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Fatty Acid Synthesis

 

 

 

 

 

 

 

 

 

 

 

One might predict that the pathway for the synthesis of fatty acids would be the reversal of the oxidation pathway. However, this would not allow distinct regulation of the two pathways to occur even given the fact that the pathways are separated within different cellular compartments.

The pathway for fatty acid synthesis occurs in the cytoplasm, whereas, oxidation occurs in the mitochondria. The other major difference is the use of nucleotide co-factors. Oxidation of fats involves the reduction of FAD and NAD+. Synthesis of fats involves the oxidation of NADPH. However, the essential chemistry of the two processes are reversals of each other. Both oxidation and synthesis of fats utilize an activated two carbon intermediate, acetyl-CoA. However, the activated form of acetyl-CoA in fat synthesis exists temporarily bound to the enzyme complex as malonyl-CoA.

The synthesis of malonyl-CoA is the first committed step of fatty acid synthesis and the enzyme that catalyzes this reaction, acetyl-CoA carboxylase (ACC), is the major site of regulation of fatty acid synthesis. Like other enzymes that transfer CO2 to substrates, ACC requires a biotin co-factor. Acetyl-CoA carboxylase is called an ABC enzyme due to the requirements for ATP, Biotin, and CO2 for the reaction. The details of the two different forms of ACC in human cells are described below in the Acetyl-CoA Carboxylase Regulation section.

Reaction catalyzed by acetyl-CoA carboxylase (ACC)

The rate of fatty acid synthesis is controlled by the equilibrium between monomeric ACC and polymeric ACC. The activity of ACC requires this polymerization process. This conformational change is enhanced by citrate and inhibited by long-chain fatty acids. ACC is also controlled through hormone mediated phosphorylation (see below).

The acetyl groups that are the products of fatty acid oxidation are linked to CoASH. As you should recall, CoA contains a phosphopantetheine group coupled to AMP. The carrier of acetyl groups (and elongating acyl groups) during fatty acid synthesis is also a phosphopantetheine prosthetic group, however, it is attached a serine hydroxyl in the synthetic enzyme complex. The carrier portion of the synthetic complex is called acyl carrier protein, ACP. This is somewhat of a misnomer in eukaryotic fatty acid synthesis since the ACP portion of the synthetic complex is simply one of many domains of a single polypeptide. The acetyl-CoA and malonyl-CoA are transferred to ACP by the action of the malonyl/acetyltransferase activity (also called acetyl-CoA transacylase and malonyl-CoA transacylase). The attachment of these carbon atoms to ACP allows them to enter the fatty acid synthesis cycle.

The synthesis of fatty acids from acetyl-CoA and malonyl-CoA is carried out by fatty acid synthase, FAS. Fatty acid synthase is encoded by the FASN gene which is located on chromosome 17q25 and is composed of 43 exons that encode a protein of 2511 amino acids. The active FAS enzyme exists as a head-to-tail homodimer. All of the reactions of fatty acid synthesis are carried out by the multiple enzymatic activities of FAS. Like fat oxidation, fat synthesis involves four primary enzymatic activities. These are (in order of reaction), β-ketoacyl-ACP synthase (contained in a domain composed of amino acids 2–404), β-ketoacyl-ACP reductase (contained in a domain composed of amino acids 1876–2112), 3-hydroxyacyl-ACP dehydratase (contained in a domain composed of amino acids 874–1106) and enoyl-CoA reductase (contained in a domain composed of amino acids 1564–1854). The two reduction reactions require NADPH oxidation to NADP+. The domain that is required for attachment and transfer of acetyl-CoA and malonyl-CoA (acyltransferase domain) is composed of amino acids 493–810. The phosphopantetheine arm of FAS is attached to a domain composed of amino acids 2111-2179.

The primary fatty acid synthesized by FAS is palmitic acid (palmitate). Palmitate is then released from the enzyme via the thioesterase activity of FAS (contained in a domain composed of amino acids 2242–2487). Once released, palmitate can then undergo separate elongation and/or unsaturation to yield other fatty acid molecules.

reactions of fatty acid synthesis by FAS

Reactions of fatty acid synthesis catalyzed by fatty acid synthase, FAS. Only half of the normal head-to-tail (head-to-foot) dimer of functional FAS is shown. Synthesis of malonyl-CoA from CO2 and acetyl-CoA is carried out by ACC as described. FAS is initially activated by the incorporation of the acetyl group from acetyl-CoA. The acetyl group is initially attached to the sulfhydryl of the 4'-phosphopantothenate of the acyl carrier protein portion of FAS (ACP-SH). This is catalyzed by malonyl/acetyl-CoA ACP transacetylase (1 and 2; also called malonyl/acetyltransferase, MAT). This activating acetyl group represents the omega (ω) end of the newly synthesized fatty acid. Following transfer of the activating acetyl group to a cysteine sulfhydryl in the β-ketoacyl-ACP synthase portion of FAS, the three carbons from a malonyl-CoA are attached to ACP-SH (3) also catalyzed by malonyl/acetyl-CoA ACP transacetylase. The acetyl group attacks the methylene group of the malonyl attached to ACP-SH catalyzed β-ketoacyl-ACP synthase (4) which also liberates the CO2 that was added to acetyl-CoA by ACC. The resulting 3-ketoacyl group then undergoes a series of three reactions catalyzed by the β-ketoacyl-ACP reductase (5), 3-hydroxyacyl-ACP dehydratase (6), and enoyl-CoA reductase (7) activities of FAS that reduce, dehydrate, and reduce the substrate. This results in a saturated four carbon (butyryl) group attached to the ACP-SH. This butyryl group is then transferred to the CYS-SH (8) as for the case of the activating acetyl group. At this point another malonyl group is attached to the ACP-SH (3b) and the process begins again. Reactions 4 through 8 are repeated another six times, each beginning with a new malonyl group being added. At the completion of synthesis the saturated 16 carbon fatty acid, palmitic acid, is released via the action of the thioesterase activity of FAS (palmitoyl ACP thioesterase) located in the C-terminal end of the enzyme. Not shown are the released CoASH groups.


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Origin of Cytoplasmic Acetyl-CoA

Acetyl-CoA is generated in the mitochondria primarily from two sources, the pyruvate dehydrogenase (PDH) reaction and fatty acid oxidation. In order for these acetyl units to be utilized for fatty acid synthesis they must be present in the cytoplasm. The shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energy diminishes. This results in reduced oxidation of acetyl-CoA in the TCA cycle and the oxidative phosphorylation pathway. Under these conditions the mitochondrial acetyl units can be stored as fat for future energy demands.

Acetyl-CoA enters the cytoplasm in the form of citrate via the tricarboxylate transport system (see Figure). In the cytoplasm, citrate is converted to oxaloacetate and acetyl-CoA by the ATP driven ATP-citrate lyase (ACLY) reaction. This reaction is essentially the reverse of that catalyzed by the TCA enzyme citrate synthase except it requires the energy of ATP hydrolysis to drive it forward. The resultant oxaloacetate is converted to malate by malate dehydrogenase (MDH).

Transport of acetylCoA from the mitochondria to the cytosol

Pathway for the movement of acetyl-CoA units from within the mitochondrion to the cytoplasm. Note that the cytoplasmic malic enzyme catalyzed reaction generates NADPH which can be used for reductive biosynthetic reactions such as those of fatty acid and cholesterol synthesis. SLC25A1 is the citrate transporter (also called the dicarboxylic acid transporter). Transport of pyruvate across the plasma membrane is catalyzed by the SLC16A1 protein (also called the monocarboxylic acid transporter 1, MCT1) and transport across the outer mitochondrial membrane involves a voltage-dependent porin transporter. Transport across the inner mitochondrial membrane requires a heterotetrameric transport complex (mitochondrial pyruvate carrier) consisting of the MPC1 gene and MPC2 gene encoded proteins.


The malate produced by this pathway can undergo oxidative decarboxylation by cytoplasmic malic enzyme. The co-enzyme for this reaction is NADP+ generating NADPH. The advantage of this series of reactions for converting mitochondrial acetyl-CoA into cytoplasmic acetyl-CoA is that the NADPH produced by the malic enzyme reaction can be a major source of reducing co-factor for the fatty acid synthase activities.

