatty acids synthesized or ingested by an organism have one of two fates, depending on the organism’s needs: incorporation into triacylglycerols for the storage of metabolic energy or incorporation into the phospholipid components of membranes. During rapid growth, synthesis of new membranes requires the production of membrane phospholipids; when an organism has a plentiful food supply but is not actively growing, it shunts most of its fatty acids into storage fats. Both pathways begin at the same point: the formation of fatty acyl esters of glycerol. In this section we examine the route to triacylglycerols and its regulation, and the production of glycerol 3-phosphate in the process of glyceroneogenesis. Triacylglycerols and Glycerophospholipids Are Synthesized from the Same Precursors Animals can synthesize and store large quantities of triacylglycerols, to be used later as fuel (see Box 17-1). Humans can store only a few hundred grams of glycogen in liver and muscle, barely enough to supply the body’s energy needs for 12 hours. However, a 70 kg human stores about 15 kg of triacylglycerol in its tissues, enough to support basal energy needs for as long as 12 weeks (see Table 23-5). Triacylglycerols have the highest energy content of all stored nutrients — more than 38 kJ/g. Whenever carbohydrate is ingested in excess of the organism’s capacity to store glycogen, the excess is converted to triacylglycerols and stored in adipose tissue. Plants also manufacture triacylglycerols as an energy-rich fuel, mainly stored in fruits, nuts, and seeds. In animal tissues, triacylglycerols and glycerophospholipids such as phosphatidylethanolamine share two precursors, fatty acyl– CoA and L-glycerol 3-phosphate, and several biosynthetic steps. The vast majority of the glycerol 3-phosphate is derived from the glycolytic intermediate dihydroxyacetone phosphate (DHAP) by the action of the cytosolic NAD-linked glycerol 3-phosphate dehydrogenase; in liver and kidney, a small amount of glycerol 3- phosphate is also formed from glycerol by the action of glycerol kinase (Fig. 21-17). The other precursors of triacylglycerols are fatty acyl–CoAs, formed from fatty acids by acyl-CoA synthetases, the same enzymes responsible for the activation of fatty acids for β oxidation (see Fig. 17-5).

FIGURE 21-17 Biosynthesis of phosphatidic acid. A fatty acyl group is activated by formation of the fatty acyl–CoA, then transferred to ester linkage with -glycerol 3-phosphate, formed in either of the two ways shown. Phosphatidic acid is shown here with the correct stereochemistry () at C-2 of the glycerol molecule. (The intermediate product with only one esterified fatty acyl group is lysophosphatidic acid.) To conserve space in subsequent figures (and in Fig. 21-14), both fatty acyl groups of glycerophospholipids, and all three acyl groups of triacylglycerols, are shown projecting to the right. The first stage in the biosynthesis of triacylglycerols is acylation of the two free hydroxyl groups of L-glycerol 3-phosphate by two molecules of fatty acyl–CoA to yield diacylglycerol 3-phosphate, more commonly called phosphatidic acid, or phosphatidate (Fig. 21-17). Phosphatidic acid is present in only trace amounts in cells but is a central intermediate in lipid biosynthesis; it can be converted either to a triacylglycerol or to a glycerophospholipid. In the pathway to triacylglycerols, phosphatidic acid is hydrolyzed by phosphatidic acid phosphatase (also called lipin) to form a 1,2-diacylglycerol (Fig. 21-18). Diacylglycerols are then converted to triacylglycerols by transesterification with a third fatty acyl– CoA. FIGURE 21-18 Phosphatidic acid in lipid biosynthesis. Phosphatidic acid is the precursor of both triacylglycerols and glycerophospholipids. The mechanisms for head-group attachment in phospholipid synthesis are described later in this section. Triacylglycerol Biosynthesis in Animals Is Regulated by Hormones In humans, the amount of body fat stays relatively constant over long periods, although there may be minor short-term changes as caloric intake fluctuates. Biosynthesis and degradation of triacylglycerols are regulated to meet the metabolic requirements of the moment. The rate of triacylglycerol biosynthesis is profoundly altered by the action of several hormones. Insulin, for example, promotes the conversion of carbohydrate to triacylglycerols (Fig. 21-19). People with severe diabetes mellitus, due to failure of insulin secretion or action, not only are unable to use glucose properly but also fail to synthesize fatty acids from carbohydrates or amino acids. If the diabetes is untreated, these individuals have increased rates of fat oxidation and ketone body formation (Chapter 17) and therefore lose weight.

FIGURE 21-19 Regulation of triacylglycerol synthesis by insulin. Insulin stimulates conversion of dietary carbohydrates and proteins to fat. Individuals with diabetes mellitus either lack insulin or are insensitive to it. This results in diminished fatty acid synthesis, and the acetyl-CoA arising from catabolism of carbohydrates and proteins is shunted instead to ketone body production. People in severe ketosis smell of acetone, so the condition is sometimes mistaken for drunkenness. Approximately 75% of all fatty acids released by triacylglycerol breakdown (lipolysis) are reesterified to form triacylglycerols rather than used for fuel. This ratio persists even under starvation conditions, when energy metabolism is shunted from the use of carbohydrate to the oxidation of fatty acids. Some of this fatty acid recycling takes place in adipose tissue, with the reesterification occurring before release into the bloodstream; some takes place via a systemic cycle in which free fatty acids are transported to the liver, recycled to triacylglycerol, exported again into the blood (transport of lipids in the blood is discussed in Section 21.4), and taken up again by adipose tissue, aer release from triacylglycerol by extracellular lipoprotein lipase (Fig. 21-20; see also Fig. 17-1). Flux through this triacylglycerol cycle between adipose tissue and liver may be low when other fuels are available and the release of fatty acids from adipose tissue is limited, but, as noted above, the proportion of released fatty acids that are reesterified remains roughly constant at 75% under all metabolic conditions. The level of free fatty acids in the blood thus reflects both the rate of release of fatty acids and the balance between the synthesis and breakdown of triacylglycerols in adipose tissue and liver. FIGURE 21-20 The triacylglycerol cycle. In mammals, triacylglycerol molecules are broken down and resynthesized in a triacylglycerol cycle during starvation. Some of the fatty acids released by lipolysis of triacylglycerol in adipose tissue pass into the bloodstream, and the remainder are used for resynthesis of triacylglycerol. Some of the fatty acids released into the blood are used for energy (in muscle, for example), and some are taken up by the liver and used in triacylglycerol synthesis. The triacylglycerol formed in the liver is transported in the blood back to adipose tissue, where the fatty acid is released by extracellular lipoprotein lipase, taken up by adipocytes, and reesterified into triacylglycerol. When the mobilization of fatty acids is required to meet energy needs, release from adipose tissue is stimulated by the hormones glucagon and epinephrine (see Figs. 17-2, 17-13). Simultaneously, these hormonal signals decrease the rate of glycolysis and increase the rate of gluconeogenesis in the liver (providing glucose for the brain, as further elaborated in Chapter 23). The released fatty acid is taken up by several tissues, including muscle, where it is oxidized to provide energy. Much of the fatty acid taken up by liver is not oxidized but is recycled to triacylglycerol and returned to adipose tissue. The function of the apparently futile triacylglycerol cycle is not well understood, but as we learn more about how the cycle is sustained via metabolism in two separate organs and is coordinately regulated, some possibilities emerge. For example, the excess capacity in the triacylglycerol cycle — the fatty acid that is eventually reconverted to triacylglycerol rather than oxidized as fuel — could represent an energy reserve in the bloodstream during fasting, one that could be more rapidly mobilized in a “fight or flight” emergency than stored triacylglycerol could be. The constant recycling of triacylglycerols in adipose tissue even during starvation raises a second question: what is the source of the glycerol 3-phosphate required for this process? As noted above, glycolysis is suppressed under these conditions by the action of glucagon and epinephrine, so little DHAP is available. And glycerol released during lipolysis cannot be converted directly to glycerol 3-phosphate in adipose tissue, which lacks glycerol kinase (Fig. 21-17). So, how is sufficient glycerol 3- phosphate produced? The answer lies in the pathway of glyceroneogenesis, discovered in the 1960s by Lea Reshef, Richard Hanson, and John Ballard, and simultaneously by Eleazar Shafrir and his coworkers. The investigators were intrigued by the presence of two gluconeogenic enzymes, pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase, in adipose tissue, where glucose is not synthesized. Yet, the importance of this pathway was not appreciated until decades later. Glyceroneogenesis is intimately linked to the triacylglycerol cycle and, in a larger sense, to the balance between fatty acid and carbohydrate metabolism. Adipose Tissue Generates Glycerol 3- Phosphate by Glyceroneogenesis Glyceroneogenesis is a shortened version of gluconeogenesis, from pyruvate to DHAP (see Fig. 14-16), followed by conversion of the DHAP to glycerol 3-phosphate by cytosolic NAD-linked glycerol 3-phosphate dehydrogenase (Fig. 21-21). Glycerol 3- phosphate is subsequently used in triacylglycerol synthesis. There is a link between glyceroneogenesis and type 2 diabetes, as we shall see.

FIGURE 21-21 Glyceroneogenesis. The pathway is essentially an abbreviated version of gluconeogenesis, from pyruvate to dihydroxyacetone phosphate (DHAP), followed by conversion of DHAP to glycerol 3-phosphate, which is used for the synthesis of triacylglycerol. Glyceroneogenesis has multiple roles. In adipose tissue, glyceroneogenesis coupled with reesterification of free fatty acids controls the rate of fatty acid release to the blood. In brown adipose tissue, the same pathway may control the rate at which free fatty acids are delivered to mitochondria for use in thermogenesis. And in fasting humans, glyceroneogenesis in the liver alone supports the synthesis of enough glycerol 3-phosphate to account for up to 65% of fatty acids reesterified to triacylglycerol. Flux through the triacylglycerol cycle between liver and adipose tissue is controlled to a large degree by the activity of PEP carboxykinase, which limits the rate of both gluconeogenesis and glyceroneogenesis. Glucocorticoid hormones such as cortisol (a biological steroid derived from cholesterol; see Fig. 21-48) and dexamethasone (a synthetic glucocorticoid) regulate the levels of PEP carboxykinase reciprocally in the liver and adipose tissue. Acting through the glucocorticoid receptor, these steroid hormones increase the expression of the gene encoding PEP carboxykinase in the liver, thus increasing gluconeogenesis and glyceroneogenesis (Fig. 21-22).

