Fatty acid oxidation and synthesis

Fatty acid β-oxidation is a multi step process by which fatty acids are broken down by various tissues to produce energy. It involves first getting the fatty acid into the cytosol and then transferring it to the mitochondria where β-oxidation takes place. β-oxidation involves activation to acyl-CoA by conjugation with coenzyme A in the cytosol, conversion to acylcarnitine for transport across the mitochondrial membrane and conversion back to acyl-CoA inside the mitochondrion where fatty acid oxidation (β-oxidation) takes place. β-oxidation involves a repeated sequence of four enzyme activities that results in the release of an acetyl-CoA unit, a molecule of FADH2 and a molecule of NADH + H+. The acetyl-CoA then enters the mitochondrial tricarboxylic acid cycle (TCA) where it is oxidized to CO2 and H2O with the generation of additional FADH2 and NADH + H+. The NADH and FADH2 produced by both fatty acid β-oxidation and the TCA cycle are used by the electron transport chain to generate ATP.

Fatty acid synthesis (lipogenesis) is the process by which end products of glucose catabolism are converted to fatty acids, which are subsequently esterified with glycerol to form the triacylglycerols that are packaged in VLDL and secreted from the liver. Lipogenesis starts with acetyl-CoA and builds up by the addition of two carbons units donated by malonyl-CoA, generated by the ATP-dependant carboxylation of acetyl-CoA. The activated donor of two carbon units is malonyl-CoA, the elongation reaction being driven by the release of CO2. Fatty acid synthesis occurs in the cytoplasm in contrast to β-oxidation which occurs in the mitochondria. In eukaryotes the enzymes for fatty acid synthesis are organized into a multienzyme complex called fatty acid synthetase. The intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups of an acyl carrier protein (ACP), whereas intermediates in fatty acid oxidation are covalently attached to the sulfhydryl group of coenzyme A. Elongation by the fatty acid synthase complex stops on formation of palmitate (C16). Further elongation and the insertion of double bonds are carried out by other enzyme systems. The reductant in fatty acid synthesis is NADPH whereas the oxidants in fatty acid degradation are NAD+ and FAD.

Enzymes involved in fatty acid oxidation

Activation and transport steps

Figure 1. Overview of fatty acid uptake and oxidation. Fatty acids are transported into the cell, activated to acyl-CoA, transported into the mitochondria as acylcarnitine by carnitine translocase (CAT), reconverted back to acyl-CoA and subject to β-oxidation. ATP is generated via the TCA cycle and the electron transport chain. See text for details. Reproduced from Fillmore et al[1] with permission. Click to enlarge.
Figure 1. Overview of fatty acid uptake and oxidation. Fatty acids are transported into the cell, activated to acyl-CoA, transported into the mitochondria as acylcarnitine by carnitine translocase (CAT), reconverted back to acyl-CoA and subject to β-oxidation. ATP is generated via the TCA cycle and the electron transport chain. See text for details. Reproduced from Fillmore et al[1] with permission. Click to enlarge.
Fatty acid β-oxidation is a multi step process by which fatty acids are broken down by various tissues to produce energy. It involves first getting the fatty acid into the cytosol and then transferring it to the mitochondria where β-oxidation takes place. On being released from chylomicrons or very-low-density lipoproteins (VLDLs) by lipoprotein lipase in the capillary beds in the body, free fatty acids primarily enter the cell via fatty acid transporters on the cell surface. Fatty acid transporters include fatty acid translocase (FAT/CD36), tissue specific fatty acid transport proteins (FATP), and plasma membrane bound fatty acid binding protein (FABPpm).[1] The processes involved in fatty acid uptake and oxidation are summarized in Figure 1.

Once inside the cell fatty acids are activated by conjugation with coenzyme A (CoA) in a reaction catalyzed by acyl-CoA synthetase (thiokinase). This enzyme is associated with the endoplasmic reticulum and outer mitochondrial membrane and requires ATP. The ATP is cleaved to AMP plus PPi in this reaction. A second enzyme inorganic pyrophosphatase then cleaves the PPi to 2 molecules of Pi which helps to drive the acylation reaction to completion[2]. Fatty acid oxidation and fatty acid synthesis require the acyl group to be covalently attached to either coenzyme A (oxidation) or acyl carrier protein (synthesis). In both cases their carboxyl groups are covalently linked to the terminal cysteine of a phosphopantetheine group, with CoA being the source of the phosphopantetheine group attached to ACP[3].