Humans express three malic enzymes, one cytoplasmic that requires NADP+ and two mitochondrial enzymes, one that requires NADP+ and one that requires NAD+. The cytoplasmic enzyme is called malic enzyme 1 and is encoded by the ME1 gene that is located on chromosome 6q12 and is composed of 14 exons that encode a protein of 572 amino acids. The NAD+-dependent mitochondrial enzyme is called malic enzyme 2 and is encoded by the ME2 gene located on chromosome 18q21 and is composed of 16 exons that generate two isoforms from alternatively spliced mRNAs. The NADP+-dependent mitochondrial enzyme is called malic enzyme 3 and is encoded by the ME3 gene located on chromosome 11cen–q22.3 and is composed of 22 exons that generate at least three alternatively spliced mRNAs that all encode the same 604 amino acid protein.

The role of the mitochondrial malic enzymes is principally to provide the cell with an alternate source of pyruvate under conditions where glycolytic flux in reduced. In these circumstances, the pyruvate generated by the actions of ME2 and/or ME3 come from fumarate precursors such as glutamine. In neurons, as well as in numerous types of tumor cells, mitochondrial malic enzymes allow for the utilization of the amino acid glutamine as a fuel source. When glutamine is de-aminated by glutaminase the resulting glutamate can also be de-aminated by glutamate dehydrogenase yielding 2-oxoglutarate (α-ketoglutarate) which can then be shunted to malate synthesis in the TCA cycle. The malate can then be decarboxylated to pyruvate via mitochondrial malic enzyme. The pyruvate can then be decarboxylated by the PDHc and the resulting acetyl-CoA can enter the TCA cycle ultimately allowing for glutamine carbons to be oxidized for ATP synthesis. Within β-cells of the pancreas, this process driven by mitochondrial malic enzyme serves as an important means for the use of amino acid carbon oxidation for the stimulated secretion of insulin. Indeed, this process is energetically equal to glucose-stimulated insulin secretion (GSIS).

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Regulation of Fatty Acid Metabolism

Regulation by Acetyl-CoA Carboxylase: ACC

One must consider the global organismal energy requirements in order to effectively understand how the synthesis and degradation of fats (and also carbohydrates) needs to be exquisitely regulated. The blood is the carrier of triacylglycerols in the form of VLDLs and chylomicrons, fatty acids bound to albumin, amino acids, lactate, ketone bodies and glucose. The pancreas is the primary organ involved in sensing the organisms dietary and energetic states via glucose concentrations in the blood. In response to low blood glucose, glucagon is secreted, whereas, in response to elevated blood glucose insulin is secreted. The regulation of fat metabolism occurs via two distinct mechanisms. One is short term regulation which is regulation effected by events such as substrate availability, allosteric effectors and/or enzyme modification.

ACC is the rate-limiting (committed) step in fatty acid synthesis. There are two major isoforms of ACC in mammalian tissues. These are identified as ACC1 (also called ACCα) and ACC2 (also called ACCβ). The ACC1 gene (symbol: ACACA) is located on chromosome 17q21 and is composed of 65 exons that undergo alternative splicing to yield at least five splice variant mRNAs that generate proteins from 2268 to 2383 amino acids in length. Transcriptional regulation of ACACA is effected by three promoters (PI, PII, and PIII), which are located upstream of exons 1, 2, and 5A, respectively. The PI promoter is a constitutive promoter, the PII promoter is regulated by various hormones, and the PIII promoter is expressed in a tissue-specific manner. The presence of the alternatively spliced exons does not alter the translation of the ACC1 protein which starts from an AUG present in exon 5. The ACC2 gene (symbol: ACACB) is located on chromosome 12q24.11 and is composed of 58 exons that encode a protein of 2,458 amino acids.

ACC1 is strictly cytosolic and is enriched in liver, adipose tissue and lactating mammary tissue. ACC2 was originally discovered in rat heart but is also expressed in liver and skeletal muscle. ACC2 has an N-terminal extension that contains a mitochondrial targeting motif and is found associated with carnitine palmitoyltransferase I (CPT I) allowing for rapid regulation of CPT I by the malonyl-CoA produced by ACC. Both isoforms of ACC are allosterically activated by citrate and inhibited by palmitoyl-CoA and other short- and long-chain fatty acyl-CoAs. Citrate triggers the polymerization of ACC1 which leads to significant increases in its activity. Although ACC2 does not undergo significant polymerization (presumably due to its mitochondrial association) it is allosterically activated by citrate. Glutamate and other dicarboxylic acids can also allosterically activate both ACC isoforms.

ACC activity can also be affected by phosphorylation. Both ACC1 and ACC2 contain at least eight sites that undergo phosphorylation. The sites of phosphorylation in ACC2 have not been as extensively studied as those in ACC1. Phosphorylation of ACC1 at three serine residues (S79, S1200, and S1215) by AMPK leads to inhibition of the enzyme. AMPK also phosphorylates and inhibits the activity of ACC2. Glucagon-stimulated increases in cAMP and subsequently to increased PKA activity also lead to phosphorylation of ACC where ACC2 is a better substrate for PKA than is ACC1. Epinephrine activation of β-adrenergic receptors also results in increased PKA activity and consequently phosphorylation and inhibition of ACC1. The activating effects of insulin on ACC are complex and not completely resolved. It is known that insulin leads to the dephosphorylation of the serines in ACC1 that are AMPK targets in the heart enzyme. This insulin-mediated effect has not been observed in hepatocytes or adipose tissues cells. At least a portion of the activating effects of insulin are related to changes in cAMP levels. Early evidence has shown that phosphorylation and activation of ACC occurs via the action of an insulin-activated kinase. However, contradicting evidence indicates that although there is insulin-mediated phosphorylation of ACC this does not result in activation of the enzyme. Activation of α-adrenergic receptors in liver and skeletal muscle cells inhibits ACC activity as a result of phosphorylation by an as yet undetermined kinase.

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Regulation by Malonyl-CoA Decarboxylase: MCD

The intracellular levels of malonyl-CoA represent a balance between its synthesis from acetyl-CoA by ACC and its utilization in fatty acid synthesis by FAS as well as by its degradation to acetyl-CoA via the action of malonyl-CoA decarboxylase (MCD). Indeed, MCD is involved in regulating malonyl-CoA levels in multiple tissues. MCD is found in the cytosol, the mitochondria, and in peroxisomes. The MCD enzyme is encoded by the MLYCD gene located on chromosome 16q24 and is composed of 5 exons encoding a protein of 493 amino acids. Inhibition of MCD results in reduced rates of fatty acid oxidation in highly oxidative tissues such as the heart. As well, MCD inhibition leads to reduced triacylglyceride content in lipid synthesizing tissues such as the liver. When hypothalamic MCD levels are experimentally increased in laboratory animals there is a dramatic increase in food intake, weight gain, and ultimately results in obesity. Conversely, inactivation of the MCD gene in mice protects the animals from high-fat diet-induced insulin resistance. Additionally, chemical inhibition of MCD leads to reduced macrophage-associated inflammation in conditions of insulin resistance. The primary mechanism for this effect appears to be via the accumulation of long-chain fatty acids which in turn activate PPARα.

Interestingly, reduced MCD activity also exerts a beneficial effect in the hypothalamus. Transcriptional regulation of the MCD gene is exerted by PPARα, a major transcription factor involved in the regulation of fatty acid oxidation. Hypothalamic PPARα has been shown to play a role in the regulation of appetite, presumably via enhanced expression of MCD. PPARα-mediated increases in MCD levels results in reduced levels of malonyl-CoA in the hypothalamus. This is associated with increased food intake in the FAS knock-out mice demonstrating that malonyl-CoA levels are indeed responsible for the hypophagic effects observed in the FAS knock-out mice. The potential therapeutic benefits to reduced MCD activity in the treatment of obesity and diabetes are currently undergoing intensive investigation.

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Additional Regulatory Processes

Control of a given pathways' regulatory enzymes can also occur by alteration of enzyme synthesis and turn-over rates. These changes are long term regulatory effects. Insulin stimulates ACC and FAS synthesis, whereas, starvation leads to decreased synthesis of these enzymes. Adipose tissue lipoprotein lipase levels also are increased by insulin and decreased by starvation. However, in contrast to the effects of insulin and starvation on adipose tissue, their effects on heart lipoprotein lipase are just the inverse. This allows the heart to absorb any available fatty acids in the blood in order to oxidize them for energy production. Starvation also leads to increases in the levels of fatty acid oxidation enzymes in the heart as well as a decrease in FAS and related enzymes of synthesis.