FIGURE 21-22 Regulation of glyceroneogenesis. (a) Glucocorticoid hormones stimulate glyceroneogenesis and gluconeogenesis in the liver, while suppressing glyceroneogenesis in adipose tissue (by reciprocal regulation of the gene expressing PEP carboxykinase (PEPCK) in the two tissues); this increases the flux through the triacylglycerol cycle. The glycerol freed by the breakdown of triacylglycerol in adipose tissue is released to the blood and transported to the liver, where it is primarily converted to glucose, although some is converted to glycerol 3-phosphate by glycerol kinase. (b) A class of drugs called thiazolidinediones is used to treat type 2 diabetes. In this disease, high levels of free fatty acids in the blood interfere with glucose utilization in muscle and promote insulin resistance. Thiazolidinediones activate a nuclear receptor called peroxisome proliferator-activated receptor γ (PPARγ), which induces the activity of PEP carboxykinase. Therapeutically, thiazolidinediones increase the rate of glyceroneogenesis, thus increasing the resynthesis of triacylglycerol in adipose tissue and reducing the amount of free fatty acid in the blood. In both panels, dashed lines identify pathways in which flux declines under the conditions indicated. Stimulation of glyceroneogenesis leads to an increase in the synthesis of triacylglycerol molecules in the liver and their release into the blood. At the same time, glucocorticoids suppress expression of the gene encoding PEP carboxykinase in adipose tissue. This results in a decrease in glyceroneogenesis in adipose tissue; recycling of fatty acids declines as a result, and more free fatty acids are released into the blood. Thus, regulation of glyceroneogenesis in the liver and adipose tissue affects lipid metabolism in opposite ways: a lower rate of glyceroneogenesis in adipose tissue leads to more fatty acid release (rather than recycling), whereas a higher rate in the liver leads to more synthesis and export of triacylglycerols. The net result is an increase in flux through the triacylglycerol cycle. When the glucocorticoids are no longer present, flux through the cycle declines as the expression of PEP carboxykinase increases in adipose tissue and decreases in the liver. Thiazolidinediones Treat Type 2 Diabetes by Increasing Glyceroneogenesis The connection between glyceroneogenesis and diabetes has stimulated new interest. High levels of free fatty acids in the blood interfere with glucose utilization in muscle and promote the insulin resistance that leads to type 2 diabetes. A class of drugs called thiazolidinediones reduces the levels of fatty acids circulating in the blood and increases sensitivity to insulin. Thiazolidinediones promote the increased expression of PEP carboxykinase in adipose tissue (Fig. 21-22), leading to increased synthesis of the precursors of glyceroneogenesis. The therapeutic effect of thiazolidinediones is thus due, at least in part, to the increase in glyceroneogenesis, which in turn increases the resynthesis of triacylglycerol in adipose tissue and reduces the release of free fatty acid from adipose tissue into the blood. Two thiazolidinediones have been available for treatment of type 2 diabetes: rosiglitazone (Avandia) and pioglitazone (Actos). Large- scale trials of rosiglitazone have indicated an increased risk of heart attack, so rosigliatazone has been withdrawn in the United Kingdom, India, South Africa, and many European countries. It remains available in the United States with limitations.

SUMMARY 21.2 Biosynthesis of Triacylglycerols Triacylglycerols are formed by reaction of two molecules of fatty acyl–CoA with glycerol 3-phosphate to form phosphatidic acid; this product is dephosphorylated to a diacylglycerol, then acylated by a third molecule of fatty acyl–CoA to yield a triacylglycerol. The synthesis and degradation of triacylglycerols are hormonally regulated. Mobilization and recycling of triacylglycerol molecules result in a triacylglycerol cycle. Triacylglycerols are resynthesized from free fatty acids and glycerol 3-phosphate even during starvation. The dihydroxyacetone phosphate precursor of glycerol 3- phosphate is derived from pyruvate via glyceroneogenesis. Thiazolidinediones stimulate glyceroneogenesis and can be used to treat type 2 diabetes. 21.3 Biosynthesis of Membrane Phospholipids In Chapter 10 we introduced two major classes of membrane phospholipids: glycerophospholipids and sphingolipids. Many different phospholipid species can be constructed by combining various fatty acids and polar head groups with the glycerol or sphingosine backbone (see Figs. 10-8, 10-11). All the biosynthetic pathways follow a few basic patterns. In general, the assembly of phospholipids from simple precursors requires (1) synthesis of the backbone molecule (glycerol or sphingosine); (2) attachment of fatty acid(s) to the backbone through an ester or amide linkage; (3) addition of a hydrophilic head group to the backbone through a phosphodiester linkage; and, in some cases, (4) alteration or exchange of the head group or the fatty acids to yield the final phospholipid product. In eukaryotic cells, phospholipid synthesis occurs primarily on the surfaces of the smooth ER and the inner mitochondrial membrane. Some newly formed phospholipids remain at the site of synthesis, but most are destined for other cellular membranes. Once they arrive, phospholipids can be remodeled within membranes to alter the fatty acid constituents. The process by which water-insoluble phospholipids move from the site of synthesis to the point of their eventual function is not fully understood, but we discuss some mechanisms that have emerged in recent years. Cells Have Two Strategies for Attaching Phospholipid Head Groups Stage 1 of glycerophospholipid synthesis is shared with the pathway to triacylglycerols, the formation of glycerol 3-phosphate by one of the two paths shown in Fig. 21-17. In stage 2, fatty acyl groups are esterified to C-1 and C-2 of L-glycerol 3-phosphate to form phosphatidic acid. Usually, the fatty acid at C-1 is saturated and the one at C-2 is unsaturated. A second route to phosphatidic acid is the phosphorylation of a diacylglycerol by a specific kinase. Eugene P. Kennedy, 1919–2011 In stages 3 and 4, the polar head group of glycerophospholipids is attached through a phosphodiester bond, in which each of two alcohol hydroxyls (one on the polar head group and one on C-3 of glycerol) forms an ester with phosphoric acid (Fig. 21-23). In the biosynthetic process, one of the hydroxyls is first activated by attachment of a nucleotide, cytidine diphosphate (CDP). Cytidine monophosphate is then displaced in a nucleophilic attack by the other hydroxyl (Fig. 21-24). Two strategies are employed by mammals. The CDP is attached either to the diacylglycerol, forming the activated phosphatidic acid CDP-diacylglycerol (strategy 1), or to the hydroxyl of the head group (strategy 2). The central importance of cytidine nucleotides in lipid biosynthesis was discovered by Eugene P. Kennedy in the late 1950s, and this pathway is commonly referred to as the Kennedy pathway. In bacteria, only strategy 1 is used to generate glycerophospholipids. FIGURE 21-23 Final stages of glycerophospholipid biosynthesis: head- group attachment. The phospholipid head group is attached to a diacylglycerol by a phosphodiester bond (shaded light red), formed when phosphoric acid condenses with two alcohols, eliminating two molecules of H2O. FIGURE 21-24 Two general strategies for forming the phosphodiester bond of phospholipids. In both cases, CDP supplies the phosphate group of the phosphodiester bond. Pathways for Phospholipid Biosynthesis Are Interrelated In eukaryotes, many phospholipids are synthesized using strategy 1 in Figure 21-24, and many of the pathways begin with CDP- diacylglycerol. The synthesis of phosphatidylglycerol provides our first example. Beginning with CDP-diacylglycerol (Fig. 21-25), displacement of CMP through nucleophilic attack by the C-1 hydroxyl of glycerol 3-phosphate yields phosphatidylglycerol 3- phosphate. Phosphatidylglycerol 3-phosphate is processed further by cleavage of the phosphate monoester (with release of Pi) to yield phosphatidylglycerol.

FIGURE 21-25 Synthesis of glycerophospholipids in eukaryotes using CDP- diacylglycerol. These glycerophospholipids are synthesized using strategy 1 in Figure 21-24. Phosphatidylglycerol is synthesized by reaction of CDP-diacylglycerol with glycerol-3-phosphate, followed by dephosphorylation. Phosphatidylglycerol can react with CDP-diacylglycerol to generate cardiolipin. Phosphatidylinositol is generated from CDP-diacylglycerol in a single step. The pathway from CDP-diacylglycerol to phosphatidylserine is used in yeast but not in mammals. The pathway from phosphatidylserine to phosphatidylethanolamine and phosphatidylcholine is common to all eukaryotes. In eukaryotes, cardiolipin is a relatively uncommon phospholipid, found almost exclusively in the inner membranes of mitochondria. As described below, cardiolipin is important in bacteria, and its presence in mitochondria is likely yet another relic of the bacterial origin of these organelles. Cardiolipin is essential for the function of some mitochondrial enzymes. It is also synthesized using strategy 1, by condensation of CDP- diacylglycerol with phosphatidylglycerol (Fig. 21-25). Phosphatidylinositol is similarly synthesized by condensation of CDP-diacylglycerol with inositol (Fig. 21-25). Specific phosphatidylinositol kinases then convert phosphatidylinositol to its phosphorylated derivatives. Phosphatidylinositol and its phosphorylated products in the plasma membrane play a central role in signal transduction in eukaryotes (see Figs. 12-11, 12-15, 12-23). Yeast (but not mammals) use a similar path to produce phosphatidylserine by condensation of CDP-diacylglycerol and serine, and they can synthesize phosphatidylethanolamine from phosphatidylserine in the reaction catalyzed by phosphatidylserine decarboxylase (Fig. 21-25). This pathway for synthesis of phosphatidylethanolamine occurs primarily in the mitochondria, although this lipid is transported from there to other cellular membranes. Phosphatidylethanolamine may be converted to phosphatidylcholine (lecithin) by the addition of three methyl groups to its amino group; S-adenosylmethionine is the methyl group donor (see Fig. 18-18) for all three methylation reactions. In mammals, strategy 2 (Fig. 21-24) is utilized for the synthesis of phosphatidylethanolamine and phosphatidylcholine in the membranes of the ER and nucleus. The activation of the head group to the CDP derivative is followed by condensation with diacylglycerol as shown for phosphatidylcholine (Fig. 21-26a). These pathways serve to salvage free ethanolamine and choline. In contrast, mammalian phosphatidylserine biosynthesis does not utilize either strategy shown in Figure 21-24; instead, it is derived from phosphatidylethanolamine or phosphatidylcholine via one of two head-group exchange reactions carried out in the ER (Fig. 21-26b). These reactions generate free ethanolamine and choline, respectively. The major sources of phosphatidylethanolamine and phosphatidylcholine in all eukaryotic cells are summarized in Figure 21-27. FIGURE 21-26 Pathways for phosphatidylserine and phosphatidylcholine synthesis in mammals. (a) Phosphatidylserine is synthesized by Ca2+-dependent head-group exchange reactions promoted by phosphatidylserine synthase 1 (PSS1) or phosphatidylserine synthase 2 (PSS2). PSS1 can use either phosphatidylethanolamine or phosphatidylcholine as a substrate. (b) The same strategy shown here for phosphatidylcholine synthesis (strategy 2 in Fig. 21-24) is also used for salvaging ethanolamine in phosphatidylethanolamine synthesis. FIGURE 21-27 Summary of the pathways for synthesis of major phospholipids and triacylglycerols in eukaryotes. Phosphatidic acid is formed by transacylation of -glycerol 3-phosphate with two fatty acyl groups donated from fatty acyl–CoA. The enzyme phosphatidic acid phosphatase (lipin) converts phosphatidic acid to diacylglycerol, which in the Kennedy pathway condenses with a CDP-activated head group (ethanolamine or choline) to form phosphatidylethanolamine or phosphatidylcholine. Alternatively, phosphatidic acid can be activated with a CDP moiety, which is displaced by condensation with a head-group alcohol — inositol, glycerol 3-phosphate, or serine, forming phosphatidylinositol, phosphatidylglycerol, or (only in yeast and fungi) phosphatidylserine. Decarboxylation of phosphatidylserine yields phosphatidylethanolamine, and methylation of phosphatidylethanolamine produces phosphatidylcholine. In mammals, phosphatidylserine and phosphatidylcholine are generated via the head-group exchange pathways, detailed in Fig. 21-26. Lysophosphatidic acid is phosphatidic acid missing one of the two fatty acyl groups. [Information from G. M. Carman and G.-S. Han, Annu. Rev. Biochem. 80:859, 2011, Fig. 2.] The most prominent phospholipids in bacteria are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin. The pathway for phosphatidylglycerol synthesis (Fig. 21-28) is identical to the path employed in mammals (compare to Fig. 21-25), beginning with CDP-diacylglycerol and using strategy 1. Phosphatidylethanolamine is produced in a similar pathway, with phosphatidylserine an intermediate. In bacteria, there are multiple biosynthetic paths to the third prominent phospholipid, cardiolipin, in which two diacylglycerols are joined through a common head group (Fig. 21-28).

