Fatty Acid, Triglyceride, Phospholipid Synthesis and Metabolism.pdf

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Fatty Acid SynthesisOrigin of Acetyl-CoA for Fat SynthesisRegulation of Fatty Acid SynthesisChREBP: Master Lipid Regulator in LiverElongation and Desaturation of Fatty AcidsTriacylglyceride SynthesisLipin Genes: TAG Synthesis and Transcriptional RegulationPhospholipid StructuresPhospholipid MetabolismPlasmalogen Synthesis

Omega-3, and -6 Polyunsaturated Fatty Acids (PUFAs)Eicosanoid MetabolismSphingolipid MetabolismCholesterol Metabolism

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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 areseparated 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 FADH+ and NAD+.Synthesis of fats involves the oxidation of NADPH. However, the essential chemistry of the two processes arereversals of each other. Both oxidation and synthesis of fats utilize an activated two carbon intermediate, acetyl-CoA.However, the 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 thisreaction, acetyl-CoA carboxylase (ACC), is the major site of regulation of fatty acid synthesis. Like other enzymes thattransfer CO2 to substrates, ACC requires a biotin co-factor.

The rate of fatty acid synthesis is controlled by the equilibrium between monomeric ACC and polymeric ACC. The

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activity of ACC requires polymerization. This conformational change is enhanced by citrateand inhibited by long-chain fatty acids. ACC is also controlled through hormone mediatedphosphorylation (see below).

The acetyl groups that are the products of fatty acid oxidation are linked to CoASH. As youshould recall, CoA contains a phosphopantetheine group coupled to AMP. The carrier of acetylgroups (and elongating acyl groups) during fatty acid synthesis is also a phosphopantetheineprosthetic 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 issomewhat of a misnomer in eukaryotic fatty acid synthesis since the ACP portion of thesynthetic complex is simply one of many domains of a single polypeptide. The acetyl-CoA andmalonyl-CoA are transferred to ACP by the action of acetyl-CoA transacylase and malonyl-CoA transacylase, respectively. The attachment of these carbon atoms to ACP allows them toenter the fatty acid synthesis cycle.

The synthesis of fatty acids from acetyl-CoA and malonyl-CoA is carried out by fatty acidsynthase, FAS. The active enzyme is a dimer of identical subunits.

All of the reactions of fatty acid synthesis are carried out by the multiple enzymaticactivities of FAS. Like fat oxidation, fat synthesis involves 4 enzymatic activities. These are, β-keto-ACP synthase, β-keto-ACP reductase, 3-OH acyl-ACP dehydratase and enoyl-CoA

reductase. The two reduction reactions require NADPH oxidation to NADP+.

The primary fatty acid synthesized by FAS is palmitate. Palmitate is then released fromthe enzyme and can then undergo separate elongation and/or unsaturation to yield other fattyacid molecules.

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 acetylgroup 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

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malonyl/acetyltransferase, MAT). This activating acetyl group represents the omega (ω) end of the newly synthesizedfatty acid. Following transfer of the activating acetyl group to a cysteine sulhydryl in the β-keto-ACP synthase portionof FAS, the three carbons from a malonyl-CoA are attached to ACP-SH (3) also catalyzed by malonyl/acetyl-CoAACP transacetylase. The acetyl group attacks the methylene group of the malonyl attached to ACP-SH catalyzed β-keto-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-keto-ACP reductase (5), 3-OH acyl-ACPdehydratase (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 transferredto the CYS-SH (8) as for the case of the activating acetyl group. At this point another malonyl group is attached to theACP-SH (3b) and the process begins again. Reactions 4 through 8 are repeated another six times, each beginningwith a new malonyl group being added. At the completion of synthesis the saturated 16 carbon fatty acid, palmiticacid, 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 bepresent in the cytoplasm. The shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energydiminishes. This results in reduced oxidation of acetyl-CoA in the TCA cycle and the oxidative phosphorylationpathway. 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 thecytoplasm, 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 theenergy of ATP hydrolysis to drive it forward. The resultant oxaloacetate is converted to malate by malatedehydrogenase (MDH).

Pathway for the movement of acetyl-CoA units from within the mitochondrion to the cytoplasm for use in lipid andcholesterol biosynthesis. Note that the cytoplasmic malic enzyme catalyzed reaction generates NADPH which canbe used for reductive biosynthetic reactions such as those of fatty acid and cholesterol synthesis.

The malate produced by this pathway can undergo oxidative decarboxylation by malic enzyme. The co-enzyme for

this reaction is NADP+ generating NADPH. The advantage of this series of reactions for converting mitochondrialacetyl-CoA into cytoplasmic acetyl-CoA is that the NADPH produced by the malic enzyme reaction can be a majorsource of reducing co-factor for the fatty acid synthase activities.