Adipose tissue contains hormone-sensitive lipase (HSL), that is activated by PKA-dependent phosphorylation leading to increased fatty acid release to the blood. The activity of HSL is also affected through the action of AMPK, however, AMPK-mediated phosphorylation of HSL is inhibitory. The phosphorylation and inhibition of HSL by AMPK may seem paradoxical since the release of fatty acids stored in triglycerides would seem necessary to promote the production of ATP via fatty acid oxidation and the major function of AMPK is to shift cells to ATP production from ATP consumption. This paradigm can be explained if one considers that if the fatty acids that are released from triglycerides are not consumed they will be recycled back into triglycerides at the expense of ATP consumption. Thus, it has been proposed that inhibition of HSL by AMPK mediated phosphorylation is a mechanism to ensure that the rate of fatty acid release does not exceed the rate at which they are utilized either by export or oxidation.

In the liver the net result of activation of HSL (due to increased acetyl-CoA levels) is the production of ketone bodies. This would occur under conditions where insufficient carbohydrate stores and gluconeogenic precursors were available in liver for increased glucose production. The increased fatty acid availability in response to glucagon or epinephrine is assured of being completely oxidized since both PKA and AMPK also phosphorylate (and as a result inhibits) ACC, thus inhibiting fatty acid synthesis.

Insulin, on the other hand, has the opposite effect to glucagon and epinephrine leading to increased glycogen and triacylglyceride synthesis. One of the many effects of insulin is to lower cAMP levels which leads to increased dephosphorylation through the enhanced activity of protein phosphatases such as PP-1. With respect to fatty acid metabolism this yields dephosphorylated and inactive hormone sensitive lipase. Insulin also stimulates certain phosphorylation events. This occurs through activation of several cAMP-independent kinases.

Regulation of fat metabolism also occurs through malonyl-CoA induced inhibition of carnitine acyltransferase I (CPTI). This functions to prevent the newly synthesized fatty acids from entering the mitochondria and being oxidized. Indeed, as pointed out above, the localization of ACC2 to the mitochondrial outer membrane allows for rapid inhibition of CPTI when fatty acid synthesis has been stimulated.

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ChREBP: Master Lipid Regulator in the Liver

When glycogen stores are maximal in the liver, excess glucose is diverted into the lipid synthesis pathway. Glucose is catabolized to acetyl-CoA and the acetyl-CoA is used for de novo fatty acid synthesis. The fatty acids are then incorporated into triglycerides and exported from hepatocytes as very-low-density lipoproteins (see the Lipoproteins page for more details) and ultimately stored as triglycerides in adipose tissue. A diet rich in carbohydrates leads to stimulation of both the glycolytic and lipogenic pathways. Genes encoding glucokinase (GK) and liver pyruvate kinase (L-PK) of glycolysis and ATP-citrate lyase (ACLY), ACC1, and FAS of lipogenesis are regulated by modulation of their transcription rates. In addition, the enzymes encoded by these genes are subject to post-translational and allosteric regulation. These genes contain glucose- or carbohydrate-response elements (ChoREs) that are responsible for their transcriptional regulation.

One transcription factor that exerts control over glucose and lipid homeostasis is sterol-response element-binding protein (SREBP), in particular SREBP-1c. The regulation and actions of SREBP are discussed in the Cholesterol Metabolism page. SREBP controls the expression of a number of genes involved in lipogenesis and its own transcription is increased by insulin and repressed by glucagon. However, SREBP activity alone cannot account for the stimulation of glycolytic and lipogenic gene expression in response to a carbohydrate rich diet. A search for potential additional regulatory factors revealed a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor which was identified as carbohydrate-responsive element-binding protein (ChREBP). ChREBP was identified as a major glucose-responsive transcription factor and it is required for glucose-induced expression of L-PK and the lipogenic genes, ACC1 and FAS.

Expression of the ChREBP gene is induced in the liver in response to increased glucose uptake. In addition to gene activation, the activity of ChREBP is regulated by post-translational modifications as well as sub-cellular localization. The kinases PKA and AMPK both phosphorylate ChREBP rendering it inactive as a transcriptional activator. PKA is known to phosphorylate ChREBP on serine 196 (S196) and threonine 666 (T666), whereas, AMPK phosphorylates ChREBP at serine 568 (S568). Under conditions of low (basal) glucose concentration, ChREBP is phosphorylated and resides in the cytosol. When glucose levels rise, protein phosphatase 2A delta (PP2Aδ) is activated by xylulose 5-phosphate which is an intermediate in the pentose phosphate pathway. PP2Aδ dephosphorylates S196 resulting in translocation of ChREBP into the nucleus. In the nucleus PP2Aδ dephosphorylates T666 which allows ChREBP to bind to specific sequence elements (ChoREs) in target genes. ChREBP does not bind to ChoREs as a typical homodimeric bHLH transcription factor. ChREBP interacts with another bHLH protein identified as MAX-like protein X (MLX). MLX is a member of the MYC/MAX/MAD family of transcription factors that serve as interacting partners in transcription factor networks. The binding of MLX to ChREBP occurs within a domain located in the C-terminal portion of ChREBP. This interaction between ChREBP and MLX is essential for DNA-binding.

Additional mechanisms of glucose-mediated regulation of ChREBP activity were made apparent when it was shown that mutations in the PKA phosphorylation sites (S196 and T666) did not completely abolish glucose-responsiveness. Further analysis of ChREBP regulation in response to glucose administration was shown to be due to domains present in the amino terminal portion of ChREBP. The glucose sensing domain (GSM) is actually composed of two distinct sub-domains identified as the low-glucose inhibitory domain (LID) and the glucose-responsive activation conserved element (GRACE). Under conditions of low glucose the LID inhibits transcriptional transactivation by the GRACE domain. This inhibition is reversed by glucose or a glucose metabolite.

Recently a newly discovered mechanism of regulated ChREBP activity involves the tissue-specific transcription of an alternatively spliced form of ChREBP. This alternative splice variant contains a novel upstream exon (identified as exon 1b) and bypasses the originally identified exon 1 (now identified as exon 1a). This novel mechanism of ChREBP activity has been shown to occur in adipose tissue and represents a potent mechanism for glucose-mediated modulation of adipose tissue fatty acid synthesis and insulin sensitivity. The original ChREBP is now referred to as ChREBP-α and the novel alternative splice form is called ChREBP-β. The means by which glucose plays a role in adipose tissue ChREBP activity is that glucose, or a metabolite, activates the transcriptional activity of ChREBP-α by the mechanisms described earlier. Then, one of the important adipose tissue targets for glucose-activated ChREBP-α is the upstream transcriptional activation site that regulates the transcription of ChREBP-β. Following adipose tissue activation of ChREBP-β expression, both ChREBP-α and ChREBP-β work in concert to dramatically alter lipogenic gene expression.

Genes whose patterns of expression are under the control of ChREBP activity include L-PK, ACC1 and FAS as indicated above. As indicated, within adipose tissue ChREBP-α and ChREBP-β function together to dramatically increase the transcription of lipogenic genes in this tissue such as FAS and ACC1. In addition, it has been shown that when expression of ChREBP is reduced the expression levels of glycerol 3-phosphate acyltransferases, GPAT (encoded by four distinct genes: GPAM, GPAT2, GPAT3, GPAT4) and Δ9-stearoly-CoA desaturase 1 (SCD1) are also reduced. The GPAT enzymes esterify glycerol-3-phospate generating lysophosphatidic acid (LPA) which is the first step in the synthesis of triacylglycerides (TAGs) as described below. SCD1 is the rate-limiting enzyme involved in the synthesis of the major monounsaturated fatty acids oleic acid (18:1) and palmitoleic acid (16:1) as described below in the section on Elongation and Desaturation.

The liver X receptors (LXRs) are members of the steroid/thyroid hormone superfamily of cytosolic ligand binding receptors that migrate to the nucleus upon ligand binding and regulate gene expression by binding to specific target sequences. There are two forms of the LXRs, LXRα and LXRβ. The LXRs form heterodimers with the retinoid X receptors (RXRs) and as such can regulate gene expression either upon binding oxysterols (e.g. 22R-hydroxycholesterol) or 9-cis-retinoic acid. LXRs are also important regulators of the lipogenic pathway. Recent evidence has demonstrated that the ChREBP gene is a direct target of LXRs and that glucose itself can bind and activate LXRs.