FIGURE 21-28 Origin of the polar head groups of phospholipids in E. coli. Initially, a head group (either serine or glycerol 3-phosphate) is attached via a CDP-diacylglycerol intermediate (strategy 1 in Fig. 21-24). For phospholipids other than phosphatidylserine, the head group is further modified, as shown here. In the enzyme names, PG represents phosphatidylglycerol; PS, phosphatidylserine. Cardiolipin can be generated from either phosphatidylglycerol or phosphatidylethanolamine via multiple pathways, as shown. One of these pathways, in which phosphatidylglycerol is condensed with CDP-diacylglycerol, is identical to the pathway used in eukaryotes. Eukaryotic Membrane Phospholipids Are Subject to Remodeling In principle, the two fatty acyl groups esterified to C-1 and C-2 in a phospholipid may vary in length and degree of desaturation, thus altering the properties of the membrane of which it is a part. Phosphatidylcholine is the major structural phospholipid of mammalian membranes, oen representing 40% to 50% of the total. Thus, much of the remodeling centers on phosphatidylcholine. The remodeling occurs largely by a process called the Lands cycle (Fig. 21-29), which replaces the polyunsaturated fatty acyl group at C-2. The fatty acyl moieties are first hydrolyzed by phospholipase A2 (Fig. 10-14) to generate 1-acyl lysophospholipids. The fatty acid is then replaced by a class of enzymes called lysophosphatidylcholine acyltransferases, or LPCATs. There are at least four LPCATs in humans, each with distinct tissue distributions and substrate specificities. FIGURE 21-29 The Lands cycle for phospholipid remodeling. Phosphatidylcholine, which is the most common phospholipid of eukaryotic membranes, is a major target of this process. Fatty acids at C-2 are removed by phospholipase A2 enzymes. A new fatty acid is then introduced by the action of a lysophosphatidylphocholine acyltransferase (LPCAT). The four mammalian LPCATs differ in tissue distribution and substrate specificity. The physiological effects of LPCAT enzymes go far beyond the alteration of the lipid composition of membranes. LPCAT3, the most widely distributed version of the enzyme, helps to regulate lipogenesis and secretion of very-low-density lipoproteins (VLDLs), described later in this chapter. Mice lacking LPCAT3 have a greatly reduced intake of fatty acids in the intestine, resulting in the release of gut hormones that control appetite. They are unable to survive on a high-fat diet, they resist eating, and they die of starvation unless the diet is changed. LPCATs play a demonstrable but still oen mysterious role in processes from atherosclerosis to obesity to cancer, making these enzymes the subjects of increasing interest as research and drug targets. Plasmalogen Synthesis Requires Formation of an Ether-Linked Fatty Alcohol The biosynthetic pathway to ether lipids, including plasmalogens and the platelet-activating factor (see Fig. 10-9), requires displacement of an esterified fatty acyl group by a long- chain alcohol to form the ether linkage (Fig. 21-30). Head-group attachment follows, by mechanisms essentially like those used in formation of the common ester-linked phospholipids. Finally, the characteristic double bond of plasmalogens is introduced by the action of a mixed-function oxidase similar to that responsible for desaturation of fatty acids (Fig. 21-13). The peroxisome is the primary site of plasmalogen synthesis. FIGURE 21-30 Synthesis of ether lipids and plasmalogens. The newly formed ether linkage is shaded light red. The intermediate 1-alkyl-2-acylglycerol 3-phosphate is the ether analog of phosphatidic acid. Mechanisms for attaching head groups to ether lipids are essentially the same as for their ester-linked analogs. The characteristic double bond of plasmalogens (shaded blue) is introduced in a final step by a mixed-function oxidase system similar to fatty acyl–CoA desaturase. Sphingolipid and Glycerophospholipid Synthesis Share Precursors and Some Mechanisms The biosynthesis of sphingolipids takes place in four stages: (1) synthesis of the 18-carbon amine sphinganine from palmitoyl-CoA and serine; (2) attachment of a fatty acid in amide linkage to yield N-acylsphinganine; (3) desaturation of the sphinganine moiety to form N-acylsphingosine (ceramide); and (4) attachment of a head group to produce a sphingolipid such as a cerebroside or sphingomyelin (Fig. 21-31). The first few steps of this pathway occur in the ER; the attachment of head groups in stage 4 occurs in the Golgi complex. The pathway shares several features with the pathways leading to glycerophospholipids: NADPH provides reducing power, and fatty acids enter as their activated CoA derivatives. In cerebroside formation, sugars enter as their activated nucleotide derivatives. Head-group attachment in sphingolipid synthesis has several novel aspects. For example, phosphatidylcholine, rather than CDP-choline, serves as the donor of phosphocholine in the synthesis of sphingomyelin. FIGURE 21-31 Biosynthesis of sphingolipids. Condensation of palmitoyl- CoA and serine, forming β -ketosphinganine, followed by reduction with NADPH, yields sphinganine, which is then acylated to N-acylsphinganine (a ceramide). The sphingosine is shaded gray. In animals, a double bond (shaded light red) is created by a mixed-function oxidase before the final addition of a head group: phosphatidylcholine, to form sphingomyelin, or glucose, to form a cerebroside. In glycolipids — the cerebrosides and gangliosides (see Fig. 10-11) — the head-group sugar is attached directly to the C-1 hydroxyl of sphingosine in glycosidic linkage rather than through a phosphodiester bond. The sugar donor is a UDP-sugar (UDP- glucose or UDP-galactose). Polar Lipids Are Targeted to Specific Cellular Membranes Membrane lipids are insoluble in water, so they cannot simply diffuse from their point of synthesis (the ER) to their point of insertion. Instead, they are transported from the ER to the Golgi complex, where additional synthesis can take place. They are then delivered in membrane vesicles that bud from the Golgi complex and then move to and fuse with the target membrane (see Fig. 11-4). Sphingolipid transfer proteins carry ceramide from the ER to the Golgi complex, where sphingomyelin synthesis occurs. Cytosolic proteins also bind phospholipids and sterols and transport them between cellular membranes (see Fig. 11-7). These mechanisms contribute to establishment of the characteristic lipid compositions of organelle membranes (see Fig. 11-5). SUMMARY 21.3 Biosynthesis of Membrane Phospholipids Beginning with diacylglycerol precursors, there are two pathways for adding head groups to phospholipids. Either the diacylglycerol (strategy 1) or the head group (strategy 2) is activated by CDP. In eukaryotes, phospholipid biosynthetic strategies vary with subcellular location. The major phospholipids phosphatidylethanolamine and phosphatidylcholine are synthesized using strategy 1 in the mitochondria and strategy 2 in the ER and nucleus. Phosphatidylserine is derived from head- group exchange with phosphatidylethanolamine or phosphatidylcholine. In mammals, phospholipids are remodeled in membranes via the Lands cycle. Remodeling is facilitated by lysophosphatidylcholine acyltransferases. The characteristic double bond in plasmalogens is introduced by a mixed-function oxidase. The head groups of sphingolipids are attached by unique mechanisms. Phospholipids travel to their intracellular destinations via transport vesicles or specific proteins. 21.4 Cholesterol, Steroids, and Isoprenoids:Biosynthesis, Regulation, and Transport Cholesterol is doubtless the most publicized lipid, notorious because of the strong correlation between high levels of cholesterol in the blood and the incidence of human cardiovascular diseases. Less well advertised is cholesterol’s crucial role as a component of cellular membranes and as a precursor of steroid hormones and bile acids. Cholesterol is an essential molecule in many animals, including humans, but is not required in the mammalian diet — all cells can synthesize it from simple precursors. The structure of this 27-carbon compound suggests a complex biosynthetic pathway, but all of its carbon atoms are provided by a single precursor — acetate. The isoprene units that are the essential intermediates in the pathway from acetate to cholesterol are also precursors to many other natural lipids, and the mechanisms by which isoprene units are polymerized are similar in all these pathways. We begin with an account of the main steps in the biosynthesis of cholesterol from acetate, and then discuss the transport of cholesterol in the blood, its uptake by cells, the normal regulation of cholesterol synthesis, and its regulation in those with defects in cholesterol uptake or transport. We next consider other cellular components derived from cholesterol, such as bile acids and steroid hormones. Finally, an outline of the biosynthetic pathways to some of the many compounds derived from isoprene units, which share early steps with the pathway to cholesterol, illustrates the extraordinary versatility of isoprenoid condensations in biosynthesis. Cholesterol Is Made from Acetyl-CoA in FourStages Cholesterol, like long-chain fatty acids, is made from acetyl-CoA. But the assembly plan of cholesterol is quite different from that of long-chain fatty acids. In early experiments, animals were fed acetate labeled with 14C in either the methyl carbon or the carboxyl carbon. The pattern of labeling in the cholesterol isolated from the two groups of animals in these tracer experiments (Fig. 21-32) provided the blueprint for working out the enzymatic steps in cholesterol biosynthesis. FIGURE 21-32 Origin of the carbon atoms of cholesterol. This was deduced from tracer experiments with acetate labeled in the methyl carbon (black) or the carboxyl carbon (red). The individual rings in the fused- ring system are designated A through D. Synthesis takes place in four stages, as shown in Figure 21-33: condensation of three acetate units to form a six-carbon intermediate, mevalonate; conversion of mevalonate to activated isoprene units; polymerization of six 5-carbon isoprene units to form the 30-carbon linear squalene; and cyclization of squalene to form the four rings of the steroid nucleus, with a further series of changes (oxidations, removal or migration of methyl groups) to produce cholesterol. FIGURE 21-33 Summary of cholesterol biosynthesis. Isoprene units in squalene are set off by dashed red lines. Stage Synthesis of Mevalonate from Acetate The first stage in cholesterol biosynthesis leads to the intermediate mevalonate (Fig. 21-34). Two molecules of acetyl-CoA condense to form acetoacetyl-CoA, which condenses with a third molecule of acetyl-CoA to yield the six-carbon compound β - hydroxy-β -methylglutaryl-CoA (HMG-CoA). These first two reactions are catalyzed by acetyl-CoA acetyl transferase and HMG-CoA synthase, respectively. Both reactions are Claisen condensations, and the standard equilibrium in each case favors degradation to acetyl-CoA. However, in cells, the synthetic reactions are facilitated by the rapid utilization of the product HMG-CoA in subsequent reactions. The cytosolic HMG-CoA synthase in this pathway is distinct from the mitochondrial isozyme that catalyzes HMG-CoA synthesis in ketone body formation (see Fig. 17-16).