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Regulation of Fatty Acid Metabolism

One must consider the global organismal energy requirements in order to effectively understand how the synthesisand degradation of fats (and also carbohydrates) needs to be exquisitely regulated. The blood is the carrier oftriacylglycerols in the form of VLDLs and chylomicrons, fatty acids bound to albumin, amino acids, lactate, ketonebodies and glucose. The pancreas is the primary organ involved in sensing the organisms dietary and energetic statesvia glucose concentrations in the blood. In response to low blood glucose, glucagon is secreted, whereas, in responseto 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 effectorsand/or enzyme modification.

ACC is the rate-limiting (committed) step in fatty acid synthesis. There are two major isoforms of ACC inmammalian tissues. These are identified as ACC1 (also called ACCα) and ACC2 (also called ACCβ). The ACC1gene (symbol = ACACA) is located on chromosome 17q12 and encodeds a 2,346 amino acid proteins. The ACACAgene spans approximately 330 kb and is composed of 64 exons which includes 7 alternatively spliced minor exons.Transcriptional regulation of ACACA is effected by 3 promoters (PI, PII, and PIII), which are located upstream of exons1, 2, and 5A, respectively. The PI promoter is a constitutive promoter, the PII promoter is regulated by varioushormones, and the PIII promoter is expressed in a tissue-specific manner. The presence of the alternatively splicedexons does not alter the translation of the ACC1 protein which starts from an ATG present in exon 5. The ACC2 gene(symbol = ACACB) is located on chromosome 12q24.11 and ecodes a protein of 2,458 amino acids.

ACC1 is strictly cytosolic and is enriched in liver, adipose tissue and lactating mammary tissue. ACC2 wasoriginally discovered in rat heart but is also expressed in liver and skeletal muscle. ACC2 has an N-terminal extensionthat 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 allostericallyactivated by citrate and inhibited by palmitoyl-CoA and other short- and long-chain fatty acyl-CoAs. Citrate triggers thepolymerization of ACC1 which leads to significant increases in its activity. Although ACC2 does not undergosignificant 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 thatundergo phosphorylation. The sites of phosphorylation in ACC2 have not been as extensively studied as those inACC1. Phosphorylation of ACC1 at three serine residues (S79, S1200, and S1215) by AMPK leads to inhibition ofthe enzyme. Glucagon-stimulated increases in cAMP and subsequently to increased PKA activity also lead tophosphorylation of ACC where ACC2 is a better substrate for PKA than is ACC1. The activating effects of insulin onACC are complex and not completely resolved. It is known that insulin leads to the dephosphorylation of the serines inACC1 that are AMPK targets in the heart enzyme. This insulin-mediated effect has not been observed in hepatocytesor 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-activatedkinase. However, contradicting evidence indicates that although there is insulin-mediated phosphorylation of ACC thisdoes not result in activation of the enzyme. Activation of α-adrenergic receptors in liver and skeletal muscle cellsinhibits ACC activity as a result of phosphorylation by an as yet undetermined kinase.

Control of a given pathways' regulatory enzymes can also occur by alteration of enzyme synthesis and turn-overrates. These changes are long term regulatory effects. Insulin stimulates ACC and FAS synthesis, whereas, starvationleads to decreased synthesis of these enzymes. Adipose tissue lipoprotein lipase levels also are increased by insulinand decreased by starvation. However, in contrast to the effects of insulin and starvation on adipose tissue, theireffects on heart lipoprotein lipase are just the inverse. This allows the heart to absorb any available fatty acids in theblood in order to oxidize them for energy production. Starvation also leads to increases in the levels of fatty acidoxidation 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 phosphorylationleading 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 AMPKmay seem paradoxical since the release of fatty acids stored in triglycerides would seem necessary to promote theproduction of ATP via fatty acid oxidation and the major function of AMPK is to shift cells to ATP production from ATPconsumption. This paradigm can be explained if one considers that if the fatty acids that are released fromtriglycerides are not consumed they will be recycled back into triglycerides at the expense of ATP consumption. Thus, ithas been proposed that inhibition of HSL by AMPK mediated-phosphorylation is a mechanism to ensure that the rateof 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 ketonebodies. This would occur under conditions where insufficient carbohydrate stores and gluconeogenic precursors wereavailable in liver for increased glucose production. The increased fatty acid availability in response to glucagon orepinephrine is assured of being completely oxidized since both PKA and AMPK also phosphorylate (and as a resultinhibits) ACC, thus inhibiting fatty acid synthesis.