While fatty acid activation occurs in the cytosol, β-oxidation occurs inside the mitochondrion. Hence the long chain acyl-CoA is transported across the impermeable mitochondrial membrane after conversion of the long chain acyl-CoA to long chain acylcarnitine by the enzyme carnitine palmitoyltransferase-1 (CPT1). CPT1, resides on the inner surface of the outer mitochondrial membrane, and is a major site of regulation of mitochondrial fatty acid uptake[1]. The fatty acylcarnitine moiety is transported across the inner mitochondrial membrane via the transport protein carnitine translocase (CAT), which exchanges long chain acylcarnitines for carnitine. An inner mitochondrial membrane carnitine palmitoyltransferase-2 (CPT2) then converts the long chain acylcarnitine back to long chain acyl-CoA which is now ready to be metabolized by the β-oxidation pathway.

β-oxidation
Figure 2. The fatty acid oxidation cycle. The example shown is for the oxidation of the C16 fatty acid palmitate. Each cycle of four reactions generates one acetyl-CoA and an acyl-CoA that is two C units shorter. See text for details. The complete oxidation of palmitate requires 7 cycles. Click to enlarge
Figure 2. The fatty acid oxidation cycle. The example shown is for the oxidation of the C16 fatty acid palmitate. Each cycle of four reactions generates one acetyl-CoA and an acyl-CoA that is two C units shorter. See text for details. The complete oxidation of palmitate requires 7 cycles. Click to enlarge
As shown in Figure 2, there are four enzymes involved in β-oxidation: acyl-CoA dehydrogenase, enoyl-CoA hydratase, hydroxy acyl-CoA dehydrogenase, and ketoacyl-CoA thiolase. In the first step acyl-CoA dehydrogenase creates a double bond between the second and third carbons down from the CoA group on acyl-CoA and in the process produces one molecule of FADH2. In the second step, enoyl-CoA hydratase adds a water molecule by removing the double bond just formed, adding a hydroxyl group to the third carbon down from the CoA group and a hydrogen to the second carbon down from the CoA group.

In the third step hydroxyacyl-CoA dehydrogenase removes the hydrogen in the hydroxyl group just attached and in the process produces a molecule of NADH. In the final step, ketoacyl-CoA thiolase splits off the terminal acetyl-CoA moiety by attaching a new CoA group to the third carbon upstream of the original CoA group, resulting in the formation of two molecules, acetyl-CoA and an acyl-CoA that is now two carbons shorter[1][2]. β-oxidation of long chain acyl CoA results in the production of one acetyl-CoA from each cycle of fatty acid β-oxidation. This acetyl-CoA then enters the mitochondrial tricarboxylic acid cycle (TCA). The NADH and FADH2 produced by both fatty acid β-oxidation and the TCA cycle are used by the electron transport chain to produce ATP[1].

A list of saturated fatty acids, their formulae and alternative terminology is shown in Table 1.

Table 1. List of some saturated fatty acids[4].

Common Name Systematic Name Structural Formula Lipid Numbers
Propionic acid Propanoic acid CH3CH2COOH C3:0
Butyric acid Butanoic acid CH3(CH2)2COOH C4:0
Valeric acid Pentanoic acid CH3(CH2)3COOH C5:0
Caproic acid Hexanoic acid CH3(CH2)4COOH C6:0
Enanthic acid Heptanoic acid CH3(CH2)5COOH C7:0
Caprylic acid Octanoic acid CH3(CH2)6COOH C8:0
Pelargonic acid Nonanoic acid CH3(CH2)7COOH C9:0
Capric acid Decanoic acid CH3(CH2)8COOH C10:0
Undecylic acid Undecanoic acid CH3(CH2)9COOH C11:0
Lauric acid Dodecanoic acid CH3(CH2)10COOH C12:0
Tridecylic acid Tridecanoic acid CH3(CH2)11COOH C13:0
Myristic acid Tetradecanoic acid CH3(CH2)12COOH C14:0
Pentadecylic acid Pentadecanoic acid CH3(CH2)13COOH C15:0
Palmitic acid Hexadecanoic acid CH3(CH2)14COOH C16:0
Margaric acid Heptadecanoic acid CH3(CH2)15COOH C17:0
Stearic acid Octadecanoic acid CH3(CH2)16COOH C18:0