ChREBP and LXR in glucose and lipid homeostasis

Role of ChREBP in the modulation of lipid and glucose homeostasis. Increased entry of glucose into the cell results in enhanced oxidation in the pentose phosphate pathway (PPP) resulting in increased levels of xylulose-5-phosphate (X5P). X5P activates the phosphatase PP2Aδ which removes inhibitory phosphorylations on ChREBP both in the cytosol and the nucleus. Active ChREBP then can turn on the expression of numerous genes involved in the homeostasis of glucose and lipid metabolism in the liver. Activation of LXRα, by lipid ligands, results in increased expression of ChREBP in the liver which in turn can lead to further modulation of lipid and glucose homeostasis. GPAT is glycerol-3-phosphate acyltransferase. SCD1 is stearoyl-CoA desaturase. L-PK is liver pyruvate kinase. ACC1 is acetyl-CoA carboxylase 1. FAS is fatty acid synthase. MUFA is monounsaturated fatty acid. The PKA and AMPK sites of phosphorylation in ChREBP are indicated where S is serine and T is threonine and the numbers refer to the specific amino acid in the ChREBP protein. The red T-lines indicate inhibition.

An emerging model of the role of ChREBP in overall glucose and lipid metabolism indicates that this transcription factor is a master regulator of glucose-mediated lipid homeostasis not only in the liver but also in adipose tissue. In the liver ChREBP controls 50% of overall lipogenesis through its concerted actions on the expression of lipogenic and glycolytic genes.

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Elongation and Desaturation

Elongation

The fatty acid product released from FAS is palmitate (via the action of palmitoyl thioesterase) which is a 16:0 fatty acid (i.e. 16 carbons and no sites of unsaturation). Elongation of fatty acids occurs in the cytosol, the mitochondria, and the endoplasmic reticulum (microsomal membranes). The predominant site of fatty acid elongation is in the ER membranes. Elongation involves condensation of fatty acyl-CoA groups with either acetyl-CoA or malonyl-CoA. The resultant product is two carbons longer (CO2 is released from malonyl-CoA as in the FAS reaction) which undergoes reduction, dehydration and reduction yielding a saturated fatty acid extended in length by two carbon atoms. The reduction reactions of elongation require NADPH as co-factor. Cytoplasmic fatty acid elongation is essentially an extension of the normal fatty acid synthesis reaction. Acetyl-CoA carboxylase (ACC) produces malonyl-CoA and then FAS utilizes acetyl-CoA and the malonyl-CoA to elongate the primary end-product of normal fatty aicd synthesis, palmitate. Mitochondrial fatty acid elongation involves a fatty acyl-CoA and acetyl-CoA units and the enzyme trans-2-enoyl-CoA reductase (encoded by the MERC gene). The mitochondrial fatty acid elongation pathway is a minor pathway in humans.

The microsomal fatty acid elongation pathway represents the major pathway for the elongation of both endogenous and dietary fatty acids. The overall pathway involves four enzymes with the substrates being fatty acyl CoAs, malonyl CoA, and NADPH. In the initial step in the pathway a 3-keto acyl-CoA synthase catalyzes the condensation of malonyl CoA with a fatty acyl-CoA precursor. In the second reaction a 3-keto acyl-CoA reductase reduces the resulting 3-keto intermediate. The third step involves the dehydration of the 3-hydroxy species and it is catalyzed by 3-hydroxyacyl-CoA dehydratase 2 (encoded by the HACD2 gene). In the fourth step the product of step 3 is reduced by trans-2,3-enoyl-CoA reductase. Humans express seven distinct 3-keto acyl-CoA synthase encoding genes. These seven genes are designated ELOVL1–ELOVL7 which stands for ELOngation of Very Long fatty acids. The two reductases, 3-keto acyl-CoA reductase and trans-2,3-enoyl-CoA reductase (encoded by the TERC gene), have been identifed in humans. The 3-keto acyl-CoA reductase activity is associated with the enzyme hydroxysteroid 17-β dehydrogenase 12 encoded by the HSD17B12 gene.

Fatty acyl-CoA substrate specificity and the rate of fatty acid elongation in the microsomes is determined by the ELOVL enzymes and not the reductases nor the dehydratase. The ELOVL1 encoded enzyme exhibits preference for saturated fatty acids in the 18-carbon (C18) to C26 length with highest activity towards the C22:0 saturated fatty acid called behenic acid. The ELOVL2 encoded enzyme elongates polyunstaturated fatty acids (PUFA) of C20 and C22 length with highest activity towards the C20:4 PUFA, arachidonic acid. The ELOVL3 encoded enzyme elongates saturated fatty acids with highest preference for stearic acid (C18:0). The ELOVL4 encoded enzyme preferentially elongates very long-chain saturated fatty acids such as lignoceric (C24:0) and cerotic (C26:0) acids. The ELOVL5 encoded enzyme elongates PUFA with highest preference for the C18:3 fatty acids, α-linolenic acid (ALA) and gamma(γ)-linolenoyl-CoA (GLA). ALA has sites of unsaturation at carbons 9, 12, and 15 and is the precursor fatty acid in the pathway of omega-3 PUFA synthesis. GLA has sites of unsaturation at carbons 6, 9, and 12 and is an intermediate in the pathway of arachidonic acid synthesis. ELOVL5 can also elongate some monounsaturated fatty acids, such as palmitoleic acid (C16:1). The ELOVL6 encoded enzyme elongates saturated fatty acids with 12, 14, and 16 carbon atoms with highest activity towards palmitic acid (C16:0). The ELOVL7 encoded enzyme has been shown to elongate a range of both saturated and unsaturated fatty acids such as the C16:0, C18:0, C18:1, C18:2, C18:3, and C20:4 fatty acids. The C18:3 substrates of ELOVL7 are the same as those used by the ELOVL5 enzyme. ELOVL7 shows no elongation activity towards fatty acids longer than 20 carbons. The ELOVL genes are expressed in tissue specific patterns as well as being subjected to dietary and hormonal regulation. ELOVL1, ELOVL2, ELOVL3, ELOVL5, and ELOVL6 are highly expressed in the liver. The heart does not express ELOVL2 but does express ELOVL1, ELOVL5, and ELOVL6.

The fatty acid elongases function in concert with with fatty acid desaturases (next section) to generate many of the long chain monounsaturated (MUFA) and PUFA that are found in cellular lipids. For example ELOVL6 and steroryl-CoA desaturase (SCD) generate the 18-carbon monounsaturated fatty acid (C18:1), oleic acid. ELOVL2 and ELOVL5 function along with the delta-5 (D5D) and delta-6 (D6D) desaturases to generate the omega-6 PUFA, arachidonic acid (C20:4) and the omega-3 PUFA, docosahexaenoic acid, DHA (C22:6).

Desaturation

The desaturation of fatty acids occurs in the ER membranes as well. In mammalian cells fatty acid desaturation involves three broad specificity fatty acyl-CoA desaturases (non-heme iron containing enzymes). These enzymes introduce unsaturation at C5, C6 or C9. The names of these enzymes are Δ5-eicosatrienoyl-CoA desaturase (D5D), Δ6-oleoyl(linolenoyl)-CoA desaturase (D6D), and Δ9-stearoyl-CoA desaturase (SCD). The D5D enzyme is encoded by the FADS1 gene located on chromosome 11q12.2 and is composed of 13 exons that encode a 501 amino acid protein. The D6D enzyme is encoded by the FADS2 gene located on the same chromosome as the FADS1 gene, 11q12.2 and is composed of 13 exons that generates three alternatively spliced mRNAs that encode three distinct isoforms of the enzyme. This chromosomal region is referred to as the FADS cluster and harbors an additional desaturase gene identified as FADS3. As yet, the precise biochemical function(s) of the FADS3 gene in humans remains unclear. The SCD enzyme is encoded by the SCD gene (also known as FADS5) on chromosome 10q24.31 and composed of 6 exons that generate at least two mRNAs that differ by alternative polyadenylation signal sequence utilization. Both SCD mRNAs encode the same 359 amino acid protein.