Insulin, on the other hand, has the opposite effect to glucagon and epi leading to increased glycogen andtriacylglyceride synthesis. One of the many effects of insulin is to lower cAMP levels which leads to increaseddephosphorylation through the enhanced activity of protein phosphatases such as PP-1. With respect to fatty acidmetabolism this yields dephosphorylated and inactive hormone sensitive lipase. Insulin also stimulates certain

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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.This functions to prevent the newly synthesized fatty acids from entering the mitochondria and being oxidized.

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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 arethen incorporated into triglycerides and exported from hepatocytes as very-low-density lipoproteins (see theLipoproteins page for more details) and ultimately stored as triglycerides in adipose tissue. A diet rich incarbohydrates 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 areregulated by modulation of their transcription rates. In addition, the enzymes encoded by these genes are subject topost-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-bindingprotein (SREBP), in particular SREBP-1c. The regulation and actions of SREBP are discussed in the CholesterolMetabolism page. SREBP controls the expression of a number of genes involved in lipogenesis and its owntranscription is increased by insulin and repressed by glucagon. However, SREBP activity alone cannot account for thestimulation of glycolytic and lipogenic gene expression in response to a carbohydrate rich diet. A search for potentialadditional regulatory factors revealed a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor which wasidentified as carbohydrate-responsive element-binding protein (ChREBP). ChREBP was identified as a majorglucose-responsive transcription factor and it is required for glucose-induced expression of L-PK and the lipogenicgenes ACC1 and FAS.

Expression of the ChREBP gene is induced in the liver in response to increased glucose uptake. In addition togene activation, the activity of ChREBP is regulated by post-translational modifications as well as sub-cellularlocalization. The kinases PKA and AMPK both phosphorylate ChREBP rendering it inactive as a transcriptionalactivator. PKA is known to phosphorylate ChREBP on serine 196 (S196) and threonine 666 (T666), wheras, AMPKphosphorylates ChREBP at serine 568 (S568). Under conditions of low (basal) glucose concentration, ChREBP isphosphorylated and resides in the cytosol. When glucose levels rise, protein phosphatase 2A delta (PP2Aδ) isactivated by xylulose 5-phosphate which is an intermediate in the pentose phosphate pathway. PP2Aδdephosphorylates S196 resulting in translocation of ChREBP into the nucleus. In the nucleus PP2Aδ dephosphorylatesT666 which allows ChREBP to bind to specific sequence elements (ChoREs) in target genes. ChREBP does not bindto ChoREs as a typical homodimeric bHLH transcription factor. ChREBP interacts with another bHLH proteinidentified as MAX-like protein X (MLX). MLX is a member of the MYC/MAX/MAD family of transcription factors thatserve as interacting partners in transcription factor networks. The binding of MLX to ChREBP occurs within a domainlocated 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 wasshown 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 todomains present in the amino terminal portion of ChREBP. The glucose sensing domain (GSM) is actually composedof two distinct sub-domains identified as the low-glucose inhibitory domain (LID) and the glucose-responsive activationconserved element (GRACE). Under conditions of low glucose the LID inhibits transcriptional transactivation by theGRACE 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 ofan alternatively spliced form of ChREBP. This alternative splice variant contains a novel upstream exon (identified asexon 1b) and bypasses the originally identified exon 1 (now identified as exon 1a). This novel mechanism of ChREBPactivity has been shown to occur in adipose tissue and represents a potent mechanism for glucose-mediatedmodulation of adipose tissue fatty acid synthesis and insulin sensitivity. The original ChREBP is now referred to asChREBP-α and the novel alternative splice form is called ChREBP-β. The means by which glucose plays a role inadipose tissue ChREBP activity is that glucose, or a metabolite, activates the transcriptional activity of ChREBP-α bythe mechanisms described earlier. Then, one of the important adipose tissue targets for glucose-activated ChREBP-αis the upstream transcriptional activation site that regulates the transcription of ChREBP-β. Following adipose tissueactivation of ChREBP-β expression, both ChREBP-α and ChREBP-β work in concert to dramatically alter lipogenicgene expression.

Genes whose patterns of expression are under the control of ChREBP activity include L-PK, ACC1 and FAS asindicated above. As indicated, within adipose tissue ChREBP-α and ChREBP-β function together to dramaticallyincrease 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 acyltransferase (GPAT) and Δ9-stearoly-CoA desaturase 1 (SCD1) are also reduced. GPAT is the enzyme that esterifies glycerol-3-phospategenerating lysophosphatidic acid which is the first step in the synthesis of triacylglycerols (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.