Short- and medium-chain fatty acids of between 4-12 carbon atoms in length are oxidized exclusively in the mitochondria whereas long-chain fatty acids C12-C16 are oxidized in both the mitochondria and peroxisomes[5]. Longer chain fatty acids (C17–C26) are preferentially oxidized in the peroxisomes rather than in mitochondria with cerotic acid (a 26:0 fatty acid) being solely oxidized in this organelle. The peroxisomes also metabolize di– and trihydroxycholestanoic acids (bile acid intermediates); long-chain dicarboxylic acids that are produced by ω-oxidation of long-chain monocarboxylic acids; pristanic acid and certain polyunsaturated fatty acids (PUFAs)[5].

Energy yield from fatty acid oxidation

Fat is the body's preferred way of storing energy owing to its high energy density (9 kCal/g compared to 4 kCal/g for carbohydrates and proteins). The amount of energy (ATP) generated per mole of stearic acid (C18) is 120 ATP molecules. In contrast, the complete oxidation of three molecules of glucose (3 x C6 = C18) via glycolysis to pyruvate, decarboxylation to acetyl-CoA and oxidation of acetylCoA via the TCA cycle and electron transfer chain generates only 90 ATP, 33% less than that generated from stearate[6].

Enzymes involved in fatty acid synthesis

Fatty acid synthesis is not simply a reversal of the oxidative pathway. The acyl intermediates are similar but the pathway consists of a new set of reactions, exemplifying the principle that synthetic and degradative pathways are usually distinct[3]. In both pathways the acyl intermediates are attached to a prosthetic group which is CoA for oxidation and acyl carrier protein (ACP) for fatty acid synthesis. Both CoA and ACP have phosphopantetheine as their reactive units to which the fatty acid moiety is attached[2][3].

Figure 3. Linking glycolysis to fatty acid synthesis involves mitochondrial and cytosolic enzymes. Pyruvate, the end product of glycolysis is transported to the mitochondria and decarboxylated to form acetyl-CoA, the key building block of fatty acid synthesis. Acetyl-CoA combines with oxaloacetate for transport to the cytoplasm as citrate. There acetyl- CoA is regenerated by ATP citrate lyase and activated to malonyl-CoA. Before the fatty acid synthesis cycle can commence the acetyl- and malonyl- groups are transferred from their linkage with CoA to a larger prosthetic group, acyl carrier protein (ACP). The oxaloacetate lost from the mitochondrial pool through the formation and transport of citrate is returned as pyruvate after reduction to malate and decarboxylation. As illustrated in Figure 4 fatty acid synthesis proceeds through the sequential addition of malonyl-ACP to the growing acy-ACP moiety. Click to enlarge
Figure 3. Linking glycolysis to fatty acid synthesis involves mitochondrial and cytosolic enzymes. Pyruvate, the end product of glycolysis is transported to the mitochondria and decarboxylated to form acetyl-CoA, the key building block of fatty acid synthesis. Acetyl-CoA combines with oxaloacetate for transport to the cytoplasm as citrate. There acetyl- CoA is regenerated by ATP citrate lyase and activated to malonyl-CoA. Before the fatty acid synthesis cycle can commence the acetyl- and malonyl- groups are transferred from their linkage with CoA to a larger prosthetic group, acyl carrier protein (ACP). The oxaloacetate lost from the mitochondrial pool through the formation and transport of citrate is returned as pyruvate after reduction to malate and decarboxylation. As illustrated in Figure 4 fatty acid synthesis proceeds through the sequential addition of malonyl-ACP to the growing acy-ACP moiety. Click to enlarge
An overview of fatty acid synthesis is shown in Figure 3. In liver cells, glycolysis converts excess glucose to pyruvate. Each molecule of glucose (a six carbon sugar) is catabolised to form two molecules of pyruvate (a 3-carbon sugar) which is transported into the mitochondria via the transport protein pyruvate translocase. There it is decarboxylated by the enzyme pyruvate dehydrogenase to form acetyl-CoA, which combines with oxaloacetate to form the tricarboxylic acid, citrate which may be oxidized further in the mitochondrion via the tricarboxylic acid cycle (Krebs Cycle). Excess citrate that is not oxidized via the tricarboxylic acid cycle is exported to the cytosol for fatty acid synthesis. There citrate is cleaved into oxaloacetate and acetyl-CoA by the enzyme ATP citrate lyase. This enzyme is an important link between the metabolism of carbohydrates and the production of fatty acids (Figure 3) as the acetyl-CoA so produced is the building block for the synthesis of fatty acids and cholesterol[7].