FIGURE 21-34 Formation of mevalonate from acetyl-CoA. The origin of C-1 and C-2 of mevalonate from acetyl-CoA is shaded light red. The third reaction is the committed step: reduction of HMG-CoA to mevalonate, for which two molecules of NADPH each donate two electrons. HMG-CoA reductase, an integral membrane protein of the smooth ER, is the major point of regulation on the pathway to cholesterol, as we shall see. Stage Conversion of Mevalonate to Two ActivatedIsoprenes In the next stage, three phosphate groups are transferred from three ATP molecules to mevalonate (Fig. 21-35). The phosphate attached to the C-3 hydroxyl group of mevalonate in the intermediate 3-phospho-5-pyrophosphomevalonate is a good leaving group; in the next step, both this phosphate and the nearby carboxyl group leave, producing a double bond in the five-carbon product, Δ3-isopentenyl pyrophosphate. This is the first of the two activated isoprenes central to cholesterol formation. Isomerization of Δ3-isopentenyl pyrophosphate yields the second activated isoprene, dimethylallyl pyrophosphate. Synthesis of isopentenyl pyrophosphate in the cytoplasm of plant cells follows the pathway described here. However, plant chloroplasts and many bacteria use a mevalonate-independent pathway. This alternative pathway does not occur in animals, so it is an attractive target for the development of new antibiotics. FIGURE 21-35 Conversion of mevalonate to activated isoprene units. Six of these activated units combine to form squalene (see Fig. 21-36). The leaving groups of 3-phospho-5-pyrophosphomevalonate are shaded light red. The bracketed intermediate is hypothetical. Both of these isoprene products are required for the next stage in cholesterol biosynthesis. Stage Condensation of Six Activated Isoprene Unitsto Form Squalene Isopentenyl pyrophosphate and dimethylallyl pyrophosphate now undergo a head-to- tail condensation, in which one pyrophosphate group is displaced and a 10-carbon chain, geranyl pyrophosphate, is formed (Fig. 21-36). (The “head” is the end to which pyrophosphate is joined.) Geranyl pyrophosphate undergoes another head-to-tail condensation with isopentenyl pyrophosphate, yielding the 15-carbon intermediate farnesyl pyrophosphate. Finally, two molecules of farnesyl pyrophosphate join head to head, with the elimination of both pyrophosphate groups, to form squalene. Squalene has 30 carbons: 24 in the main chain and 6 in the form of methyl group branches. FIGURE 21-36 Formation of squalene. This 30-carbon structure arises through successive condensations of activated isoprene (five-carbon) units. The common names of these intermediates derive from the sources from which they were first isolated. Geraniol, a component of rose oil, has the aroma of geraniums, and farnesol is an aromatic compound found in flowers of the Farnese acacia tree. Many natural scents of plant origin are synthesized from isoprene units. Squalene was first isolated from the liver of sharks (genus Squalus). Stage Conversion of Squalene to the Four-RingSteroid Nucleus When the squalene molecule is represented as in Figure 21-37, the relationship of its linear structure to the cyclic structure of the sterols becomes apparent. All sterols have the four fused rings that form the steroid nucleus, and all are alcohols, with a hydroxyl group at C-3 — thus the name “sterol.” The action of squalene monooxygenase adds one oxygen atom from O2 to the end of the squalene chain, forming an epoxide. This enzyme is another mixed-function oxidase; NADPH reduces the other oxygen atom of O2 to H2O. The double bonds of the product, squalene 2,3-epoxide, are positioned so that a remarkable concerted reaction can convert the linear squalene epoxide to a cyclic structure. In animal cells, this cyclization results in the formation of lanosterol, which contains the four rings characteristic of the steroid nucleus. Lanosterol is finally converted to cholesterol in a series of about 20 reactions that include the migration of some methyl groups and the removal of others. Elucidation of this extraordinary biosynthetic pathway, one of the most complex known, was accomplished by Konrad Bloch, Feodor Lynen, John Cornforth, and George Popják in the late 1950s. FIGURE 21-37 Ring closure converts linear squalene to the condensed steroid nucleus. The first step in this sequence is catalyzed by a mixed-function oxygenase, for which the cosubstrate is NADPH. The product is an epoxide, which in the next step is cyclized to the steroid nucleus. The final product of these reactions in animal cells is cholesterol; in other organisms, slightly different sterols are produced, as shown. Cholesterol is the sterol characteristic of animal cells; plants, fungi, and protists make other, closely related sterols instead. They use the same synthetic pathway as far as squalene 2,3-epoxide, at which point the pathways diverge slightly, yielding other sterols, such as stigmasterol in many plants and ergosterol in fungi (Fig. 21-37). WORKED EXAMPLE 21-1 Energetic Cost ofSqualene Synthesis What is the energetic cost of the synthesis of squalene from acetyl-CoA, in number of ATPs per molecule of squalene synthesized? SOLUTION: In the pathway from acetyl-CoA to squalene, ATP is consumed only in the steps that convert mevalonate to the activated isoprene precursors of squalene. Three ATP molecules are used to create each of the six activated isoprenes required to construct squalene, for a total cost of 18 ATP molecules. Cholesterol Has Several Fates Most of the cholesterol synthesis in vertebrates takes place in the liver. A small fraction of the cholesterol made there is incorporated into the membranes of hepatocytes, but most of it is exported in one of three forms: as bile acids, as biliary cholesterol, or as cholesteryl esters (Fig. 21-38). Small quantities of oxysterols such as 25- hydroxycholesterol are formed in the liver and act as regulators of cholesterol synthesis (see below). In other tissues, cholesterol is converted into steroid hormones (in the adrenal cortex and gonads, for example; see Fig. 10-18) or into vitamin D hormone (in the liver and kidney; see Fig. 10-19). Such hormones are extremely potent biological signals acting through nuclear receptor proteins. FIGURE 21-38 Metabolic fates of cholesterol. Modifications of the cholesterol structure are shown in red. Esterification converts cholesterol to an even more hydrophobic form for storage and transport; each of the other modifications yields a less hydrophobic product. Bile acids, one of the three forms of cholesterol exported from the liver, are the principal components of bile, a fluid stored in the gallbladder and excreted into the small intestine to aid in the digestion of fat-containing meals. Bile acids and their salts are relatively hydrophilic cholesterol derivatives that serve as emulsifiers in the intestine, converting large particles of fat into tiny micelles and thereby greatly increasing the surface at which digestive lipases can act (see Fig. 17-1). Bile also contains much smaller amounts of cholesterol (biliary cholesterol). Bile helps remove excess cholesterol from the intestine and facilitates excretion. Dietary fiber can enhance this effect by binding to bile and interfering with bile reabsorption in the intestines, leading to increased bile excretion in the feces. More cholesterol is then used to make bile. The soluble fiber available in oats (oatmeal) and barley is especially effective. Cholesteryl esters are formed in the liver through the action of acyl-CoA–cholesterol acyltransferase (ACAT). This enzyme catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group of cholesterol (Fig. 21-38), converting the cholesterol to a more hydrophobic form that is no longer sufficiently amphipathic to function appropriately in membranes. Cholesteryl esters are transported in secreted lipoprotein particles to other tissues that use cholesterol, or they are stored in the liver in lipid droplets. Cholesterol and Other Lipids Are Carried onPlasma Lipoproteins Cholesterol and cholesteryl esters, like triacylglycerols and phospholipids, are essentially insoluble in water, yet they must be moved from the tissue of origin to the tissues in which they will be stored or consumed. They are carried in the blood plasma as plasma lipoproteins, macromolecular complexes of specific carrier proteins, called apolipoproteins, and various combinations of phospholipids, cholesterol, cholesteryl esters, and triacylglycerols. Apolipoproteins (“apo” designates the protein in its lipid-free form) combine with lipids to form several classes of lipoprotein particles, spherical complexes with hydrophobic lipids in the core and hydrophilic amino acid side chains at the surface (Fig. 21-39a, b). Different combinations of lipids and proteins produce particles of different densities, ranging from chylomicrons to high-density lipoproteins. These particles can be separated by ultracentrifugation (Table 21-1) and visualized by electron microscopy (Fig. 21-39c). FIGURE 21-39 Lipoproteins. (a) Structure of a chylomicron. Apolipoprotein B-48 (apoB-48) defines the chylomicron. Through the life cycle of a chylomicron other apolipoproteins, including apoC-II, apoC-III, and apoE, become part of the particle, acting as signals in the uptake and metabolism of chylomicron contents. Chylomicrons range from about 100 to 500 nm in diameter. (b) Structure of a low-density lipoprotein (LDL). Apolipoprotein B-100 (apoB-100) is one of the largest single polypeptide chains known, with 4,636 amino acid residues (Mr512,000). One particle of LDL contains a core with about 1,500 molecules of cholesteryl esters, surrounded by a shell composed of about 500 more molecules of cholesterol, 800 molecules of phospholipids, and one molecule of apoB-100. (c) Four classes of lipoproteins, visualized in the electron microscope aer negative staining. The chylomicrons shown here are 50 to 200 nm in diameter; VLDL, 28 to 70 nm; LDL, 20 to 25 nm; and HDL, 8 to 11 nm. Particle sizes given are those measured for these samples; particle sizes vary considerably in different preparations. For properties of lipoproteins, see Table 21-1. [(b) Data for apoB-100 from A. Johs et al., J. Biol. Chem. 281:19,732, 2006. (c) Robert Hamilton, Jr., PhD.] TABLE 21-1 Major Classes of Human Plasma Lipoproteins: Some Properties Composition (wt %) Lipoprotein Density (g/mL) Protein Phospholipids Free cholesterol Cholesteryl esters Triacylglycerols Chylomicrons        <1.006    2    9    1    3 85 VLDL 0.95–1.006 10 18 7 12 50 LDL 1.006– 1.063 23 20 8 37 10 HDL 1.063– 1.210 55 24 2 15    4 Data from D. Kritchevsky, Nutr. Int. 2:290, 1986. Each class of lipoprotein has a specific function, determined by its point of synthesis, lipid composition, and apolipoprotein content. At least 10 distinct apolipoproteins are found in the lipoproteins of human plasma (Table 21-2), distinguishable by their size, their reactions with specific antibodies, and their characteristic distribution in the lipoprotein classes. These protein components act as signals, targeting lipoproteins to specific tissues or activating enzymes that act on the lipoproteins. Figure 21-40 provides an overview of the formation and transport of the lipoproteins in mammals. The numbered steps in the following discussion refer to this figure. TABLE 21-2 Apolipoproteins of the Human Plasma