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The liver X receptors (LXRs) are members of the steroid/thyroid hormone superfamily of cytosolic ligand bindingreceptors that migrate to the nucleus upon ligand binding and regulate gene expression by binding to specific targetsequences. There are two forms of the LXRs, LXRα and LXRβ. The LXRs form heterodimers with the retinoid Xreceptors (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. Recentevidence has demonstrated that the ChREBP gene is a direct target of LXRs and that glucose itself can bind andactivate LXRs.

Role of ChREBP in the modulation of lipid and glucose homeostasis. Increased entry of glucose into the cellresults 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 ChREBPboth in the cytosol and the nucleus. Active ChREBP then can turn on the expression of numerous genes involved inthe homeostasis of glucose and lipid metabolism in the liver. Activation of LXRα, by lipid ligands, results in increasedexpression of ChREBP in the liver which in turn can lead to furhter 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 andAMPK sites of phosphorylation in ChREBP are indicated where S is serine and T is threonine and the numbers referto the specific amino acid in the ChREBP protein.

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

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Elongation and Desaturation

The fatty acid product released from FAS is palmitate (via the action of palmitoyl thioesterase) which is a 16:0 fattyacid, i.e. 16 carbons and no sites of unsaturation. Elongation and unsaturation of fatty acids occurs in both themitochondria and endoplasmic reticulum (microsomal membranes). The predominant site of these processes is in theER membranes. Elongation involves condensation of acyl-CoA groups with malonyl-CoA. The resultant product is twocarbons longer (CO2 is released from malonyl-CoA as in the FAS reaction) which undergoes reduction, dehydration

and reduction yielding a saturated fatty acid. The reduction reactions of elongation require NADPH as co-factor just asfor the similar reactions catalyzed by FAS. Mitochondrial elongation involves acetyl-CoA units and is a reversal ofoxidation except that the final reduction utilizes NADPH instead of FADH2 as co-factor.

The desaturation of fatty acids occurs in the ER membranes as well. In mammalian cells fatty acid desaturationinvolves 3 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, Δ6-

oleoyl(linolenoyl)-CoA desaturase and Δ9-stearoyl-CoA desaturase. The latter desaturase (SCD) is the rate-limitingenzyme catalyzing the synthesis of monounsaturated fatty acids, primarily oleate (18:1) and palmitoleate (16:1). These

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two monounsaturated fatty acids represent the majority of monounsaturated fatty acids present in membranephospholipids, triglycerides, and cholesterol esters. The expression of SCD is under the control of the transcriptionfactor ChREBP as discussed above. The ratio of saturated to monounsaturated fatty acids in membranephospholipids 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 importantphysiological significance.

The electrons transferred from the oxidized fatty acids during desaturation are transferred from the desaturases tocytochrome b5 and then NADH-cytochrome b5 reductase. These electrons are un-coupled 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 toas 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 offatty 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 triacylglycerols (TAGs) in all cells, but primarily in adipocytes of adiposetissue. TAGs constitute molecules of glycerol to which three fatty acids have been esterified. The fatty acids present inTAGs are predominantly saturated. The major building block for the synthesis of TAGs, in tissues other than adiposetissue, is glycerol. Adipocytes lack glycerol kinase, therefore, dihydroxyacetone phosphate (DHAP), produced duringglycolysis, is the precursor for TAG synthesis in adipose tissue. This means that adipocytes must have glucose tooxidize in order to store fatty acids in the form of TAGs. DHAP can also serve as a backbone precursor for TAGsynthesis in tissues other than adipose, but does so to a much lesser extent than glycerol.

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Phosphatidic acid Synthesis Triglyceride Synthesis

The glycerol backbone of TAGs is activated by phosphorylation at the C-3 position by glycerol kinase. Theutilization of DHAP for the backbone is carried out through either of two pathways depending upon whether thesynthesis of triglycerides is carried out in the mitochondria and ER or the ER and the peroxisomes. In the former casethe action of glycerol-3-phosphate dehydrogenase, a reaction that requires NADH (the same reaction as that used inthe glycerol-phosphate shuttle), converts DHAP to glycerol-3-phosphate. Glycerol-3-phosphate acyltransferase (GPAT)then esterifies a fatty acid to glycerol-3-phosphate generating the monoacylglycerol phosphate structure calledlysophosphatidic acid. The expression of the GPAT gene is under the influence of the transcription factor ChREBP asdescribed above.

