Fatty acid metabolism

Lipid diversity
(KEGG summary pathway)

The term lipids includes a divers group of structurally distinct hydrophobic and amphipathic molecules.

Fatty acids

Fatty acids are a group of negatively charged, linear hydrocarbon chains of various length and degrees of oxidation states. The negative charge is located at a carboxyl end group that is completely deprotonated at physiological pH values (pK ~ 2-3). The length of the fatty acid 'tail' determines its water solubility (or rather insolubility) and amphipathic characteristics. Fatty acids are components of phospholipids and sphingolipids which are part of biological membranes, fats (triglycerides) which are used as energy storage devices inside cells (adipose tissue), function as fat transport vehicle in the blood (high and low density lipoproteins), and through covalent attachment are used for protein modification. Important members of this groups of fats are palmitic acid (C16) and stearic acid (C18) and the respective monounsaturated C18 oleic acid(C18:1;D9).

An unsaturated (oxidized) fatty acid contains a C=C bond in a cis conformation with the D9  indicating the unsaturated bond between C9 and C10.

Fats (triglycerides) Fats are the mono-, di-, and triacylglycerol forms of fatty acids. Triglycerides (triacylglycerols) are neutrally charged, entirely hydrophobic, i.e. reduced molecules. The mono- and diacylglycerides are metabolic intermediates in phospholipid synthesis, while the triacylglycerol (or triglycerides) are the fat molecules used to store chemical energy in a water free, compact state. This latter is the main function of adipocytes (fat storage cells) in adipose tissue of mammals. Fat storage provides an anhydrous form (devoid of water) because of the hydrophobic packing of fully saturated fatty acid chains. The energy content per gram fat is 9 kcal as compared to 4kcal/g for carbohydrates.
Phospholipids Phospholipids are diacylglycerol derivatives with a hydrophilic, zwitter ionic, often charged headgroup at position C3 of the glycerol backbone. Fatty acyl chains (R groups in figure below) of most membrane phospholipids are C16 and C18 members, with the acyl chain R2 usually unsaturated. Phosphatidic acid (C00416) is the biosynthetic precursor of all phospholipids used for cell membrane synthesis and triglycerides.
Steroids (and steroid hormones) Steroids (hormones) are signaling molecules derived from cholesterol. They readily diffuse across biological membranes. They form a class of hormones with target receptors inside the cell. Examples of steroid hormones are Testosterone and Progesterone, the male and female sex determining hormones in higher animals. Non hormonal steroids are cholesterol and their derivatives like ergosterol and cholic acid all of which serve as lipid components of eukaryotic membranes. Specific steroid membrane lipids are unique for different groups of organisms. Cholesterol (C00187; the parent structure of all steroids), for example, is a membrane component found in large quantities in mammals only, while plants are devoid of this particular lipid as membrane component.
Eicosanoids Eicosanoids are specialized fatty acid derivatives in cell membranes that function in processes like inflammation. Prostaglandins (C02064) and Leukotrienes are the two major groups of this class of lipids. Their role is part of the immune response of higher organisms (mammals).
Fat-soluble vitamins Fat-soluble vitamins (vitamins A, D, E, K) are a mixed group of linear and cyclic p -electron systems. Vitamin D is synthesized as part of the sterol biosynthetic pathway MAP00100. The vitamin A group is synthesize by the retinol metabolism

Lipids and their role in metabolism

About 40% of the bodies caloric intake is derived from lipids and almost all of these calories come from fats, the triacylglycerols. The fatty acid composition in terms of saturation (oxidation forms) is not uniform but varies with the origin. Plant fats contain more polyunsaturated fatty acids and animal fats contain more saturated fatty acids as well as cholesterol. Polyunsaturated fats are essential for humans because animals are not able to synthesize those on their own. Most lipids, however, have metabolic functions contributing to membrane structures and signaling. Arachidonic acid (C20:4) is a fatty acid which plays a central role as precursor for prostaglandin synthesis. Phospholipids are synthesized from diacylgycerolphosphate, a negatively charged phospholipid precursor and signaling molecule itself, carrying various hydrophilic and/or charged headgroups that determine the surface charge and chemical properties of biological membrane surfaces.

Fatty acid degradation
(KEGG  pathway MAP00071)

The use of fatty acids for energy production and inter conversion of biosynthetic precursors is predominantly performed in liver. The liver may play a modifying part in fat storage and retrieval. The major source of lipids entering the liver does so in free fatty acid form released form adipose tissue and transported in the systemic blood plasma complexed with albumin. Fatty acid oxidation yields twice the usable chemical energy that carbohydrates can deliver. As an example, 130 mols of ATP result from the oxidation of one mol of palmitic acid (C16:0), as compared to 38 mols of ATP from one mol of glucose. On a weight basis, the caloric yield from fatty acids is about double that from carbohydrates; 9kcal/g from fat vs. 4kcal/g from carbohydrate or protein).