The oxaloacetate employed in the transfer of acetyl groups to the cytosol needs to be returned to the mitochondria. Since the inner mitochondrial membrane is impermeable to oxaloacetate a series of bypass reactions occurs in the cytosol: malate dehydrogenase reduces oxaloacetate to malate and the NADP-linked enzyme, malate enzyme, decarboxylates the newly formed malate to pyruvate, generating one molecule of NADH + H+ in the process (Figure 3). The pyruvate formed in this reaction readily enters the mitochondria, where it is carboxylated to oxaloacetate by pyruvate carboxylase. The NADH + H+ generated is required for the reductive steps in fatty acid synthesis (discussed below).

As shown in Figure 3, fatty acid synthesis starts with the carboxylation of acetyl-CoA to malonyl-CoA by the enzyme acetyl-CoA carboxylase (ACC). This irreversible reaction, which requires one molecule of ATP is the committed step in Figure 4. The fatty acid synthesis cycle. The example shown is for the generation of the C16 fatty acid palmitate. Each cycle of four reactions consumes one malonyl-ACP and produces an acyl-ACP that is two C units longer. Seven cycles results in the production of the C16 intermediate palmitoyl-ACP which is hydrolysed to palmitate and ACP by the enzyme palmitoyl thioesterase. Further elongation and the insertion of double bonds are carried out by other enzyme systems[3]. Click to enlarge
Figure 4. The fatty acid synthesis cycle. The example shown is for the generation of the C16 fatty acid palmitate. Each cycle of four reactions consumes one malonyl-ACP and produces an acyl-ACP that is two C units longer. Seven cycles results in the production of the C16 intermediate palmitoyl-ACP which is hydrolysed to palmitate and ACP by the enzyme palmitoyl thioesterase. Further elongation and the insertion of double bonds are carried out by other enzyme systems[3]. Click to enlarge
fatty acid synthesis and ACC is the essential regulatory enzyme for fatty acid metabolism[3]. The intermediates involved in the subsequent steps in fatty acid synthesis are linked to an acyl carrier protein (ACP) via the sulfhydryl terminus of a phosphopantetheine group, which, in turn, is attached to a serine residue of the acyl carrier protein. ACP is a single polypeptide chain of 77 residues and can be regarded as a giant prosthetic group, a “macro CoA.”. In mammals the generation of acetyl-ACP and malonyl-ACP are catalysed by a single bifunctional protein domain, malonyl-acetyl transferase (MAT)[8].

The subsequent pathway of fatty acid synthesis from acetyl-ACP and malonyl-ACP involves the repetition of a 4-step reaction sequence: condensation, reduction, dehydration, and reduction (Figure 4).

Condensation - In the condensation reaction, a four-carbon unit acetoacetyl-ACP is formed from a two carbon unit acetyl-ACP and a three-carbon unit malonyl-ACP by the enzyme 3-ketoacyl-ACP synthetase (also called acyl-malonyl-ACP condensing enzyme) and CO2 is released. The reason acetyl-ACP and malonyl-ACP are involved rather than two molecules of acetyl-ACP is that the equilibrium for the synthesis of acetoacetyl-ACP from two molecules of acetyl-ACP is highly unfavorable[2]. In contrast, the equilibrium is favorable if malonyl-ACP is a reactant because its decarboxylation contributes a substantial decrease in free energy[3]. In effect ATP, drives the condensation reaction even though it does not directly participate. Rather, ATP is used in the earlier step of carboxylation of acetyl-CoA to malonyl-CoA. It is the free energy thus stored in malonyl-CoA that is released in the decarboxylation reaction accompanying the formation of acetoacetyl-ACP. Although HCO3- is required for fatty acid synthesis, its carbon atom does not appear in the product (Figure 4). Rather, all the carbon atoms of fatty acids containing an even number of carbon atoms are derived from acetyl-CoA[3].