Lipoproteins Apolipoprotein Polypeptide molecular weight Lipoprotein association Function (if known) ApoA-I 28,100 HDL Activates LCAT; interacts with ABC transporter ApoA-II 17,400 HDL Inhibits LCAT ApoA-IV 44,500 Chylomicrons, HDL Activates LCAT; cholesterol transport/clearance ApoB-48 242,000 Chylomicrons Cholesterol transport/clearance ApoB-100 512,000 VLDL, LDL Binds to LDL receptor ApoC-I    7,000 VLDL, HDL ApoC-II    9,000 Chylomicrons, VLDL, HDL Activates lipoprotein lipase ApoC-III    9,000 Chylomicrons, VLDL, HDL Inhibits lipoprotein lipase ApoD 32,500 HDL ApoE 34,200 Chylomicrons, VLDL, HDL Triggers clearance of VLDL and chylomicron remnants ApoH 50,000 Possibly VLDL, binds phospholipids such as cardiolipin Roles in coagulation, lipid metabolism, apoptosis, inflammation Information from D. E. Vance and J. E. Vance (eds), Biochemistry of Lipids and Membranes, 5th edn, Elsevier Science Publishing, 2008. FIGURE 21-40 Lipoproteins and lipid transport. Lipids are transported in the bloodstream as lipoproteins, which exist as several variants that have different functions, different protein and lipid compositions (see Tables 21-1 and 21-2), and thus different densities. Numbered steps are described in the text. In the exogenous pathway (blue arrows), dietary lipids are packaged into chylomicrons; fatty acids from triacylglycerol (TAG) are released by lipoprotein lipase to adipose and muscle tissues, during transport through capillaries. Chylomicron remnants (containing largely protein and cholesterol) are taken up by the liver. Bile salts produced in the liver aid in dispersing dietary fats and are then reabsorbed in the enterohepatic pathway (green arrows). In the endogenous pathway (red arrows), lipids synthesized or packaged in the liver are delivered to peripheral tissues by VLDL. Extraction of lipid from VLDL (along with loss of some apolipoproteins) gradually converts some of it to LDL, which delivers cholesterol to extrahepatic tissues or returns to the liver. Excess cholesterol in extrahepatic tissues is transported back to the liver as HDL in reverse cholesterol transport (purple arrows). C represents cholesterol; CE, cholesteryl ester. Chylomicrons, discussed in Chapter 17 in connection with the movement of dietary triacylglycerols from the intestine to other tissues, are the first of four classes of lipoproteins we will discuss. These are the largest of the lipoproteins and the least dense, containing a high proportion of triacylglycerols. Chylomicrons are synthesized from dietary fats in the ER of enterocytes, epithelial cells that line the small intestine. The chylomicrons then move through the lymphatic system and enter the bloodstream via the le subclavian vein. The apolipoproteins of chylomicrons include apoA-IV, apoB-48 (unique to this class of lipoproteins), apoE, apoC-II, and apoC- III (Table 21-2). ApoC-II activates lipoprotein lipase in the capillaries of adipose, heart, skeletal muscle, and lactating mammary tissues, allowing the release of free fatty acids (FFA) to these tissues. Chylomicrons thus carry dietary fatty acids to tissues where they will be consumed or stored as fuel. The remnants of chylomicrons, depleted of most of their triacylglycerols but still containing cholesterol, apoE, and apoB-48, move through the bloodstream to the liver. Receptors in the liver bind to the apoE in the chylomicron remnants and mediate uptake of these remnants by endocytosis. In the liver, the remnants release their cholesterol and are degraded in lysosomes. This pathway from dietary cholesterol to the liver is the exogenous pathway (blue arrows in Fig. 21-40). Very-low-density lipoprotein (VLDL) is the second of the four classes. When the diet contains more fatty acids and cholesterol than are needed immediately as fuel or as precursors to other molecules, they are converted to triacylglycerols or cholesteryl esters in the liver and packaged with specific apolipoproteins into VLDL. Excess carbohydrate in the diet can also be converted to triacylglycerols in the liver and exported as VLDL. In addition to triacylglycerols and cholesteryl esters, VLDL contains apoB-100, apoC-I, apoC-II, apoC-III, and apoE (Table 21-2). VLDL is transported in the blood from the liver to muscle and adipose tissue. In the capillaries of these tissues, apoC-II activates lipoprotein lipase, which catalyzes the release of free fatty acids from triacylglycerols in the VLDL. Adipocytes take up these fatty acids, reconvert them to triacylglycerols, and store the products in intracellular lipid droplets; myocytes, in contrast, primarily oxidize the fatty acids to supply energy. When the insulin level is high (aer a meal), VLDL serves primarily to convey lipids from the diet to adipose tissue for storage. In the fasting state between meals, the fatty acids used to produce VLDL in the liver originate primarily from adipose tissue, and the principal VLDL target is myocytes of the heart and skeletal muscle. Low-density lipoprotein (LDL), the third class of lipoprotein, is formed when triacylglycerol loss converts some VLDL to VLDL remnants, also called intermediate- density lipoprotein (IDL). Further removal of triacylglycerol from IDL (remnants) produces LDL. Rich in cholesterol and cholesteryl esters, and containing apoB-100 as its major apolipoprotein, LDL carries cholesterol to extrahepatic tissues such as muscle, adrenal glands, and adipose tissue. These tissues have plasma membrane LDL receptors that recognize apoB-100 and mediate uptake of cholesterol and cholesteryl esters. LDL also delivers cholesterol to macrophages, sometimes converting them into foam cells (see Fig. 21-46). LDL not taken up by peripheral tissues and cells returns to the liver and is taken up via LDL receptors in the hepatocyte plasma membrane. Cholesterol that enters hepatocytes by this path may be incorporated into membranes, converted to bile acids, or reesterified by ACAT (Fig. 21-38) for storage within cytosolic lipid droplets. This pathway, from VLDL formation in the liver to LDL return to the liver, is the endogenous pathway of cholesterol metabolism and transport (red arrows in Fig. 21-40). Accumulation of excess intracellular cholesterol is prevented by reducing the rate of cholesterol synthesis when sufficient cholesterol is available from LDL in the blood. Regulatory mechanisms to accomplish this are described below. HDL Carries Out Reverse Cholesterol Transport High-density lipoprotein (HDL), the fourth major lipoprotein in mammals, originates in the liver and small intestine as small, protein-rich particles that contain relatively little cholesterol and no cholesteryl esters (Fig. 21-40). HDLs contain primarily apoA-I and other apolipoproteins (Table 21-2). They also contain the enzyme lecithin-cholesterol acyltransferase (LCAT), which catalyzes the formation of cholesteryl esters from lecithin (phosphatidylcholine) and cholesterol (Fig. 21-41). LCAT on the surface of nascent (newly forming) HDL particles converts the cholesterol and phosphatidylcholine of chylomicron and VLDL remnants encountered in the bloodstream to cholesteryl esters, which begin to form a core, transforming the disk- shaped nascent HDL to a mature, spherical HDL particle. Nascent HDL can also pick up cholesterol from cholesterol-rich extrahepatic cells (including macrophages and foam cells, formed from macrophages; see below). Mature HDL then returns to the liver, where the cholesterol is unloaded via the scavenger receptor SR-BI. Some of the cholesteryl esters in HDL can also be transferred to LDL by the cholesteryl ester transfer protein. The HDL circuit is reverse cholesterol transport (purple arrows in Fig. 21-40). Much of this cholesterol is converted to bile salts by enzymes sequestered in hepatic peroxisomes; the bile salts are stored in the gallbladder and excreted into the intestine when a meal is ingested. Bile salts are reabsorbed by the liver and recirculate through the gallbladder in this enterohepatic circulation (green arrows in Fig. 21-40). FIGURE 21-41 Reaction catalyzed by lecithin-cholesterol acyltransferase (LCAT). This enzyme is present on the surface of HDL and is stimulated by the HDL component apoA-I. Cholesteryl esters accumulate within nascent HDLs, converting them to mature HDLs. The unloading of sterols via SR-BI receptors in liver and other tissues does not occur by endocytosis, the mechanism used for LDL uptake. Instead, when HDL binds to SR-BI receptors in the plasma membranes of hepatocytes or steroidogenic tissues such as the adrenal gland, these receptors mediate partial and selective transfer of cholesterol and other lipids in HDL into the cell. Depleted HDL then dissociates to recirculate in the bloodstream and extract more lipids from remnants of chylomicrons and VLDL, and from cells overloaded with cholesterol, as described below. Cholesteryl Esters Enter Cells by Receptor-Mediated Endocytosis Each LDL particle in the bloodstream contains apoB-100, which is recognized by LDL receptors present in the plasma membranes of cells that need to take up cholesterol. Figure 21-42 shows such a cell. LDL receptors are synthesized in the ER and transported to the plasma membrane, aer modification in the Golgi complex. At the plasma membrane, they are available to bind apoB-100. Binding of LDL to an LDL receptor initiates endocytosis, which conveys the LDL and its receptor into the cell within an endosome. The receptor-containing portions of the endosome membrane bud off and are returned to the cell surface, to function again in LDL uptake. The endosome fuses with a lysosome, which contains enzymes that hydrolyze the cholesteryl esters, releasing cholesterol and fatty acids into the cytosol. The apoB-100 protein is also degraded to amino acids that are released to the cytosol. ApoB-100 is also present in VLDL, but its receptor-binding domain is not available for binding to the LDL receptor; conversion of VLDL to LDL exposes the receptor-binding domain of apoB-100. FIGURE 21-42 Uptake of cholesterol by receptor-mediated endocytosis. This pathway for the transport of cholesterol in blood and its receptor-mediated endocytosis by target tissues was elucidated by Michael Brown and Joseph Goldstein. They discovered that individuals with the genetic disease familial hypercholesterolemia (FH) have mutations in the LDL receptor that prevent the normal uptake of LDL by liver and peripheral tissues. The result of defective LDL uptake is very high blood levels of LDL (and of the cholesterol it carries). Individuals with FH have a greatly increased probability of developing atherosclerosis, a disease of the cardiovascular system in which blood vessels are occluded by cholesterol-rich plaques (see Fig. 21-46). Michael Brown and Joseph Goldstein Niemann-Pick type-C (NPC) disease is an inherited defect in lipid storage. In this disorder, cholesterol is not transported out of the lysosomes and instead accumulates in lysosomes of liver, brain, and lung, bringing about early death. NPC is the result of a mutation in either of two genes, NPC1 and NPC2, essential to moving cholesterol out of the lysosome and into the cytosol, where it can be further metabolized. NPC1 encodes a transmembrane lysosomal protein, and NPC2 encodes a soluble protein. These proteins act in tandem to transfer cholesterol out of the lysosome and into the cytosol for further processing or metabolism.