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme catalyzing the synthesis of monounsaturated fatty acids (MUFAs), primarily oleate (18:1; a physiologically significant omega-9 fatty acid) and palmitoleate (16:1). These two monounsaturated fatty acids represent the majority of monounsaturated fatty acids present in membrane phospholipids, triglycerides, and cholesterol esters. The expression of SCD is under the control of the transcription factor ChREBP as discussed above. The ratio of saturated to monounsaturated fatty acids in membrane phospholipids is critical to normal cellular function and alterations in this ratio have been correlated with diabetes, obesity, cardiovascular disease, and cancer. Thus, regulation of the expression and activity of SCD has important physiological significance.

Physiological and pathophysiological significance is associated with the activities of D5D and D6D. The activity of D6D requires pyridoxal phosphate (PLP, derived from vitamin B6), Mg2+, and Zn2+ ions and its activity is, therefore, affected by nutritional status. Both of D5D and D6D exhibit reduced and inhibited activity in conditions associated with hyperglycemia such as is typical of type 2 diabetes. Additional factors impairing D5D and D6D activity include alcohol consumption, hypercholesterolemia, and the processes of aging. Conversely, normal insulin responsiveness results in increased D5D and D6D activity as does a caloric restriction diet.

The electrons transferred from the oxidized fatty acids during desaturation are transferred from the desaturases to cytochrome b5 and then NADH-cytochrome b5 reductase. These electrons are uncoupled from mitochondrial oxidative-phosphorylation and, therefore, do not yield ATP.

Since these enzymes cannot introduce sites of unsaturation beyond C9 they cannot synthesize either linoleate (18:2Δ9,12) or linolenate (18:3Δ9,12,15). These fatty acids must be acquired from the diet and are, therefore, referred to as essential fatty acids. Linoleic is especially important in that it required for the synthesis of arachidonic acid. Arachindonate is a precursor for the eicosanoids (the prostaglandins, thromboxanes, and leukotrienes). It is this role of fatty acids in eicosanoid synthesis that leads to poor growth, wound healing and dermatitis in persons on fat free diets. Also, linoleic acid is a constituent of epidermal cell sphingolipids that function as the skins' water permeability barrier.

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Synthesis of Triglycerides

Fatty acids are stored for future use as triacylglycerides (TAG; triglycerides, TG) in all cells, but primarily in adipocytes of adipose tissue. TAGs constitute molecules of glycerol to which three fatty acids have been esterified. The fatty acids present in TAGs are predominantly saturated. The major building block for the synthesis of TAGs, in tissues other than adipose tissue, is glycerol. Adipocytes lack glycerol kinase, therefore, dihydroxyacetone phosphate (DHAP), produced during glycolysis, is the precursor for TAG synthesis in adipose tissue. This means that adipocytes must have glucose to oxidize in order to store fatty acids in the form of TAGs. DHAP can also serve as a backbone precursor for TAG synthesis in tissues other than adipose, but does so to a much lesser extent than glycerol. The fatty acids incorporated into TAGs are all activated to acyl-CoAs through the action of various acyl-CoA synthetases.

Pathways of triglyceride synthesis

Pathways for triacylglyceride synthesis. Triacylglycerides (triglycerides) are synthesized by virtually all cells. The major tissues for TAG synthesis are the small intestine, the liver, and adipocytes. Except for the intestine and adipocytes, TAG synthesis begins with glycerol. Glycerol is first phosphorylated by glycerol kinase and then activated fatty acids (fatty acyl-CoAs) serve as substrates for fatty acid addition generating phosphatidic acid. The phosphate group is then removed and the last fatty acid is added. In the small intestine, dietary TAGs are hydrolyzed to free fatty acids and monoacylglycerides (MAGs) prior to uptake by the enterocytes. The enterocyte MAGs serve as substrates for acylation in a two-step process yielding a TAG. Within adipose tissue there is no expression of glycerol kinase so the building block for TAG in this tissue is the glycolytic intermediate, dihydroxyacetone phosphate, DHAP. The DHAP is reduced to glycerol-3-phosphate by cytosolic glycerol-3-phosphate dehydrogenase and the remaining reaction of TAG synthesis are the same as for all other tissues. The enzyme acronyms are explained in the text below.


The glycerol backbone of TAGs is activated by phosphorylation at the C-3 (sn-3) position by glycerol kinase. Glycerol kinase is encoded by the GK gene which is located on the X chromosome (Xp21.3) and composed of 23 exons that generate four alternatively spliced mRNAs. The acylation of glycerol-3-phosphate is the major pathway for triglyceride synthesis in most human cells. Once formed, glycerol-3-phosphate is acylated at the sn-1 position by one of a family of glycerol-3-phosphate acyltransferase (GPAT) enzymes that are expressed in either the mitochondria or in the endoplasmic reticulum, ER. The products of the acylation reactions are lysophosphatidic acids, LPAs. Lysophosphatidic acids are themselves potent bioactive lipids. The GPAT family includes the two mitochondrial enzymes (encoded by the GPAM and GPAT2 genes) and the two ER enzymes (encoded by the GPAT3 and GPAT4 genes). The GPAM gene is located on chromosome 10q25.2 and is composed of 23 exons that generate two alternatively spliced mRNAs encoding the same 828 amino acid precursor protein. The GPAT2 gene is located on chromosome 2q11.1 and is composed of 22 exons that encode a 795 amino acid protein. The GPAT3 gene is located on chromosome 4q21.23 and is composed of 16 exons that generate three alternatively spliced mRNAs all of which encode the same 434 amino acid protein. The GPAT4 gene is located on chromosome 8p11.21 and is composed of 17 exons that encode a 456 amino acid protein.

Adipocytes lack glycerol kinase and, therefore, triglyceride synthesis requires the utilization of DHAP for the backbone in these cells. Within the adipocyte cytosol, DHAP is converted to glycerol-3-phosphate via the action of the cytoplasmic version of glycerol-3-phosphate dehydrogenase which is encoded by the GPD1 gene.

Following formation of a lysophosphatidic acid, various acylglycerol-3-phosphate acyltransferases (AGPAT) add another CoA-activated fatty acid to the sn-2 position generating 1,2-diacylglycerol phosphates which are commonly identified as phosphatidic acids (PA). Humans express five AGPAT encoding genes identified as AGPAT1–AGPAT5. All of the AGPAT enzymes are integral membrane proteins with the AGPAT1 and AGPAT2 enzymes specifically localized to the ER. The AGPAT1 gene is located on chromosome 6p21.3 and is composed of 11 exons that generate two alternatively spliced mRNAs both of which encode the same 283 amino acid protein. The AGPAT2 gene is located on chromosome 9q34.3 and is composed of 6 exons that generate two alternatively spliced mRNAs encoding two different isoforms of the enzyme. Mutations in the AGPAT2 gene result in one type of Berardinelli-Seip congenital generalized lipodystrophy (CGL). This disorder is an autosomal recessive disorder characterized by marked lack of adipose tissue at birth, severe insulin resistance, hypertriglyceridemia, hepatic steatosis, and early onset of diabetes. The AGPAT3 gene is located on chromosome 21q22.3 and is composed of 18 exons that generate two alternatively spliced mRNAs both of which encode the same 376 amino acid protein. The AGPAT4 gene is located on chromosome 6q26 and is composed of 10 exons that encode a 378 amino acid protein. The AGPAT5 gene is located on chromosome 8p23.1 and is composed of 8 exons that encode a 364 amino acid protein.

Intestinal monoacylglycerides (MAG), derived from the hydrolysis of dietary fats, can also serve as substrates for the synthesis of triglycerides (TAGs) which can then be incorporated into chylomicrons. The intestinal synthesis of a TAG from a MAG first involves MAG acylation carried out by one of a family of monoacylglycerol O-acyltransferases (MOGAT). Humans express three MOGAT genes identified as MOGAT1, MOGAT2, and MOGAT3. The MOGAT1 gene is located on chromosome 21q22.3 and is composed of 18 exons that encode a 335 amino acid protein. The MOGAT2 gene is located on chromosome 11q13.5 and is composed of 7 exons that encode a 334 amino acid protein. The MOGAT3 gene is located on chromosome 7q22.1 and is composed of 7 exons that generate two alternatively spliced mRNAs encoding two distinct isoforms of the enzyme. The resultant DAGs, in intestinal enterocytes, are then converted to TAGs via the action of the DGAT enzymes outlined in the next paragraph.