Reduction – In the next step, acetoacetyl-ACP is reduced to d-3-hydroxybutyryl-ACP by the enzyme 3-ketoacyl-ACP reductase (or β-ketoacyl reductase) which reduces the carbon 3 ketone to a hydroxyl group. This reaction differs from the corresponding one in fatty acid degradation (discussed earlier) in two respects: (i) the ‘d’ rather than the ‘l’ isomer is formed; and (ii) NADPH is the reducing agent, whereas NAD+ is the oxidizing agent in β oxidation. This difference exemplifies the general principle that NADPH is consumed in biosynthetic reactions, whereas NADH is generated in energy-yielding reactions[3].

Dehydration and further reduction - The d-3-hydroxybutyryl-ACP is dehydrated (removes H2O) by the enzyme 3-hydroxyacyl-ACP dehydratase to form crotonyl-ACP, which is a trans-Δ2-enoyl-ACP. The final step in the cycle, carried out by the enzyme enoyl-ACP reductase, reduces the C2-C3 double bond converting crotonyl-ACP to butyryl-ACP. NADPH is again the reductant, whereas FAD is the oxidant in the corresponding reaction in β-oxidation (see earlier). These last three reactions - a reduction, a dehydration, and a second reduction - convert acetoacetyl-ACP into butyryl-ACP, which completes the first elongation cycle[3]. Thus the first round of fatty acid synthesis from acetylCoA and malonylCoA results in the formation of a 4-carbon moiety[3].

In the second round of fatty acid synthesis, butyryl-ACP condenses with a new molecule of malonyl-ACP to form a C6-β-ketoacyl-ACP. Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl-ACP into a C6-acyl-ACP (caproyl-ACP), which is ready for a third round of elongation. A further five successive rounds of synthesis, consuming additional molecules of malonyl-CoA, lead to the formation of 8-, 10-, 12-, 14- and 16-carbon moieties respectively, the elongation cycles continuing until the C16-palmitoyl-ACP is formed. This intermediate is a good substrate for the enzyme palmitoyl thioesterase which hydrolyzes C16- palmitoyl-ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length[3]. Further elongation and the insertion of double bonds are carried out by other enzyme systems[3].

Overall the 7 cycles required for the synthesis of the C16 fatty acid palmitate require 8 molecules of acetyl-CoA (as one molecule of acetyl-CoA and 7 molecules of malonyl-CoA), 14 molecules of NADPH (given there are two reductive steps per cycle), and 7 molecules of ATP (required to generate the 7 molecules of malonyl-CoA from 7 molecules of acetyl-CoA). As discussed earlier, since one molecule of NADPH is generated for each molecule of acetyl-CoA that is formed by the action of ATP citrate lyase on each molecule of citrate that has moved from the mitochondrion to the cytosol, then eight molecules of NADPH will be formed when the eight molecules of acetyl-CoA required to form one molecule of palmitate are produced in this way. The additional six molecules of NADPH required for this process (14 required given 7 cycles with 2 reductive steps per cycle) come from the pentose phosphate pathway[3].

Further elongation and the insertion of double bonds are carried out by other enzyme systems and fatty acids with an odd number of carbon atoms are synthesized starting with propionyl-ACP rather than acetyl-ACP[3]. Propionyl-ACP is formed from propionyl-CoA by the enzyme malonyl-acetyl transferase (MAT)[8].

Organization of the enzymes of fatty acid synthesis in mammals

The enzyme system in mammals that catalyzes the synthesis of saturated long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NAPDH is called fatty acid synthase where the seven component enzymes involved are linked in a large polypeptide chain[3]. Mammalian fatty acid synthase is a dimer of identical 260-kd subunits. Each chain is folded into three domains joined by flexible regions. Domain 1, the substrate entry and condensation unit, contains acetyl transacylase, malonyl transacylase, and 3-ketoacyl-ACP synthetase (condensing enzyme). Domain 2, the reduction unit, contains the acyl carrier protein (ACP), and the three enzymes 3-ketoacyl reductase, 3-hydroxyacyl-ACP dehydratase, and enoyl-ACP reductase. Domain 3, the palmitate release unit, contains the thioesterase. Thus, seven different catalytic sites are present on a single polypeptide chain improving coordination of the synthetic activity of different enzymes[3][8].