Cholesterol Synthesis and Transport AreRegulated at Several Levels Cholesterol synthesis is a complex and energy-expensive process. Excess cholesterol cannot be catabolized for use as fuel and must be excreted. Therefore, it is clearly advantageous to an organism to regulate the biosynthesis of cholesterol to complement dietary intake. In mammals, cholesterol production is regulated by intracellular cholesterol concentration, by the supply of ATP, and by the hormones glucagon and insulin. The committed step in the pathway to cholesterol (and a major site of regulation) is the conversion of HMG-CoA to mevalonate (Fig. 21-34), the reaction catalyzed by HMG-CoA reductase. Short-term (minute-to-minute) regulation of the activity of existing HMG-CoA reductase is accomplished by reversible covalent alteration: phosphorylation by the AMP- dependent protein kinase (AMPK), which senses high AMP concentration (indicating low ATP concentration). Thus, when ATP levels drop, the synthesis of cholesterol slows, and catabolic pathways for the generation of ATP are stimulated (Fig. 21-43). Hormones that mediate global regulation of lipid and carbohydrate metabolism also act on HMG- CoA reductase; glucagon stimulates its phosphorylation (inactivation), and insulin promotes dephosphorylation, activating the enzyme and favoring cholesterol synthesis. These covalent regulatory mechanisms are probably not as important, quantitatively, as the mechanisms that affect the synthesis and degradation of the enzyme. FIGURE 21-43 Regulation of cholesterol formation balances synthesis with dietary uptake and energy state. Insulin promotes dephosphorylation (activation) of HMG-CoA reductase; glucagon promotes its phosphorylation (inactivation); and the AMP-dependent protein kinase AMPK, when activated by low [ATP] relative to [AMP], phosphorylates and inactivates HMG-CoA reductase. Oxysterol metabolites of cholesterol (for example, 24(S)-hydroxycholesterol) stimulate proteolysis of HMG-CoA reductase. In the longer term, the number of molecules of HMG-CoA reductase is increased or decreased in response to cellular concentrations of cholesterol. Regulation of HMG- CoA reductase synthesis by cholesterol is mediated by an elegant system of transcriptional regulation of the HMG-CoA gene (Fig. 21-44). This gene, along with more than 20 other genes encoding enzymes that mediate the uptake and synthesis of cholesterol and unsaturated fatty acids, is controlled by a small family of proteins called sterol regulatory element-binding proteins (SREBPs). When newly synthesized, these proteins are embedded in the ER. Only the soluble regulatory domain fragment of an SREBP functions as a transcription activator, through mechanisms discussed in Chapter 28. When cholesterol and oxysterol levels are high, SREBPs are held in the ER in a complex with another protein called SREBP cleavage-activating protein (SCAP), which in turn is anchored in the ER membrane by its interaction with a third membrane protein, Insig (insulin-induced gene protein) (Fig. 21-44a). SCAP and Insig act as sterol sensors. When sterol levels are high, the Insig-SCAP-SREBP complex is retained in the ER membrane. When the level of sterols in the cell declines (Fig. 21- 44b), the SCAP-SREBP complex is escorted by secretory proteins to the Golgi complex. There, two proteolytic cleavages of SREBP release a regulatory fragment, which enters the nucleus and activates transcription of its target genes, including those for HMG-CoA reductase, the LDL receptor protein, and other proteins needed for lipid synthesis. When sterol levels increase sufficiently, the proteolytic release of SREBP amino- terminal domains is again blocked, and proteasome degradation of the existing active domains results in rapid shutdown of the gene targets. FIGURE 21-44 Regulation of cholesterol synthesis by SREBP. Sterol regulatory element-binding proteins (SREBPs) are embedded in the ER when first synthesized, in a complex with the protein SREBP cleavage-activating protein (SCAP), which is in turn bound to Insig. (N and C represent the amino and carboxyl termini of the proteins.) (a) Under normal circumstances (high [sterol]), SCAP and Insig are bound to SREBPs and the SREBPs are inactive. (b) When sterol levels decline, sterol-binding sites on Insig and SCAP are unoccupied, and Insig is targeted for degradation by the attachment of several ubiquitin molecules. The remaining SCAP-SREBP complex migrates to the Golgi complex, and SREBP is cleaved (arrows) to produce a regulatory domain fragment. (c) This domain acts in the nucleus to increase the transcription of sterol-regulated genes. [Information from R. Raghow et al., Trends Endocrinol. Metab. 19:65, 2008, Fig. 2.] The level of HMG-CoA reductase is also regulated by proteolytic degradation of the enzyme itself. High levels of cellular cholesterol are sensed by Insig, which triggers attachment of ubiquitin molecules to HMG-CoA reductase, leading to its degradation by proteasomes. Liver X receptor (LXR) is a nuclear transcription factor activated by oxysterol ligands (reflecting high cholesterol levels), which integrates the metabolism of fatty acids, sterols, and glucose. LXRα is expressed primarily in liver, adipose tissue, and macrophages; LXRβ is present in all tissues. When bound to an oxysterol ligand, LXRs form heterodimers with a second type of nuclear receptor, the retinoid X receptors (RXR), and the LXR-RXR dimer activates transcription from a set of genes (Fig. 21-45), including those for acetyl-CoA carboxylase, the first enzyme in fatty acid synthesis; fatty acid synthase; the cytochrome P-450 enzyme CYP7A1, required for sterol conversion to bile acids; apoproteins that participate in cholesterol transport (apoC-I, apoC-II, apoD, and apoE); the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, required for reverse cholesterol transport (see below); GLUT4, the insulin-stimulated glucose transporter of muscle and adipose tissue; and an SREBP called SREBP1C. The transcriptional regulators LXR and SREBP therefore work together to achieve and maintain cholesterol homeostasis; SREBPs are activated by low levels of cellular cholesterol, and LXRs are activated by high cholesterol levels. FIGURE 21-45 Action of RXR-LXR dimer on expression of genes for lipid and glucose metabolism. When their ligands are absent, RXR and LXR associate with a corepressor protein, preventing transcription of the genes associated with the LXR element (LXRE). When their respective ligands are present — 9-cis-retinoic acid for RXR, cholesterol or oxysterols for LXR — the dimer dissociates from the corepressor, then associates with a coactivator protein. This complex binds to the LXR element and turns on expression of the associated genes. Regulation of gene expression is a topic discussed in more detail in Chapter 28. [Information from A. C. Calkin and P. Tontonoz, Nat. Rev. Mol. Cell Biol. 13:213, 2012, Fig. 1.] Regulation by LXRs is complemented by the activity of farnesoid X receptor (FXR), which also forms a heterodimer with RXR, with an effect that is oen reciprocal to that of LXR-RXR. Although farnesol is a ligand for this receptor, FXR responds primarily to bile acids. High levels of bile acids can be toxic. FXR, expressed mainly in the intestine, liver, kidney, and adrenal glands, provides essential control of bile acid levels by increasing or decreasing expression of multiple genes. FXR represses many of the genes that are activated by LXR. Finally, two other regulatory mechanisms influence cellular cholesterol level: (1) high cellular concentrations of cholesterol activate ACAT, which increases esterification of cholesterol for storage, and (2) high cellular cholesterol levels diminish (via SREBP) transcription of the gene that encodes the LDL receptor, reducing production of the receptor and thus the uptake of cholesterol from the blood. Dysregulation of Cholesterol Metabolism CanLead to Cardiovascular Disease As noted earlier, cholesterol cannot be catabolized by animal cells. Excess cholesterol can be removed only by excretion or by conversion to bile salts. When the sum of cholesterol synthesized and cholesterol obtained in the diet exceeds the amount required for the synthesis of membranes, bile salts, and steroids, pathological accumulations of cholesterol (plaques) can obstruct blood vessels, a condition called atherosclerosis. Heart failure due to occluded coronary arteries is a leading cause of death in industrialized societies. Atherosclerosis is linked to high levels of cholesterol in the blood, and particularly to high levels of LDL-cholesterol (“bad cholesterol”); there is a negative correlation between HDL (“good cholesterol”) levels and arterial disease. Plaque formation in blood vessels is initiated when LDL containing partially oxidized fatty acyl groups adheres to and accumulates in the extracellular matrix of epithelial cells lining arteries (Fig. 21-46). Immune cells (monocytes) are attracted to regions with such LDL accumulations, and they differentiate into macrophages, which take up the oxidized LDL and the cholesterol they contain. Macrophages cannot limit their uptake of sterols, and with increasing accumulation of cholesteryl esters and free cholesterol, the macrophages become foam cells (they appear foamy when viewed under the microscope). As excess free cholesterol accumulates in foam cells and their membranes, the cells undergo apoptosis. Over long periods of time, arteries become progressively occluded as plaques consisting of extracellular matrix material, scar tissue formed from smooth muscle tissue, and foam cell remnants gradually become larger. Within the cholesterol-rich plaques, cholesterol can crystallize (Fig. 21-46, inset). Occasionally, a plaque breaks loose from the site of its formation and is carried through the blood to a narrowed region of an artery in the brain or the heart, causing a stroke or a heart attack. Cholesterol crystals that break loose from plaques can cause vascular injury. FIGURE 21-46 Formation of atherosclerotic plaques. Excess lipid derived from the diet is deposited on arterial walls, a process facilitated by the conversion of monocytes to foam cells and incorporation of foam cells into growing plaques. Some of this deposition is countered by HDL and reverse cholesterol transport. The LCAT reaction is shown in Figure 21- 41. The inset shows a polarized light micrograph of cholesterol crystals. [Ralph C. Eagle, Jr./Science Source.] In familial hypercholesterolemia, blood levels of cholesterol are extremely high and severe atherosclerosis develops in childhood. Affected individuals have a defective LDL receptor and lack receptor-mediated uptake of cholesterol carried by LDL. Consequently, cholesterol is not cleared from the blood; it accumulates in foam cells and contributes to the formation of atherosclerotic plaques. Endogenous cholesterol synthesis continues despite the excessive cholesterol in the blood, because extracellular cholesterol cannot enter cells to regulate intracellular synthesis (Fig. 21-44). Drugs in a class called statins, some isolated from natural sources and some synthesized industrially, are used to treat patients with elevated serum cholesterol caused by familial hypercholesterolemia and other conditions. The statins resemble mevalonate (Box 21-2) and are competitive inhibitors of HMG-CoA reductase. BOX 21-2 MEDICINEThe Lipid Hypothesis and the Development of Statins Coronary heart disease is the leading cause of death in developed countries. The coronary arteries that bring blood to the heart become narrowed due to the formation of fatty deposits called atherosclerotic plaques, containing cholesterol, fibrous proteins, calcium deposits, blood platelets, and cell debris. Developing the link between artery occlusion (atherosclerosis) and blood cholesterol levels was a project of the twentieth century, triggering a dispute that was resolved only with the development of effective cholesterol-lowering drugs. The Framingham Heart Study, a longitudinal study begun in 1948 and continuing today, was aimed at identifying factors correlated with cardiovascular disease. About 5,000 participants from the city of Framingham, Massachusetts, underwent periodic physical examinations and lifestyle interviews. By 2002, participants of the third generation were included in the study. This monumental study led to the identification of risk factors for cardiovascular disease, including smoking, obesity, physical inactivity, diabetes, high blood pressure, and high blood cholesterol. In 1913, N. N. Anitschkov, an experimental pathologist in Saint Petersburg, Russia, published a study showing that rabbits fed a diet rich in cholesterol developed lesions very similar to the atherosclerotic plaques seen in aging humans. Anitschkov continued his work over the next few decades, publishing it in prominent western journals. Nevertheless, the work was not accepted as a model for human atherosclerosis, due to a prevailing view that the disease was simply a consequence of aging and could not be prevented. The link between serum cholesterol and atherosclerosis (the lipid hypothesis) was gradually strengthened, however, until researchers in the 1960s openly suggested that therapeutic intervention might be helpful. Controversy persisted until the results of a large study of cholesterol lowering, sponsored by the U.S. National Institutes of Health, was published in 1984: the Coronary Primary Prevention Trial. This study conclusively showed a statistically significant decrease in heart attacks and strokes as a result of decreasing blood cholesterol level. The study made use of a bile acid–binding resin, cholestyramine, to control cholesterol. The results triggered a search for more effective therapeutic interventions. Some controversy persisted until development of the statins in the late 1980s and 1990s. Dr. Akira Endo, working at the Sankyo company in Tokyo, discovered the first statin and reported the work in 1976. Endo had been interested in cholesterol metabolism for some time, and he speculated in 1971 that the fungi being screened at that time for new antibiotics might also contain an inhibitor of cholesterol synthesis. Over a period of several years, he screened more than 6,000 fungal cultures until a positive result emerged. The compound that resulted was named compactin (Fig. 1). This compound eventually proved effective in reducing cholesterol levels in dogs and monkeys, and the work came to the attention of Michael Brown and Joseph Goldstein at the University of Texas–Southwestern Medical School. Brown and Goldstein began to work with Endo, and they confirmed his results. Some dramatic results in the first limited clinical trials convinced several pharmaceutical firms to join the hunt for statins. A team