Fatty acid oxidation occurs in three major steps. In a first energy consuming step, the fatty acid is activated by forming a high energy thioester bond with HS-CoA. These activated fatty acid units can be used for the synthesis of phospholipids and fats (triglycerides). Second, the activated acyl-CoA is oxidized and C2 units removed in the form of Acetyl-CoA in a cyclic process called beta-oxidation. Third, acetyl-CoA moieties are funneled into the citric acid cycle for a final round of decarboxylation-oxidation reactions. This pathway is located in the mitochondrial matrix directly coupling the reducing equivalents in the form of NADH + H+ to oxidative phosphorylation and thus ATP synthesis.

Fatty acids are found in free form in the cytoplasm and need to be transported across the inner mitochondrial membrane in a controlled fashion. The activated fatty acid is first transferred by a mitochondrial outer membrane protein carnitine acyl transferase I  (1) complex from acyl-CoA to acyl-carnitine. The latter is transported across the inner membrane into the matrix compartment by carnitine acyl translocase. There, the fatty acid is reesterified with an CoA unit by the matrix enzyme complex carnitine acyl transferase II (2).

Beta oxidation of fatty acids

The process of beta oxidation is named after the carbon atom in the beta position of the fatty acyl-CoA which becomes the most oxidized during the cyclic redox reactions that remove C2 units in form of acetyl-CoA from the fatty acyl chain. The beta carbon becomes the new carboxyl end of the shortened (n-2) fatty acyl-CoA.  The oxidation steps are strictly analogous to the reaction steps in the citric acid cycle converting succinyl-CoA to oxaloacetate involving an initial oxidation by acyl-CoA dehydrogenase (EC; driven by FAD reduction), an hydration by enoyl-CoA hydratase (EC, and a second oxidation by hydroxyacyl-CoA dehydrogenase (EC driven by NAD+ reduction). A C2 unit is released by beta-ketothiolase (EC to produce acetyl-CoA and a shortened acyl(n-2)-CoA. The latter is recycled until the acyl chain is shortened to its acetyl-CoA end product and oxidized by the citric acid cycle enzymes.

The acyl-CoA dehydrogenase is specific for the length of the acyl chain being oxidized. Three types of the dehydrogenase exist in mitochondria; type I (EC; long chain) which oxidizes C12-C18 fatty acids, type II (EC which oxidizes C4-C14 fatty acids, and type III (EC; butyryl dehydrogenase) which oxidizes C4 and C6 acyl-CoA substrates. The energy yield per cycle is 5 mols of ATP for each round, 2 mols per FADH2 (goes into complex II) and 3 mols per NADH/H+ (goes into complex I). The energy balance for palmitic acid (sixteen carbon atoms) is:

CH3(CH2)14CO-S-CoA + 7H2O +7CoA + 7FAD + 7NAD+
                                                                        Þ 8CH3-CO-S-CoA + 7FADH2 + 7NADH + 7H+

The completion of the degradation process (coenzyme oxidation) requires the citric acid cycle which yields an additional 96 mols of ATP for all 8 acetyl-CoA units oxidized in the process. The total energy yield of palmitic acid oxidation results in some 130 mols of ATP, 34 units from the beta-oxidation cycle and 96 form the citric acid cycle.

Beta oxidation of odd numbered fatty acids yields a (C2) acetyl-CoA and (C3) propionyl-CoA end product during the very last cycle. Propionyl-CoA cannot be further oxidized as such and is converted to methyl-malonyl-CoA by propionyl carboxylase (EC This is an energy consuming step using 1 mol of ATP per mol of propionyl-CoA. The net reaction is:

            propionyl-CoA + CO2 + ATP  = (D/L)-methylmalonyl-CoA + ADP + Pi

The methylmalonyl is formed as racemic mixture containing equal amounts of the D- and L-enantiomer. The (D)methylmalonyl-CoA is isomerized to succinyl-CoA by methylmalonyl CoA mutase:

(D)-methylmalonyl-CoA = succinyl-CoA

Note that this reaction is also used for the degradation of hydrocarbon side chains of the amino acids valine, leucine, and isoleucine; (S)-methylmalonyl is the same as L-methylmalonyl; from KEGG pathway MAP00280)

Similarly, unsaturated fatty acids need special enzymes to provide the beta oxidation intermediate trans-D2-enoyl-CoA, the substrate of enoyl-CoA hydratase. Desaturation can result in two unusual degradation intermediates: the cis-D3- and the cis-D4-enoyl-CoA. The beta oxidation intermediate, however is a trans-D2 isomer. The cis-D3-enoyl-CoA intermediate is isomerized to the trans-D2 form by isomerase (EC The cis-D4-enoyl-CoA form is converted in two steps to cis-D3-enoyl-CoA intermediate. The first step is catalyzed by acyl-CoA dehydrogenase (the first enzyme in the beta oxidation cycle) to form 2,4-dienoyl-CoA which in turn is isomerized by 2,4-dienoyl-CoA reductase to cis-D3-enoyl-CoA.