Figure 5. Structural overview of porcine fatty acid synthase. (A) Side view cartoon representation of fatty acid synthase, colored by domains as indicated. Linkers and linker domains are depicted in gray. Bound NADP+ cofactors and the attachment sites for the disordered C-terminal ACP/TE domains are shown as blue and black spheres, respectively. The position of the pseudo-twofold dimer axis is depicted by an arrow at the top of the side view; domains of the second chain are indicated by an appended prime. The lower panel (front view) shows a corresponding schematic diagram. (B) Top (upper panel) and bottom (lower panel) views, demonstrating the “S” shape of the modifying (upper) and condensing (lower) parts of fatty acid synthase. The pseudo-twofold axis is indicated by an ellipsoid. (C) Linear sequence organization of fatty acid synthase, at approximate sequence scale. Note the C-terminal ACP and thioesterase domains of the fatty acid synthase polypeptide were not seen in the crystal structure presumably because of their flexibility. Protein flexibility may facilitate transfer of ACP-attached reaction intermediates among the several active sites in each half of the complex. Reproduced from Maier et al., 2008[8] with permission. Click to enlarge
Figure 5. Structural overview of porcine fatty acid synthase. (A) Side view cartoon representation of fatty acid synthase, colored by domains as indicated. Linkers and linker domains are depicted in gray. Bound NADP+ cofactors and the attachment sites for the disordered C-terminal ACP/TE domains are shown as blue and black spheres, respectively. The position of the pseudo-twofold dimer axis is depicted by an arrow at the top of the side view; domains of the second chain are indicated by an appended prime. The lower panel (front view) shows a corresponding schematic diagram. (B) Top (upper panel) and bottom (lower panel) views, demonstrating the “S” shape of the modifying (upper) and condensing (lower) parts of fatty acid synthase. The pseudo-twofold axis is indicated by an ellipsoid. (C) Linear sequence organization of fatty acid synthase, at approximate sequence scale. Note the C-terminal ACP and thioesterase domains of the fatty acid synthase polypeptide were not seen in the crystal structure presumably because of their flexibility. Protein flexibility may facilitate transfer of ACP-attached reaction intermediates among the several active sites in each half of the complex. Reproduced from Maier et al., 2008[8] with permission. Click to enlarge
The crystal structure of porcine fatty acid synthase is shown in Figure 5 and reveals a complex architecture of alternating linkers and enzymatic domains[8]. The enzyme assembles into an intertwined dimer approximating an “X” or back-to-back ‘ϽС’ shape. It is segregated into a lower condensing portion, containing the condensing β-ketoacyl synthase (KS) and the malonyl-acetyl transferase (MAT) domains, and an upper portion including the dehydratase (DH), NADPH-dependent enoyl reductase (ER), and NADPH–dependent β-ketoreductase (KR) domains responsible for β-carbon modification. The condensing and modifying parts of mammalian fatty acid synthase are loosely connected and form only tangential contacts. Substrate shuttling is facilitated by flexible tethering of the acyl carrier protein domain and by the limited contact between the condensing and modifying portions of the multienzyme, which are mainly connected by linkers rather than direct interaction. The structure identifies two additional nonenzymatic domains: (i) a pseudo-ketoreductase (ΨKR) and (ii) a peripheral pseudo-methyltransferase (ΨME) that is probably a remnant of an ancestral methyltransferase domain. The structural organization of domains deviates dramatically from their linear arrangement in sequence (Figure 5A and 5C) [8].

To summarise, in the priming step, the acetyl transferase loads acetyl-CoA onto the terminal thiol of the phosphopantetheine cofactor of the acyl carrier protein (ACP), which passes the acetyl moiety over to the active site cysteine of the β-ketoacyl synthase (KS). Malonyl transferase (MT) transfers the malonyl group of malonyl-CoA to ACP, and the KS catalyzes the decarboxylative condensation of the acetyl and malonyl moieties to an ACP-bound β-ketoacyl intermediate. The β-carbon position is then modified by sequential action of the NADPH–dependent β-ketoreductase (KR), a dehydratase (DH), and the NADPH-dependent enoyl reductase (ER) to yield a saturated acyl product elongated by two carbon units. This acyl group functions as a starter substrate for the next round of elongation, until the growing fatty acid chain reaches a length of 16 to 18 carbon atoms and is released from ACP. In mammalian fatty acid synthase, the malonyl and acetyl transferase reactions are catalyzed by a single bifunctional protein domain, the malonyl-acetyl transferase (MAT), and the products are released from ACP as free fatty acids by a thioesterase (TE) domain[8].

A multienzyme complex consisting of covalently joined enzymes is more stable than one formed by noncovalent attractions and intermediates can be efficiently handed from one active site to another without leaving the assembly. It seems likely that multifunctional enzymes such as fatty acid synthase arose in eukaryotic evolution by exon shuffling because each of the component enzymes is recognizably homologous to its bacterial counterpart[3].