The phosphate of phosphatidic acid is removed, by phosphatidic acid phosphatase (PAP1), to yield 1,2-diacylglycerols (DAG). The PAP1 enzyme is encoded by a gene that is a member of the lipin family (see next section) identified as the LPIN1 gene. The DAG, along with a fatty acyl-CoA, serve as substrates for a diacylglycerol O-acyltransferase (DGAT) enzyme forming a triglyceride, TAG. Humans express two DGAT encoding genes identified as DGAT1 and DGAT2. The DGAT1 gene is located on chromosome 8q24.3 and is composed of 18 exons that encode a 488 amino acid protein. The DGAT2 gene is located on chromosome 11q13.5 and is composed of 9 exons that generate two alternatively spliced mRNAs encoding two isoforms of the enzyme.

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Lipin Genes: TAG Synthesis and Transcriptional Regulation

In addition to its role in overall TAG and phospholipid homeostasis, phosphatidic acid phosphatase (PAP1: encoded by the LPIN1 gene) has been shown to possess additional important activities. The lipin-1 gene (symbol: LPIN1) was originally identified as being mutated in a line of mice possessing a fatty liver dystrophy syndrome. This mouse line is identified as the fld mouse. Subsequent to the identification of the LPIN1 gene in the fld mouse, two additional related genes were identified. There are three lipin genes (LPIN1, LPIN2, and LPIN3) expressed in humans that collectively encode five lipin proteins. The LPIN1 gene encodes three protein isoforms derived through alternative splicing. These three lipin-1 isoforms are identified as lipin-1α, lipin-1β, and lipin-1γ. All five lipin proteins possess phosphatidic acid phosphatase activity that is dependent upon Mg2+ or Mn2+ and phosphatidic acid as the substrate.

Mutations in the LPIN2 gene are associated with Majeed syndrome which is characterized by chronic recurrent osteomyelitis, cutaneous inflammation, recurrent fever, and congenital dyserythropoietic anemia.

In addition to the obvious role of lipin-1 in TAG synthesis, evidence indicates that the protein is also required for the development of mature adipocytes, coordination of peripheral tissue glucose and fatty acid storage and utilization, and serves as a transcriptional co-activator. The latter function has significance to diabetes as it has been shown that some of the effects of the thiazolidinedione (TZD) class of drugs used to treat the hyperglycemia associated with type 2 diabetes are exerted via the effects of lipin-1. Although the lipin proteins do not contain DNA-binding motifs they have protein-interaction domains that allow them to form complexes with nuclear receptors and function as transcriptional regulators. Lipin-1 has been shown to interact with peroxisome proliferator-activated receptor-γ [PPARγ] co-activator 1α (PGC-1α) and PPARα leading to enhanced gene expression. Lipin-1 also is known to interact with additional members of the nuclear receptor family including the glucocorticoid receptor (GR) and hepatocyte nuclear factor-4α (HNF-4α). Lipin-1 also induces the expression of the adipogenic transcription factors PPARγ and CCAAT-enhancer-binding protein α (C/EBPα). The functions of lipin-1α and lipin-1β appear to be complimentary with respect to adipocyte differentiation. Lipin-1α induces genes that promote adipocyte differentiation while lipin-1β induces the expression of lipid synthesizing genes such as fatty acid synthase (FAS) and diacylglycerol acyltransferase (DGAT). The interactions of lipin-1 with PPARα and PGC-1α leads to increased expression of fatty acid oxidizing genes such as carnitine palmitoyltransferase-1 (CPT-1: encoded by the CPT1A gene), acyl-CoA oxidases (peroxisomal fatty acid oxidation pathway), and medium-chain acyl-CoA dehydrogenase (MCAD: encoded by the ACADM gene).

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Phospholipid Structures

Phospholipids are synthesized by esterification of an alcohol to the phosphate of phosphatidic acid (1,2-diacylglycerol 3-phosphate). Most phospholipids have a saturated fatty acid on C-1 and an unsaturated fatty acid on C-2 of the glycerol backbone. The most commonly added alcohols (serine, ethanolamine and choline) also contain nitrogen that may be positively charged, whereas, glycerol and inositol do not. The major classifications of phospholipids are the phosphatidylserines (PS), the phosphatidylethanolamines (PE), the phosphatidylcholines (PC; often referred to as lecithins), the phosphatidylinositols (PI), phosphatidylglycerols (PG; major components of pulmonary surfactant), and the diphosphatidylglycerols (DPG; more commonly called the cardiolipins).

structures of the major phospholipid classes


Phospholipid Composition of Various Mammalian Membranes (% total phospholipid)

  Membrane Type
  Plasma Mitochondrial Nuclear ER Golgi Lysosomal
Phospholipid Class  
Phosphatidylserine 4% 1% inner; 1% outer 6% 4% 4% 1%
Phosphatidylethanolamine 21% 38% inner; 34% outer 25% 21% 17% 21%
Phosphatidylcholine 43% 41% inner; 49% outer 52% 57% 4% 42%
Phosphatidylinositol 4% 2% inner; 9% outer 4% 9% 9% 6%
Cardiolipin 0% 16% inner; 5% outer 0% 0% 0% 0%

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Phospholipid Synthesis

Phosphatidylcholine, PC

This class of phospholipids is also called the lecithins. At physiological pH, phosphatidylcholines are neutral zwitterions. They contain primarily palmitic or stearic acid at carbon 1 and primarily oleic acid (18:1), linoleic acid (18:2), or linolenic acid (18:3) at carbon 2. The specific phosphatidylcholine with both C-1 and C-2 esterified with palmitic acid (16:0) is commonly identified as dipalmitoyllecithin. Dipalmitoyllecithin is a component of pulmonary surfactant and is the major (80%) phospholipid found in the extracellular lipid layer lining the pulmonary alveoli.

A significant cause of death in premature infants and, on occasion, in full term infants is respiratory distress syndrome (RDS) or hyaline membrane disease. This condition is caused by an insufficient amount of pulmonary surfactant. Under normal conditions the surfactant is synthesized by type II endothelial cells and is secreted into the alveolar spaces to prevent atelectasis following expiration during breathing. Surfactant is comprised primarily of dipalmitoyllecithin; additional lipid components include phosphatidylglycerol and phosphatidylinositol along with proteins of 18 and 36 kDa (termed surfactant proteins). During the third trimester the fetal lung synthesizes primarily sphingomyelin, and type II endothelial cells convert the majority of their stored glycogen to fatty acids and then to dipalmitoyllecithin. Fetal lung maturity can be determined by measuring the ratio of lecithin to sphingomyelin (L/S ratio) in the amniotic fluid. An L/S ratio less than 2.0 indicates a potential risk of RDS. The risk is nearly 75-80% when the L/S ratio is 1.5.

In humans, phosphatidylcholines are primarily synthesized via the CDP-choline pathway (also called the Kennedy pathway). Choline is first activated by phosphorylation and then by coupling to CDP prior to attachment to a 1,2-diacylglycerol. The phosphorylation of choline is catalyzed by choline kinase-α (encoded by the CHKA gene). Humans express two choline kinase genes, CHKA and CHKB (choline kinase-β; formerly called choline kinase-like, CHKL). The CHKA encoded enzyme is also responsible for the phosphorylation and activation of ethanolamine.

Phosphocholine is then converted to CDP-choline by the enzymes of the cytidylyltransferase family. Humans express three genes in this family, PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), PCYT1B (phosphate cytidylyltransferase 1, choline, beta), and PCYT2 (phosphate cytidylyltransferase 2, ethanolamine). The PCYT1A and PCYT1B encoded enzymes are involved in CDP-choline synthesis while the PCYT2 encoded enzyme is invoved in CDP-ethanolamine synthesis (see next section). The PCYT1A enzyme contains a nuclear localization signal and thus, it is predominantly found in this compartment.