Fatty acid synthesis

(KEGG  pathway MAP00061 and MAP00062)

Apart from two polyunsaturated fatty acids (linoleic acid, C18:2; and alpha-linolenic acid, C18:3) the human body is able to synthesize all other fatty acids required either for structural lipids in membranes or for storage purpose. Fatty acid synthesis and their further use for phospholipids and triglycerides is referred to as lipogenesis. Any metabolite that yields acetyl-CoA during its degradation is a potential supplier for lipogenesis, the most important being carbohydrates. In general, it can be understood that excess carbohydrates beyond the body's energy needs will be converted into fat. Lipogenesis is not a simple reversal of beta oxidation, but uses an entirely different pathway for the regeneration of fatty acids from acetyl-CoA precursors. The reductive synthesis of fatty acids is a cytoplasmic process carried out by a multienzyme complex called the acyl carrier protein (ACP). The acyl chain is covalently linked to the sulfhydryl prosthetic group of ACP. The reduction-oxidation steps require NADPH (rather than FADH2 and NADH found during beta-oxidation).

There are three major processes involved in the reductive synthesis of fatty acids. First, acetyl-CoA has to be transported across the inner mitochondrial membrane into the cytoplasm. Second, the true substrate for ACP is malonyl-CoA, a C3 acyl thioester that is formed by the carboxylation of acetyl-CoA to malonyl-CoA. Third, the first end product and intermediate for further lipid biosynthesis is palmitic acid, the C16 acyl derivative. For all other lipogenetic processes, protein complexes other than ACP are required.

Fatty acid synthesis is a cytoplasmic process. All acetyl-CoA must be exported from the mitochondrial matrix via citrate (first step in Krebs cycle). In the cytoplasm, citrate is split into acetyl-CoA and oxaloacetate. The latter, if not used for gluconeogenesis, is transported back into the mitochondrial matrix. Oxaloacetate concentration, thus, plays a crucial role in connecting carbohydrate, lipid, and energy metabolism. In the cytoplasm acetyl-CoA is activated by acetyl-CoA-carboxylase (EC to malonyl-CoA (C00083). This C3 acyl intermediate is the immediate precursor for fatty acid synthesis. The enzyme contains biotin as prosthetic group catalyzing the following two reactions:

                       CO2 + biotin-Enz + ATP Þ carboxybiotin-Enz + ADP + Pi
                    carboxybiotin-Enz + acetyl-CoA Þ malonyl-CoA + biotin-Enz

This reaction is analogous to that catalyzed by pyruvate carboxylase. This committed step reaction of malonyl-CoA formation is a major control point of fatty acid synthesis. Activated acyl units are now transferred from CoA to a phosphopantetheine sulfhydryl group (C01134) linked to a serine residue on ACP for subsequent chain elongation reaction.

ACP is a subunit of a larger enzyme complex, the fatty acid synthetase (EC The flexibility and length (2nm) of the phosphopantetheine unit are critical for the function of the fatty acid synthetase complex. In yeast, the complex contains 12 subunits, with six alpha and six beta subunits. In mammals, the complex is a dimer with 3 domains and 7 catalytic sites. Once covalently linked to the ACP subunit, the activated substrate is not released but moved by the pantetheine arm from catalytic site to catalytic site through the cycle as described below. The first step uses an acetyl-ACP and a malonyl-ACP to form the C4 acyl unit acetoacyl-CoA, and releasing CO2. The carbon dioxide unit is stripped off from malonyl-ACP during acyl condensation reaction. In fact, the carbon atom released in this step is the same as the one incorporated into malonyl-CoA. The subsequent steps in fatty acid synthesis are the reversal of the beta oxidation steps. The keto group in the beta position is first reduced by NADPH to form a hydroxyl group. A dehydration step introduces a C=C which is reduced by a second NADPH to the fully saturated hydrocarbon (C4) unit butyryl-CoA. The synthesis cycle repeats itself with the condensation of a second malonyl-ACP (from a second acetyl-CoA) to the C4 acyl to form a unsaturated beta-keto-acyl-ACP. The following reduction, hydration, reduction reactions form a fully saturated C6 acyl-ACP. This cycle repeats until the C16 palmitoyl-ACP is formed. At this point the fatty acid is released from the acyl carrier protein as palmitoyl-CoA.