Regulation of fatty acid oxidation and synthesis

Acetyl-CoA carboxylase - ACC is a central enzyme involved in fatty acid β-oxidation and fatty acid biosynthesis. ACC catalyzes the carboxylation of acetyl-CoA producing malonyl-CoA, which can be used by fatty acid synthase for fatty acid biosynthesis[5]. While malonyl-CoA is used as a substrate for fatty acid biosynthesis, malonyl-CoA is also a potent inhibitor of mitochondrial fatty acid uptake secondary to inhibition of CPT1. There are two forms of ACC, a 265 kDa ACC1 isoform, which is highly expressed in the liver and adipose tissue, and a 280 kDa ACC2 isoform which is more specific to highly metabolic organs such as skeletal muscle and the heart. AMPK plays a major role in ACC1 and ACC2 regulation by phosphorylating and inhibiting ACC activity. In situations of increased energy demand, AMPK is activated, where it then phosphorylates and inactivates both isoforms of ACC. The adipocyte hormones adiponectin[9] and leptin[10] exert their effects on food intake, energy homeostasis, stimulation of fatty acid oxidation and glucose uptake by stimulating the phosphorylation and activation of AMPK in skeletal muscle (and liver – adiponectin). The inhibition of ACC by AMPK leads to a reduction of the molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. ACC2 inhibition can lead to an increase in fatty acid β-oxidation, while fatty acid biosynthesis decreases when ACC1 is inhibited[1].

Several transcriptional factors can regulate ACC gene expression, including sterol regulatory element binding protein (SREBP1a and SREBP1c) and carbohydrate response element binding protein (ChREBP). SREBP is regulated by insulin, which promotes the endoplasmic reticulum SREBP1c to be cleaved and translocated to the nucleus, leading to stimulation of ACC expression. ChREBP expression can be induced by high glucose concentrations, resulting in the activated ChREBP promoting the expression of ACC1 and fatty acid synthase. Nuclear respiratory factor-1 (NRF-1) is a principal modulator of mitochondrial protein expression and mitochondrial biogenesis, both of which are important for higher mitochondrial fatty acid β-oxidation capacity[1].

Malonyl-CoA decarboxylase - MCD is the enzyme responsible for decarboxylation of malonyl-CoA to acetyl-CoA. Generally, the level of malonyl-CoA is decreased when MCD activity is increased, resulting in an elevated rate of fatty acid oxidation. It has been reported that protein kinases that phosphorylate and inhibit ACC might activate MCD[1]. However, MCD appears to be primarily regulated by transcriptional means. Therefore, MCD and ACC appear to work in harmony to regulate the pool of malonyl-CoA that can inhibit CPT1[1].

Membrane transport proteins - Regulation can occur at the level of fatty acid entry in to the cell. AMPK, PKC, and PPARγ positively regulate the activity of the fatty acid translocase CD36/FATP.

Carnitine palmitoyltransferase 1 - The CPT isoform, CPT1, resides on the inner surface of the outer mitochondrial membrane, and is a major site of regulation of mitochondrial fatty acid uptake. CPT1 is potently inhibited by malonyl-CoA, the product of ACC that binds to the cytosolic side of CPT1. Mammals express three isoforms of CPT1, which are encoded by different genes - the liver isoform (CPT1α), the muscle isoform (CPT1β), and a third isoform of CPT1 (CPT1c), which is primarily expressed in the brain and testis. More specifically, the heart expresses two isoforms of CPT1, an 82 KDa (CPT1α) isoform and the predominant 88 KDa (CPT1β) isoform (that has the highest sensitivity to malonyl-CoA inhibition). Insulin and thyroid hormone can regulate the sensitivity of CPT1α in the liver; whereas the muscle isoform CPT1β is not affected. Levels of malonyl-CoA are inversely correlated with fatty acid β-oxidation rates and studies on ACC2 knockout mice suggest two separate cellular malonyl-CoA pools, malonyl-CoA produced by ACC1 (used mainly for lipogenesis), and a cytosolic pool of malonyl-CoA produced by ACC2 involved in the regulation of CPT1 and fatty acid β-oxidation[1].