Phosphatidylcholines are then synthesized from CDP-choline and a 1,2-diacylglycerol with concomitant release of CMP. The 1,2-diacylglycerols are derived from the action of enzymes of the phospholipid phosphatase family, such as that encoded by the LPIN1 gene, on phosphatidic acid. Other members of the family include phospholipid phosphatase 1 (encoded by the PLPP1 gene) and phospholipid phosphatase 3 (encoded by the PLPP3 gene). The last reaction of PC synthesis is catalyzed by diacylglycerol cholinephosphotransferase which is encoded by the CHPT1 (choline phosphotransferase 1) gene. The CHPT1 encoded enzyme is often referred to as CDP-choline:1,2-diacylglycerol cholinephosphotransferase.

An additional pathway for the synthesis of PC involves the trimethylation of PE using S-adenosylmethionine (SAM; or AdoMet) as methyl group donor. This second PC synthesis pathway only occurs to a significant degree in hepatocytes and is catalyzed by phosphatidylethanolamine N-methyltransferase which is encoded by the PEMT gene. Although an additional PC biosynthesis pathway is known that involves the addition of choline to CDP-activated 1,2-diacylglycerol, this pathway is not known to occur in human cells..

Phosphatidylethanolamine, PE

The phosphatidylethanolamines are neutral zwitterions at physiological pH. They contain primarily palmitic or stearic acid on carbon 1 and a long chain unsaturated fatty acid (e.g. 18:2, 20:4 and 22:6) on carbon 2. Synthesis of PE in humans is carried out by at least two distinct pathways and they account for the bulk of PE synthesis . These two pathways operate either in the ER or in the mitochondria.

One of the two major PE synthesis pathway occurs in the ER and involves the phosphorylation of ethanolamine via the action of CHKA encoded enzyme described in the section above. Phosphoethanolamine is then converted to CDP-ethanolamine via the activity of the PCYT2 encoded enzyme described in the previous section. Formation of PE from CDP-ethanolamine and a 1,2-diacylglycerol is catalzyed by selenoprotein I which is encoded by the SELENOI gene. The encoded enzyme is more commonly referred to as CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase and also as diacylglycerol ethanolaminephosphotransferase. The second major pathway for PE synthesis occurs in the inner mitochondrial membrane and involves the decarboxylation of PS. The decarboxylation reaction is catalyzed by phosphatidylserine decarboxylase which is encoded by the PISD gene.

Another minor PE synthesis reaction involves the fatty acylation of a lysophosphatidylethanolamine (lyso-PE) converting it to PE which is catalyzed by an enzyme called lyso-PE acyltransferase which is encoded by the MBOAT2 (membrane bound O-acyltransferase domain containing 2) gene.

Phosphatidylserine, PS

Phosphatidylserines will carry a net charge of –1 at physiological pH and are composed of fatty acids similar to the phosphatidylethanolamines. The pathway for PS synthesis involves base exchange reactions of serine for the ethanolamine in PE or serine for the choline in PC. These exchange reactions can be catalyzed by a single enzyme, phosphatidylserine synthase 1, which is encoded by the PTDSS1 gene. The enzyme encoded by the PTDSS1 gene exhibits a high degree of preference for PC and is, therefore, the primary ezyme for generation of PS via the base exchange reaction. A related gene, PTDSS2, encodes phosphatidylserine synthase 2 and this enzymes appears to be exclusive for the exchange of serine for ethanolamine in PE generating PS. Also, as indicated above, PS can serve as a source of PE through a decarboxylation reaction cataltyzed by the PISD encoded enzyme.

Pathways of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine synthesis

Pathways of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Phosphatidic acid serves as precursor building block for the synthesis of PS, PE, and PC. The phosphate is removed from phosphatidic acid through the action of phospholipid phosphatases such as the LPIN1 encoded enzyme (shown). Additional enzymes are encoded by the PLPP1 and PLPP3 genes (not shown). Phosphatidylcholines and phosphatidylethanolamines begin with phosphate activated choline and ethanolamine generated via the action of choline kinase-α encoded by the CHKA gene. Phosphocholine and phosphoethanolamine are then attached to CDP via the actions of the PCYT1A (or PCYT1B) and PCYT2 encoded genes, respectively. Phosphatidylcholines are synthesized from CDP-choline and a 1,2-diacylglycerol via the action of diacylglycerol cholinephosphotransferase which is encoded by the CHPT1 gene. Phosphatidylethanolamines are synthesized from CDP-ethanolamine and a 1,2-diacylglycerol via the action of selenoprotein I which is encoded by the SELENOI gene. Phosphatidylserines are generated from PE and PC via base exchange reactions catalyzed by the enzymes encoded by the PTDSS1 and PTDSS2 genes. Phosphatidylserine can be used to make PE through a decarboxylation reaction catalyzed by the PISD encoded enzyme. Phosphatidylethanolamine can serve as the precursor for PC via methylation reactions catalyzed by the PEMT encoded enzyme. SAM and AdoMet are S-adenosylmethionine. SAH and AdoHcy are S-adenosylhomocysteine.


Phosphatidylinositols, PI

The phosphatidylinositols contain almost exclusively stearic acid at carbon 1 and arachidonic acid at carbon 2. Phosphatidylinositols composed exclusively of non-phosphorylated inositol exhibit a net charge of –1 at physiological pH. These molecules exist in membranes with various levels of phosphate esterified to the hydroxyls of the inositol. Molecules with phosphorylated inositol are termed polyphosphoinositides. The polyphosphoinositides are important intracellular transducers of signals emanating from the plasma membrane.

In humans, the synthesis of PI (as well as PG) begins with the formation of CDP-diacylglycerol from phosphatidic acid and CTP. The synthesis of CDP-diacylglycerol is catalyzed by at least three enzymes that includes CDP-diacylglycerol synthase 1 and CDP-diacylglycerol synthase 2, which are encoded by the CDS1 and CDS2 genes, respectively. The third enzyme that can synthesize CDP-diacylglycerol is a bifunctional enzyme that is also responsible for the condensation of a CDP-diacylglycerol with myo-inositol forming a PI. This latter enzyme is called CDP-diacylglycerol--inositol 3-phosphatidyltransferase and it is encoded by the CDIPT gene. The CDIPT encoded enzyme is also referred to simply as phosphatidylinositol synthase, PIS. In humans the major enzyme responsible for the synthesis of CDP-diacylglycerol used for PI synthesis is likely to be the CDS1 encoded enzyme.

Phosphatidylinositols subsequently undergo a series of phosphorylations of the hydroxyls of inositol leading to the production of polyphosphoinositides. One particular polyphosphoinositide, phosphatidylinositol (4,5)-bisphosphate, PIP2, is a critically important membrane phospholipid involved in the transmission of signals for cell growth and differentiation. Another critically important PI is phosphatidylinositol (3,4,5)-trisphosphate (PIP3) which is generated from PIP2 via the action of the phosphatidylinositol-3-kinases, PI3K. PIP3 activates the kinase called phosphoinositide-dependent kinase-1 (PDK1) which serves as a master regulatory kinase in the activation of the kinases PKB/AKT, PKC, S6K (p70-S6 kinase 1), and the Ser/Thr kinases (SGK1, SGK2, and SGK3).

Phosphatidylglycerols, PG

Phosphatidylglycerols exhibit a net charge of –1 at physiological pH. These molecules are found in high concentration in mitochondrial membranes and as components of pulmonary surfactant. Phosphatidylglycerols are synthesized in a two-step process that begins with CDP-diacylglycerol and glycerol-3-phosphate. The first reaction yields phosphatidylglycerol phosphate and this reaction is catalyzed by the ER-localized enzyme identified as phosphatidylglycerophosphate synthase 1 (encoded by the PGS1 gene). The CDP-diacylglycerol is synthesized by the CDS1, CDS2, or CDIPT encoded enzymes as described in the previous section. Phosphatidylglycerol phosphates are then converted to phosphatidylglycerols (PG) via removal of phosphate most likely by the action of the PGS1 encoded enzyme although the activity has been called PGP phosphatase, phosphatidylglycerol phosphate phosphatase, and phosphatidylglycerophosphatase. Phosphatidylglycerols are also the precursors for the synthesis of the diphosphatidylglycerols (DPG) which are more commonly called the cardiolipins. The cardiolipins are major lipid components of the inner mitochondrial membrane.

Diphosphatidylglycerols: Cardiolipins

The cardiolipins are very acidic, exhibiting a net charge of –2 at physiological pH. They are found almost exclusively in the inner mitochondrial membrane. The cardiolipins are synthesized by the condensation of a CDP-diacylglycerol with a PG in a reaction catalyzed by cardiolipin synthase 1 which is encoded by the CRLS1 gene.