The net reaction and energy consumption of forming one molecule of palmitoyl-CoA from acetyl-CoA units is:

8 acetyl-CoA + 7ATP +14NADPH +14H+ Þ
          palmitoyl-CoA + 7ADP + 7Pi + 7CoA + 14NADP+ + 7H2O

Further elongation and oxidation can occur in either the mitochondrion or endoplasmatic reticulum. Palmitic acid (C16:0) and the C2 elongated form stearic acid (18:0) can be unsaturated to their respective monounsaturated forms palmitoleic acid (C16:1) and oleic acid (C18:1). This process is catalyzed by the ER membrane and provides fatty acids for phospholipid biosynthesis. Fatty acids (palmitic acid) may be transported back to the mitochondrial matrix for oxidation.

Regulation of fatty acid metabolism

Fatty acid metabolism requires a balance between degradation and synthesis according to the energy need of cells and an organism as a whole. It is obviously dependent on the metabolism of carbohydrates and proteins. In humans the liver plays a role in lipid blood homeostasis analogous to its role as glucostat organ. For the control of beta oxidation the major regulatory mechanism is the availability of substrate. In liver, fatty acids come from three major sources: (i) as fatty acids released by extra cellular lipase from triglycerides coming from fat absorption in the form of chylomicrons, (ii) as unesterified (free) fatty acids in blood plasma (complexed with albumin) originating from adipose tissue (adipocyte lipase), and (iii) from intracellular triglycerides where a liver specific lipase deesterifies triglycerides releasing free fatty acids. The cellular lipases of adipocytes and hepatocytes are under hormonal control, with glucagon activating the lipase and insulin inhibiting it. Other hormones that activate lipase are epinephrine and nor-epinephrine, and ACTH (C02017; adrenocorticotropic hormone). These positive stimulators all work through cAMP mediated kinase phosphorylation.

A second regulatory mechanism is metabolic control related to the energy charge of the cell. Beta oxidation is strictly coupled to oxidative phosphorylation and depends on the ADP/ATP ratio, with a high ratio (low energy charge) stimulating degradation. High ATP levels stimulates both fatty acid synthesis as well as phosphatidic acid synthesis. ADP however accelerates beta oxidation and the oxidation of acetyl-CoA in the citric acid cycle. The regenerated ATP will help shift the balance back to synthesis mode.

A third control mechanism is the transport of fatty acids across mitochondrial inner membranes. Fatty acid transport is mediated by the carnitine acyl-transferase system. Here is a link between carbohydrate and fatty acid metabolism. The cell avoids beta oxidation while synthesizing fatty acids from glycolysis (high energy charge). Malonyl -CoA functions as an inhibitor of the mitochondrial carnitine-acyltransferase I in the mitochondrial outer membrane. If glucose is abundant (red control function in figure), malonyl-CoA levels are high inhibiting fatty acid transport into mitochondria and engaging acetyl-CoA for fatty acid synthesis.

Ketone bodies
(KEGG pathway MAP00072)

The reason for the latter control of fatty acid entry into mitochondria lies in the fact that fatty acids, once inside the matrix compartment, will be oxidized to acetyl-CoA which can be used for TCA oxidation, retro-transport into the cytoplasm or, when abundant, is transformed into acetoacetate (C00164), the precursor of so called ketone bodies. Ketone body excess are formed in liver when glucose levels are low and fatty acid concentrations are up. Low pyruvate concentration reduces the availability of oxaloacetate and thus overloads the citric acid cycle with acetyl-CoA from both pyruvate and fatty acid degradation. Acetyl-CoA molecules are then combined to acetoacetate and is in turn converted to either beta-hydroxybutyrate (the reduced form of acetoacetate) or acetone, the product of a spontaneous decarboxylation reaction of acetoacetate in the presence of protons. In liver, ketone bodies cannot be metabolized any further and are secreted into the blood pool where they are catabolized in muscle and brain cells alongside glucose. Excess ketone bodies found in the blood are removed by the kidney in form of ketonuria, which is an indicator of metabolic acidosis, a disorder resulting from high levels of ketone bodies in the blood.

The heart and brain are organs that rely on ketone bodies as fuel molecule. While skeletal muscle under strenuous activity uses glycolysis as major energy production pathway, resting muscles primarily burn fatty acids and ketone bodies, while neurons burn glucose and ketone bodies. Both organs strictly depend on respiration.

The ketone body beta hydroxybutyrate is oxidized in neurons and cardiac muscle mitochondria to produce NADH and acetoacetate. The latter can be activated by succinyl-CoA to acetoacetyl-CoA and succinate. Acetoacetyl-CoA is then cleaved by thiolase to two units of acetyl-CoA for further oxidation in the citric acid cycle. The cardiac muscle is optimized to derive most of its energy from the oxidation of acetoacetate.

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