Allosteric control of β-oxidation enzymes - The activity of the enzymes of fatty acid β-oxidation is affected by the level of the products of their reactions. Each of the β-oxidation enzymes are inhibited by the specific fatty acyl-CoA intermediate it produces. Interestingly, 3-ketoacyl-CoA can also inhibit enoyl-CoA hydratase and acyl-CoA dehydrogenase. β-oxidation can also be allosterically regulated by the ratio of NADH/NAD+ and acetyl-CoA/CoA. A rise in the NADH/NAD+ or acetyl-CoA/CoA ratios results in inhibition of fatty acid β-oxidation. Increases in the acetyl-CoA/CoA ratio have specifically been shown to lead to feedback inhibition of ketoacyl-CoA thiolase[1].

Transcriptional and post-transcriptional regulation – The proteins involved in fatty acid β-oxidation are regulated by both transcriptional and post-transcriptional mechanisms. PGC-1α, a transcription factor co-regulator, and the transcription factor PPARα act in the nucleus to increase transcription of mitochondrial genes, fatty acid utilization genes, and other transcription factors[1].

There are a number of transcription factors that regulate the expression of these proteins. The peroxisome proliferator-activated receptors (PPARs) and a transcription factor co-activator PGC-1α are the most well known transcriptional regulators of fatty acid β-oxidation. Examples of proteins involved in fatty acid β-oxidation that are transcriptionally regulated by the PPARs include FATP, acyl-CoA synthetase (ACS), CD36/FAT, MCD, CPT1, long chain acyl-CoA dehydrogenase, and medium chain acyl-CoA dehydrogenase. Estrogen-related receptor α (ERRα) has also been implicated in the regulation of fatty acid β-oxidation, having been shown to also regulate transcription of the gene encoding medium chain acyl-CoA dehydrogenase. Ligands that bind to and modulate the activity of PPARα, δ, and γ include fatty acids[1].

The transcriptional co-activator PGC-1α binds to and increases the activity of PPARs and ERRα to regulate fatty acid β-oxidation. PGC-1α modulates the activity of a number of transcription factors that can increase the expression of proteins involved in fatty acid β-oxidation, the TCA cycle, and the electron transport chain. Increasing PGC-1α protein expression induces massive mitochondrial biogenesis in skeletal muscle[1].

PGC-1α is regulated at both the gene and protein level. AMPK increases the activity of pre-existing PGC-1α protein and increases PGC-1α mRNA levels by regulating the binding of transcription factors to specific sequences located in the PGC-1α gene promoter. Free fatty acids can also regulate PGC-1α protein expression as a high fat diet can elevate levels of PGC-1α in rat skeletal muscle[1].

References

  1. ^ Fillmore N, Osama Abo Alrob OA, Lopaschuk GD, 2011, Fatty acid oxidation http://lipidlibrary.aocs.org/animbio/fa-oxid/index.htm

  2. ^ Fatty Acid Oxidation - Oregon State University oregonstate.edu/dept/biochem/hhmi/.../2kjan14lecturenotes.html

  3. ^ Berg JM, Tymoczko JL, Stryer L, 2002, Chapter 22: ‘Fatty Acid Metabolism’ In: Biochemistry 5th Edition, New York: WH Freeman.

  4. ^ List of saturated fatty acids http://en.wikipedia.org/wiki/List_of_saturated_fatty_acids

  5. ^ King M, Lipolysis and the oxidation of fatty acids. http://themedicalbiochemistrypage.org/fatty-acid-oxidation.php

  6. ^ Lecture 34 - Fatty acid oxidation, key concepts - Overview of ... http://www.biochem.arizona.edu/miesfeld/Miesfeld-Fall2008Lecs/Lec34-Fall08/Lec34-F08-Handout.pdf

  7. ^ Postic C, Girard J, The role of the lipogenic pathway in the development of hepatic steatosis. Diabetes & amp; Metabolism 2008; 34: 643-648. http://www.em-consulte.com/en/article/200090

  8. ^ Maier T, Leibundgut M, Ban N, 2008, The crystal structure of a mammalian fatty acid synthase, Science 321, 1315-1322.

  9. ^ Yamauchi T et al., Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nature Medicine, 2002; 8:1288-95. doi:10.1038/nm788

  10. ^ Minokoshi Y et al., Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature, 2002; 415:339-43. doi:10.1038/415339a;

Comments

Nobody has commented on this article

Commenting is only available for registered Diapedia users. Please log in or register first.