Pathways of phosphatidylinositol, phosphatidylglycerol, and cardiolipin synthesis

Pathways of phosphatidylinositol, phosphatidylglycerol, and cardiolipin synthesis. Phosphatidylinositols are synthesized via a condensation between CDP-activated 1,2-diacylglycerols (CDP-diacylglycerol) and myo-inositol via the actions of CDP-diacylglycerol--inositol 3-phosphatidyltransferase which is a bifunctional enzyme encoded by the CDIPT gene. The CDIPT encoded enzyme can gererate CDP-diacylglycerol from phosphatidic acid and CTP, a reaction that is also catalyzed by at least two additional CDP-diacylglycerol synthases encoded by the CDS1 and CDS2 genes. The CDIPT encoded enzyme is also referred to simply as phosphatidylinositol synthase, PIS. Various phosphatidylinositol kinases phosphorylate the hydroxyls on the inositol moiety of phopshatidylinositols generating various polyphosphoinositides such as the important phosphatidylinositol-4,5-bisphosphate, PIP2. Phosphatidylglycerols are synthesized in a two-step process that begins with CDP-diacylglycerol and glycerol-3-phosphate followed by removal of phosphate. Both reactions are catalyzed by phosphatidylglycerophosphate synthase 1 which is encoded by the PGS1 gene. Phosphatidylglycerols and CDP-diacylglycerols are combined through the actions of cardiolipin synthase 1 to form diphosphatidylglycerols (DPG) which are more commonly called cardiolipins. Cardiolipin synthase 1 is encoded by the CRLS1 gene.

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Phospholipases and Phospholipid Remodeling

The fatty acid distribution at the C–1 and C–2 positions of glycerol within phospholipids is continually in flux, owing to phospholipid degradation and the continuous phospholipid remodeling that occurs while these molecules are in membranes. Phospholipid degradation results from the action of numerous different phospholipases. There are various phospholipases that exhibit substrate specificities for different positions in phospholipids.

In many cases the acyl group which was initially transferred to glycerol, by the action of the acyl transferases, is not the same acyl group present in the phospholipid when it resides within a membrane. The remodeling of acyl groups in phospholipids is the result of the action of enzymes of the phospholipase A1 (PLA1) and phospholipase A2 (PLA2) families.

Actions of the phospholipases

Sites of action of the phospholipases A1, A2, C and D. The products of these various phospholipases are called lysophospholipids many of which are potent bioactive lipids. Lysophospholipids can be substrates for acyl transferases utilizing different acyl-CoA groups reforming the original or different fatty acid containing phospholipids.

Outside of the context of phospholipid remodeling PLA2 is an important enzyme, whose activity is responsible for the release of polyunsaturated fatty acids (PUFAs) such as arachidonic acid from the C2 (sn-2) position of membrane phospholipids. The released arachidonate is then a substrate for the synthesis of the eicosanoids. In fact there is not just a single PLA2 enzyme. At least 30 enzymes have been identified with PLA2 activity. For more details on the PLA2 family of lipases visit the Bioactive Lipids page. There are 10 isozymes that are in the secretory pathway and these PLA2 isozymes are abbreviated sPLA2. These secretory enzymes are low molecular weight proteins that are Ca2+-dependent and are involved in numerous processes including modification of eicosanoid generation, host defense, and inflammation. The cytosolic PLA2 family (cPLA2) comprises three isozymes with cPLA2α being an essential component of the initiation of arachidonic acid metabolism. Like the sPLA2 enzymes, the cPLA2 enzymes are tightly regulated by Ca2+. In addition, this class of PLA2 enzyme is regulated by phosphorylation. An additional family of two PLA2 isozymes that are Ca2+-independent for activity are identified as iPLA2. This latter class of enzyme is involved primarily with the remodeling of phospholipids. Finally, a class of PLA2 enzymes, whose original member was identified as being involved in the hydrolysis and inactivation of platelet activating factor, PAF (see the section below), contains at least four members. The original activity was called PAF-acetylhydrolase (PAF-AH). The PAF hydrolyzing PLA2 isozymes are Ca2+-independent like the iPLA2 family. Because this latter family was shown to not only hydrolyze PAF but also oxidized phospholipids and to be associated with lipoprotein particles in the circulation they are identified as the lipoprotein-associated PLA2 (Lp-PLA2) family. More details on the functions of the Lp-PLA2 family of enzymes can be found in the Lipoproteins page.

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Plasmalogens

Plasmalogens are glycerol ether phospholipids. They are of two types, alkyl ether (–O–CH2–) and alkenyl ether (–O–CH=CH–). Dihydroxyacetone phosphate serves as the glycerol precursor for the synthesis of glycerol ether phospholipids. Three major classes of plasmalogens have been identified: choline, ethanolamine and serine plasmalogens. Ethanolamine plasmalogen is prevalent in myelin. Choline plasmalogen is abundant in cardiac tissue. The initiation of plasmalogen synthesis takes place inside the lumen of the peroxisome using dihydroxyacetone phosphate (DHAP) as the building block. The first enzyme in the synthesis of plasmalogens is glycerone phosphate O-acyltransferase encoded by the GNPAT gene (also known as dihydroxyacetone phosphate acyltransferase, DHAPAT). The GNPAT gene is located on chromosome 1q42.2 and is composed of 17 exons that generate two alternatively spliced mRNAs encoding proteins of 680 amino acids (isoform 1) and amino acids 619 (isoform 2). The GNPAT enzyme adds a fatty acyl group to the sn-1 position of DHAP. The next step in the synthesis pathway is catalyzed by acylglycerone phosphate synthase encoded by the AGPS gene. The AGPS gene is located on chromosome 2q31.2 and is composed of 21 exons that encode a 658 amino acid precursor protein. The AGPS enzyme exchanges the acyl group added by GNPAT for an alkyl group. The alky-DHAP is then reduced by a reductase found in both the peroxisomes and the ER. The remaining reactions of plasmalogen synthesis occur within the ER and include acylation reactions at the sn-2 position and the removal of the phosphate group by one of the phosphatidic acid phosphatase family of enzymes.

One particular type of choline plasmalogen (1-O-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine) has been identified as an extremely powerful biological mediator, capable of inducing cellular responses at concentrations as low as 10–11M. This molecule is called platelet activating factor, PAF. PAF functions as a mediator of hypersensitivity, acute inflammatory reactions and anaphylactic shock. PAF is synthesized in response to the formation of antigen-IgE complexes on the surfaces of basophils, neutrophils, eosinophils, macrophages and monocytes. The synthesis and release of PAF from cells leads to platelet aggregation and the release of serotonin from platelets. PAF also produces responses in liver, heart, smooth muscle, and uterine and lung tissues.

Structure of platelet activating factor (PAF)

Most common PAF structure. Platelet activating factor exists in several molecular species dependent upon the size of the fatty alkyation at the sn-1 position. The most common form is that with a 16-carbon alkyl moiety at the sn-1 position. Despite the potential for variable sized alkyl groups, PAF is always acetylated at the sn-2 position.


Clinical significance of the plasmalogen biosynthesis pathway is evidenced by the severe phenotypes associated with deficiencies in enzymes of the pathway. Deficiencies in both GNPAT and AGPS result in the peroxisomal disorders of the rhizomelic chondrodysplasia punctata (RCDP) family. These disorders are identified as RCDP1, RCDP2, RCDP3, and RCDP5. The various RCDP disorders are characterized by disproportionately short stature primarily affecting the proximal parts of the extremities, a typical facial appearance including a broad nasal bridge, epicanthus, high-arched palate, dysplastic external ears, and micrognathia, congenital contractures, characteristic ocular involvement, dwarfism, and severe mental retardation with spasticity. Almost all RCDP infants die within the first year of life. The most commonly occurring RCDP is RCDP1. Whereas RCDP1 and RCDP5 are classified as peroxisomal biogenesis disorders, RCDP2 and RCDP3 are classified as single peroxisomal enzyme deficiencies. Mutations in the PEX7 gene are the causes of RCDP1 and mutation in the PEX5 gene are the causes of RCDP5. Mutations in the GNPAT gene result in RCDP2, whereas mutations in the AGPS gene result in RCDP3.

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Last modified: November 8, 2017