at Merck, led by Alfred Alberts and P. Roy Vagelos, began screening fungal cultures and found a positive result aer screening just 18 cultures. The new statin was eventually called lovastatin (Fig. 1). In 1980, a rumor that compactin, at very high doses, was carcinogenic in dogs almost sidelined the race to develop statins, but the benefits to people with familial hypercholesterolemia were already evident. Aer much consultation with experts around the world and with the U.S. Food and Drug Administration, Merck proceeded carefully to develop lovastatin. Extensive testing over the next two decades revealed no carcinogenic effects from lovastatin, or from the newer generations of statins that have appeared since. FIGURE 1 Statins as inhibitors of HMG-CoA reductase. A comparison of the structures of mevalonate and four pharmaceutical compounds (statins) that inhibit HMG-CoA reductase. Akira Endo Alfred Alberts (1931–2018) P. Roy Vagelos Statins inhibit HMG-CoA reductase, in part, by mimicking the structure of mevalonate (Fig. 1), and thus inhibit cholesterol synthesis. Lovastatin treatment lowers serum cholesterol by as much as 30% in individuals with hypercholesterolemia resulting from one defective copy of the gene for the LDL receptor. When combined with an edible resin that binds bile acids and prevents their reabsorption from the intestine, the statin is even more effective. Statins are now the most widely used drugs for lowering serum cholesterol levels. Side effects are always a concern with drugs, but in the case of statins, many of the side effects are positive. These drugs can improve blood flow, enhance the stability of atherosclerotic plaques (so they don’t rupture and obstruct blood flow), reduce platelet aggregation, and reduce vascular inflammation. In patients taking statins for the first time, some of these effects occur before cholesterol levels drop and may be related to a secondary inhibition of isoprenoid synthesis. Not all effects of statins are positive. Some individuals, usually among those taking statins in combination with other cholesterol-lowering drugs, experience muscle pain or weakness that can become severe and even debilitating. For these patients, the effects may sometimes be ameliorated by diet supplementation with coenzyme Q10, a coenzyme that statin therapy depletes in the serum. A fairly long list of other side effects has been documented; most are rare. However, for the vast majority of people, the statin-mediated decrease in risks associated with coronary heart disease can be dramatic. As with all medications, statins should be used only in consultation with a physician. An alternative approach to controlling serum cholesterol levels is to activate LXRs, which has the overall effect of decreasing cholesterol absorption and promoting its excretion. This is the mode of action of a drug called ezetimibe. Ezetimibe is not as effective in lowering serum cholesterol as statins. However, in combination with statins, it can lower risk of stroke or heart attack in high-risk patients. Because LXR activation also activates SREBP1C, causing the liver to increase its production of fatty acids and triacylglycerols, new classes of drugs that target only intestinal LXRs are being developed. Reverse Cholesterol Transport by HDL CountersPlaque Formation and Atherosclerosis HDL plays a critical role in the reverse cholesterol transport pathway (Fig. 21-47), reducing the potential damage from foam cell buildup. Depleted HDL (low in cholesterol) picks up cholesterol stored in extrahepatic tissues, including foam cells at nascent plaques, and carries it to the liver. FIGURE 21-47 Reverse cholesterol transport. ApoA-I and HDLs pick up excess cholesterol (C) from peripheral cells, with the participation of ABCA1 and ABCG1 transporters, and return it to the liver. In individuals with genetically defective ABCA1, the failure of reverse cholesterol transport leads to severe and early cardiovascular diseases: Tangier disease and familial HDL deficiency disease. CE, cholesteryl esters; TAG, triacylglycerols. [Information from A. R. Tall et al., Cell Metab. 7:365, 2008, Fig. 1.] Cholesterol movement out of cells requires transporters. The human genome encodes 48 transporters of the ATP-binding cassette (ABC) class, and about half of these promote lipid transport. Two of them transport cholesterol out of cells. In this process, apoA-I interacts with the transporter ABCA1 in a cholesterol-rich cell. ABCA1 transports a load of cholesterol from inside the cell to the outer surface of the plasma membrane, where lipid-free or lipid-poor apoA-I picks it up, then transports it to the liver. Another transporter, ABCG1, interacts with mature HDL, facilitating the movement of cholesterol out of the cell and into the HDL. This efflux process is particularly critical to reverse cholesterol transport away from foam cells at the sites of plaques in the blood vessels of individuals with cardiovascular disease. In familial HDL deficiency, HDL levels are very low, and in Tangier disease they are almost undetectable (Fig. 21-47). Both genetic disorders are the result of mutations in the ABCA1 protein. ApoA-I in cholesterol-depleted HDL cannot take up cholesterol from cells that lack ABCA1 protein, and apoA-I and cholesterol-poor HDL are rapidly removed from the blood and destroyed. Both familial HDL deficiency and Tangier disease are very rare (worldwide, fewer than 100 families with Tangier disease are known), but the existence of these diseases establishes a role for ABCA1 and ABCG1 proteins in the regulation of plasma HDL levels. Steroid Hormones Are Formed by Side-ChainCleavage and Oxidation of Cholesterol Humans derive all their steroid hormones from cholesterol (Fig. 21-48). Two classes of steroid hormones are synthesized in the cortex of the adrenal gland: mineralocorticoids, which control the reabsorption of inorganic ions (Na+,Cl−, and HCO−3) by the kidney, and glucocorticoids, which help regulate gluconeogenesis and reduce the inflammatory response. Sex hormones are produced in male and female gonads and the placenta. They include progesterone, which regulates the female reproductive cycle, and androgens (such as testosterone) and estrogens (such as estradiol), which influence the development of secondary sexual characteristics in males and females, respectively. Steroid hormones are effective at very low concentrations and are therefore synthesized in relatively small quantities. In comparison with the bile salts, their production consumes relatively little cholesterol.

FIGURE 21-48 Some steroid hormones derived from cholesterol. The structures of some of these compounds are shown in Figure 10-18. Synthesis of steroid hormones requires removal of some or all of the carbons in the “side chain” on C-17 of the D ring of cholesterol. Side-chain removal takes place in the mitochondria of steroidogenic tissues. Removal requires the hydroxylation of two adjacent carbons in the side chain (C-20 and C-22) followed by cleavage of the bond between them (Fig. 21-49). Formation of the various hormones also requires the introduction of oxygen atoms. All the hydroxylation and oxygenation reactions in steroid biosynthesis are catalyzed by mixed-function oxygenases (see Box 21-1) that use NADPH, O2, and mitochondrial cytochrome P-450. FIGURE 21-49 Side-chain cleavage in the synthesis of steroid hormones. Cytochrome P-450 acts as electron carrier in this monooxygenase system that oxidizes adjacent carbons. The process also requires the electron-transferring proteins adrenodoxin and adrenodoxin reductase. This system for cleaving side chains is found in mitochondria of the adrenal cortex, where active steroid production occurs. Pregnenolone is the precursor of all other steroid hormones (see Fig. 21-48). Intermediates in Cholesterol Biosynthesis HaveMany Alternative Fates In addition to its role as an intermediate in cholesterol biosynthesis, isopentenyl pyrophosphate is the activated precursor of a huge array of biomolecules with diverse biological roles (Fig. 21-50). They include vitamins A, E, and K; plant pigments such as carotene and the phytol chain of chlorophyll; natural rubber; many essential oils, such as the fragrant principles of lemon oil, eucalyptus, and musk; insect juvenile hormone, which controls metamorphosis; dolichols, which serve as lipid-soluble carriers in complex polysaccharide synthesis; and ubiquinone and plastoquinone, electron carriers in mitochondria and chloroplasts. Collectively, these molecules are called isoprenoids. More than 20,000 different isoprenoid molecules have been discovered in nature, and hundreds of new ones are reported each year. FIGURE 21-50 Overview of isoprenoid biosynthesis. The structures of most of the end products shown here are given in Chapter 10. Prenylation, the covalent attachment of an isoprenoid (see Fig. 27-35), is a common mechanism by which proteins are anchored to the inner surface of cellular membranes in mammals (see Fig. 11-16). In some of these proteins, the attached lipid is the 15- carbon farnesyl group; others have the 20-carbon geranylgeranyl group. Different enzymes attach the two types of lipids. It is possible that prenylation targets proteins to different membranes, depending on which lipid is attached. Protein prenylation is another important role for the isoprene derivatives of the pathway to cholesterol. SUMMARY 21.4 Cholesterol, Steroids, andIsoprenoids: Biosynthesis, Regulation, andTransport Cholesterol is formed from acetyl-CoA in a complex series of reactions, through the intermediates β -hydroxy-β -methylglutaryl-CoA (HMG-CoA), mevalonate, and two activated isoprenes, dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Condensation of isoprene units produces the noncyclic squalene, which is cyclized to yield the steroid ring system and side chain. Synthesized primarily in the liver, cholesterol is exported as bile acids, biliary cholesterol, or cholesteryl esters. Cholesterol and cholesteryl esters are carried in the blood as plasma lipoproteins. VLDL carries cholesterol, cholesteryl esters, and triacylglycerols from the liver to other tissues, where the triacylglycerols are degraded by lipoprotein lipase, converting VLDL to LDL. Cholesterol scavenging and transport back to the liver are mediated by HDL. Much of the lipid carried to the liver is utilized in bile salts. The LDL, rich in cholesterol and its esters, is taken up by receptor-mediated endocytosis, in which the apolipoprotein B-100 of LDL is recognized by receptors in the plasma membrane. Cholesterol synthesis and transport are under complex regulation by hormones, cellular cholesterol content, and energy level (AMP concentration). HMG-CoA reductase is regulated allosterically and by covalent modification. Furthermore, a complex of three proteins—Insig, SCAP, and SREBP — sense cholesterol levels and trigger increased synthesis or degradation of HMG-CoA reductase in response. The number of LDL receptors per cell is also regulated by cholesterol content. Dietary conditions or genetic defects in cholesterol metabolism may lead to atherosclerosis and heart disease. In reverse cholesterol transport, HDL removes cholesterol from peripheral tissues, carrying it to the liver. By reducing the cholesterol content of foam cells, HDL protects against atherosclerosis. The steroid hormones (glucocorticoids, mineralocorticoids, and sex hormones) are produced from cholesterol by alteration of the side chain and introduction of oxygen atoms into the steroid ring system. In addition to cholesterol, a wide variety of isoprenoid compounds are derived from mevalonate through condensations of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Prenylation of certain proteins targets them for association with cellular membranes and is essential for their biological activity. Chapter Review KEY TERMS Terms in bold are defined in the glossary. malonyl-CoA acetyl-CoA carboxylase fatty acid synthase acyl carrier protein (ACP) 4′-phosphopantetheine fatty acid elongation systems fatty acyl–CoA desaturase mixed-function oxidases stearoyl-ACP desaturase (SCD) essential fatty acids arachidonate eicosatetraenoate prostaglandin (PG) cyclooxygenase (COX) prostaglandin H2 synthase mixed-function oxygenases cytochrome P-450 thromboxane synthase thromboxane (TX) leukotriene (LT) catabasis specialized pro-resolving mediator (SPM) lipoxin glycerol 3-phosphate dehydrogenase phosphatidic acid triacylglycerol cycle glyceroneogenesis thiazolidinediones phosphatidylglycerol cardiolipin phosphatidylinositol phosphatidylserine phosphatidylethanolamine phosphatidylcholine Lands cycle lysophosphatidylcholine acyltransferases (LPCATs) plasmalogen platelet-activating factor cerebroside sphingomyelin ganglioside isoprene mevalonate β -hydroxy-β -methylglutaryl-CoA (HMG-CoA) HMG-CoA synthase HMG-CoA reductase squalene bile acids cholesteryl esters apolipoprotein chylomicron exogenous pathway very-low-density lipoprotein (VLDL) low-density lipoprotein (LDL) LDL receptors endogenous pathway high-density lipoprotein (HDL) reverse cholesterol transport enterohepatic circulation receptor-mediated endocytosis sterol regulatory element-binding proteins (SREBPs) SREBP cleavage-activating protein (SCAP) insulin-induced gene protein (Insig) liver X receptor (LXR) retinoid X receptor (RXR) farnesoid X receptor (FXR) atherosclerosis foam cell statin PROBLEMS 1. Pathway of Carbon in Fatty Acid Synthesis Using your knowledge of fatty acid biosynthesis, provide an explanation for the two experimental observations. a. A biochemist adds uniformly labeled [14C]acetyl-CoA to a soluble liver fraction, which yields palmitate uniformly labeled with 14C. b. In a second experiment, the biochemist adds a trace of uniformly labeled [14C]acetyl-CoA in the presence of an excess of unlabeled malonyl-CoA to a soluble liver fraction, which yields palmitate labeled with 14C only in C-15 and C-16. 2. Synthesis of Fatty Acids from Glucose Aer a person has ingested large amounts of sucrose, the body transforms the glucose and fructose that exceed caloric requirements to fatty acids for triacylglycerol synthesis. This fatty acid synthesis consumes acetyl-CoA, ATP, and NADPH. How do cells produce acetyl-CoA, ATP, and NADPH from glucose? 3. Net Equation of Fatty Acid Synthesis Write the net equation for the biosynthesis of palmitate in rat liver, starting from mitochondrial acetyl-CoA and cytosolic NADPH, ATP, and CO2. 4. Pathway of Hydrogen in Fatty Acid Synthesis A researcher has prepared a solution that contains all the enzymes and cofactors necessary for fatty acid biosynthesis from added acetyl-CoA and malonyl-CoA. a. She then adds [2-2H]acetyl-CoA (labeled with deuterium, the heavy isotope of hydrogen) and an excess of unlabeled malonyl-CoA as substrates.

How many deuterium atoms incorporate into every molecule of palmitate? What are their locations? Explain. b. In a separate experiment, the researcher adds unlabeled acetyl-CoA and [2-2H]malonyl-CoA as substrates. How many deuterium atoms incorporate into every molecule of palmitate? What are their locations? Explain. 5. Energetics of β -Ketoacyl-ACP Synthase The condensation reaction catalyzed by β -ketoacyl-ACP synthase (see Fig. 21-6) synthesizes a four-carbon unit by combining a two-carbon unit and a three-carbon unit, with the release of CO2. What is the thermodynamic rationale for this process relative to one that simply combines two two-carbon units? 6. Modulation of Acetyl-CoA Carboxylase Acetyl-CoA carboxylase is the principal regulation point in the biosynthesis of fatty acids. Following are some of the properties of the enzyme: a. Addition of citrate or isocitrate raises the Vm ax of the enzyme as much as 10-fold. b. The enzyme exists in two interconvertible forms that differ markedly in their activities: Protom er(inactive)⇌ filam entouspolym er(active) Citrate and isocitrate bind preferentially to the filamentous form, and palmitoyl-CoA binds preferentially to the protomer. Explain how these properties are consistent with the regulatory role of acetyl-CoA carboxylase in the biosynthesis of fatty acids. 7. Shuttling of Acetyl Groups across the Inner Mitochondrial Membrane The acetyl group shuttle transfers acetyl-CoA, produced by oxidative decarboxylation of pyruvate in the mitochondrion, to the cytosol as outlined in Figure 21-10. a. Write the overall equation for the transfer of one acetyl group from the mitochondrion to the cytosol. b. What is the cost of this process in ATPs per acetyl group? c. In Chapter 17 we encountered an acyl group shuttle in the transfer of fatty acyl–CoA from the cytosol to the mitochondrion in preparation for β oxidation (see Fig. 17-6). One result of that shuttle is separation of the mitochondrial and cytosolic pools of CoA. Does the acetyl group shuttle also accomplish this? Explain. 8. Oxygen Requirement for Desaturases The biosynthesis of palmitoleate (see Fig. 21-12), a common unsaturated fatty acid with a cis double bond in the Δ 9 position, uses palmitate as a precursor. Can palmitoleate synthesis be carried out under strictly anaerobic conditions? Explain. 9. Energy Cost of Triacylglycerol Synthesis Use a net equation for the biosynthesis of tripalmitoylglycerol (tripalmitin) from glycerol and palmitate to show how many ATPs are required per molecule of tripalmitin formed. 10. Turnover of Triacylglycerols in Adipose Tissue A researcher adds [14C]glucose to the balanced diet of adult rats. She finds no increase in the total amount of stored triacylglycerols, but the triacylglycerols become labeled with 14C. Explain. 11. Energy Cost of Phosphatidylcholine Synthesis Write the sequence of steps and the net reaction for the biosynthesis of phosphatidylcholine by the salvage pathway from oleate, palmitate, dihydroxyacetone phosphate, and choline. Starting from these precursors, what is the cost (in number of ATPs) of the synthesis of phosphatidylcholine by the salvage pathway? 12. Salvage Pathway for Synthesis of Phosphatidylcholine A young rat maintained on a diet deficient in methionine fails to thrive unless choline is included in the diet. Explain. 13. Energetics of Acetyl-CoA Condensation to Form Acetoacetyl-CoA The formation of a thioester of acetoacetate is catalyzed by fatty acid synthase during fatty acid synthesis, and by acetyl-CoA acetyltransferase in the first step of cholesterol biosynthesis. Both are Claisen condensations. However, in fatty acid synthesis, malonyl-CoA forms in an earlier step so that decarboxylation facilitates the condensation. In the cholesterol biosynthesis pathway, the condensation occurs between two acetyl-CoA molecules, and no decarboxylation occurs to facilitate the reaction. Suggest a reason why the thermodynamic augmentation of decarboxylation is needed in fatty acid synthesis, but not in the first steps of cholesterol biosynthesis. 14. Synthesis of Isopentenyl Pyrophosphate A researcher adds [2-14C]acetyl-CoA to a rat liver homogenate that is synthesizing cholesterol. Where will the 14C label appear in Δ 3-isopentenyl pyrophosphate, the activated form of an isoprene unit? 15. Activated Donors in Lipid Synthesis In the biosynthesis of complex lipids, components are assembled by transfer of the appropriate group from an activated donor. For example, the activated donor of acetyl groups is acetyl-CoA. For each of the following groups, give the form of the activated donor: a. phosphate; b. D-glucosyl; c. phosphoethanolamine; d. D-galactosyl; e. fatty acyl; f. methyl; g. the two-carbon group in fatty acid biosynthesis; h. Δ 3-isopentenyl. 16. Importance of Fats in the Diet When young rats are placed on a completely fat-free diet, they grow poorly, develop a scaly dermatitis, lose hair, and soon die. These symptoms can be prevented if linoleate or plant material is included in the diet. What makes linoleate an essential fatty acid? Why can plant material be substituted? 17. Regulation of Cholesterol Biosynthesis Cholesterol in humans can be obtained from the diet or synthesized de novo. An adult human on a low-cholesterol diet typically synthesizes 600 mg of cholesterol per day in the liver. If the amount of cholesterol in the diet is large, de novo synthesis of cholesterol is drastically reduced. How is this regulation brought about? 18. Lowering Serum Cholesterol Levels with Statins Patients treated with a statin drug generally exhibit a dramatic lowering of serum cholesterol. However, the amount of the enzyme HMG-CoA reductase present in cells can increase substantially. Suggest a simple explanation for this effect. 19. Roles of Thiol Esters in Cholesterol Biosynthesis Draw a mechanism for each of the three reactions shown in Figure 21-34, detailing the pathway for the synthesis of mevalonate from acetyl-CoA. 20. Potential Side Effects of Treatment with Statins Although the benefits of taking statins have become clear, side effects have yet to be documented in detail. Some physicians have suggested that patients being treated with statins should also take a supplement of coenzyme Q. Suggest a rationale for this recommendation. DATA ANALYSIS PROBLEM 21. Engineering E. coli to Produce Large Quantities of an Isoprenoid There are more than 20,000 naturally occurring isoprenoids, some of which are medically or commercially important and produced industrially. The production methods include in vitro enzymatic synthesis, an expensive and low-yield process. In 1999, Wang, Oh, and Liao reported on their experiments to engineer the easily grown bacterium E. coli to produce large amounts of astaxanthin, a commercially important isoprenoid. Astaxanthin is a red- orange carotenoid pigment (an antioxidant) produced by marine algae. Marine animals such as shrimp, lobster, and some fish that feed on the algae get their orange color from the ingested astaxanthin. Astaxanthin is composed of eight isoprene units; its molecular formula is C40H52O4. a. Circle the eight isoprene units in the astaxanthin molecule. Hint: Use the projecting methyl groups as a guide. Astaxanthin is synthesized by the pathway shown on the next page, starting with Δ 3-isopentenyl pyrophosphate (IPP). Steps and are shown in Figure 21-36, and the reaction catalyzed by IPP isomerase is shown in Figure 21-35. b. In step of the pathway, two molecules of geranylgeranyl pyrophosphate are linked to form phytoene. Is this a head-to-head joining or is it a head- to-tail joining? (See Figure 21-36 for details.) c. Briefly describe the chemical transformation in step . d. The synthesis of cholesterol (Fig. 21-37) includes a cyclization (ring closure) that requires a net oxidation by O2. Does the cyclization in step of the astaxanthin synthetic pathway require a net oxidation of the substrate (lycopene)? Explain your reasoning. E. coli does not make large quantities of many isoprenoids, and does not synthesize astaxanthin. It is known to synthesize small amounts of IPP, DMAPP, geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Wang and colleagues cloned several of the E.coli genes that encode enzymes needed for astaxanthin synthesis, in plasmids that allowed their overexpression. These genes included idi, which encodes IPP isomerase, and ispA, which encodes a prenyl transferase that catalyzes steps and . To engineer an E. coli capable of the complete astaxanthin pathway, Wang and colleagues cloned several genes from other bacteria into plasmids that would allow their overexpression in E. coli. These genes included crtE from Erwinia uredovora, which encodes an enzyme that catalyzes step ; and crtB, crtI, crtY, crtZ, and crtW from Agrobacterium aurantiacum, which encode enzymes for steps , , , , and , respectively. The investigators also cloned the gene gps from Archaeoglobus fulgidus, overexpressed this gene in E. coli, and extracted the gene product. When this extract was reacted with [14C]IPP and DMAPP or geranyl pyrophosphate or farnesyl pyrophosphate, only 14C- labeled geranylgeranyl pyrophosphate was produced in all cases. e. Based on these data, which step(s) in the pathway are catalyzed by the enzyme encoded by gps? Explain your reasoning. Wang and coworkers then constructed several E. coli strains overexpressing different genes; they measured the orange color of the colonies (wild-type E. coli colonies are off-white) and the amount of astaxanthin produced (as measured by its orange color). Their results are shown below (ND indicates not determined). Strain Gene(s) overexpressed Orange color Astaxanthin yield (μ g/g dry weight) 1 crtBIZYW − ND 2 crtBIZYW, ispA − ND 3 crtBIZYW, idi − ND 4 crtBIZYW, idi, ispA − ND 5 crtBIZYW, crtE + 32.8 6 crtBIZYW, crtE, ispA + 35.3 7 crtBIZYW, crtE, idi ++ 234.1 8 crtBIZYW, crtE, idi, ispA + + + 390.3 9 crtBIZYW, gps + 35.6 10 crtBIZYW, gps, idi + + + 1,418.8 f. Comparing the results for strains 1 through 4 with those for strains 5 through 8, what can you conclude about the expression level of an enzyme capable of catalyzing step 3 of the astaxanthin synthetic pathway in wild-type E. coli? Explain your reasoning. g. Based on the data above, which enzyme is rate- limiting in this pathway, IPP isomerase or the enzyme encoded by idi? Explain your reasoning. Reference Wang, C.-W., M.-K. Oh, and J.C. Liao. 1999. Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli. Biotechnol. Bioeng. 62:235–241.