The second reaction pathway utilizes the peroxisomal enzyme DHAP acyltransferase to fatty acylate DHAP toacyl-DHAP which is then reduced by the NADPH-requiring enzyme acyl-DHAP reductase. An interesting feature of thelatter pathway is that DHAP acyltransferase is one of only a few enzymes that are targeted to the peroxisomes throughthe recognition of a peroxisome targeting sequence 2 (PTS2) motif in the enzyme. Most peroxisomal enzymes containa PTS1 motif. For more information on peroxisome enzymes see the Zellweger syndrome page.

The fatty acids incorporated into TAGs are activated to acyl-CoAs through the action of acyl-CoA synthetases.Two molecules of acyl-CoA are esterified to glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonlyidentified as phosphatidic acid). The phosphate is then removed, by phosphatidic acid phosphatase (PAP1), to yield1,2-diacylglycerol, the substrate for addition of the third fatty acid. Intestinal monoacylglycerols, derived from thehydrolysis of dietary fats, can also serve as substrates for the synthesis of 1,2-diacylglycerols.

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Lipin Genes: TAG Synthesis and Transcriptional Regulation

Recent studies have identified a critical role for the enzyme PAP1 in overall TAG and phospholipid homeostasis.In the yeast Saccharomyces cerevisiae, the PAP1 gene was identified as Smp2p and the encoded protein wasshown to be the yeast ortholog of the mammalian protein called lipin-1. The fission yeast lipin-1 ortholog is identified asNed1p. Lipin-1 is only one of five lipin proteins identified in mammals. The lipin-1 gene (symbol = LPIN1) was originallyidentified in a mutant mouse called the fatty liver dystrophy (fld) mouse. The mutation causing this disorder was foundto reside in the LPIN1 gene. There are three lipin genes (LPIN1, LPIN2, and LPIN3) with the LPIN1 gene encodingthree 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 have recently been associated with Majeed syndrome which is characterized bychronic 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 forthe 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 thatsome of the effects of the thiazolidinedione (TZD) class of drugs used to treat the hyperglycemia associated with type2 diabetes are exerted via the effects of lipin-1. Although the lipin proteins do not contain DNA-binding motifs they haveprotein-interaction domains that allow them to form complexes with nuclear receptors and function as transcriptionalregulators. Lipin-1 has been shown to interact with peroxisome proliferator-activated receptor-γ [PPARγ] co-activator1α (PGC-1α) and PPARα leading to enhanced gene expression. Lipin-1 also is known to interact with additionalmembers 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 toadipocyte differentiation. Lipin-1α induces genes that promote adipocyte differentiation while lipin-1β induces theexpression 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 suchas carnitine palmitoyl transferase-1, acyl CoA oxidase, and medium-chain acylCoA dehydrogenase (MCAD).

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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 containnitrogen that may be positively charged, whereas, glycerol and inositol do not. The major classifications ofphospholipids are:

Phosphatidylcholine (PC)

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Phosphatidylethanolamine (PE)

Phosphatidylserine (PS)

Phosphatidylinositol (PI)

Phosphatidylglycerol (PG)

Cardiolipin (diphosphatidylglycerol, DPG)

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Phospholipid Synthesis

Phospholipids can be synthesized by two mechanisms. One utilizes a CDP-activated polar head group forattachment to the phosphate of phosphatidic acid. The other utilizes CDP-activated 1,2-diacylglycerol and aninactivated polar head group.

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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, linoleic or linolenic acidat carbon 2. The lecithin dipalmitoyllecithin is a component of lung or pulmonary surfactant. It contains palmitateat both carbon 1 and 2 of glycerol and is the major (80%) phospholipid found in the extracellular lipid layer liningthe pulmonary alveoli. Choline is activated first by phosphorylation and then by coupling to CDP prior toattachment to phosphatidic acid. PC is also synthesized by the addition of choline to CDP-activated 1,2-diacylglycerol. A third pathway to PC synthesis, involves the conversion of either PS or PE to PC. Theconversion of PS to PC first requires decarboxylation of PS to yield PE; this then undergoes a series of threemethylation reactions utilizing S-adenosylmethionine (SAM) as methyl group donor.

PE:These molecules 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 canoccur by two pathways. The first requires that ethanolamine be activated by phosphorylation and then by couplingto CDP. The ethanolamine is then transferred from CDP-ethanolamine to phosphatidic acid to yield PE. Thesecond involves the decarboxylation of PS.

PS:Phosphatidylserines will carry a net charge of –1 at physiological pH and are composed of fatty acids similarto the phosphatidylethanolamines. The pathway for PS synthesis involves an exchange reaction of serine forethanolamine in PE. This exchange occurs when PE is in the lipid bilayer of the a membrane. As indicatedabove, PS can serve as a source of PE through a decarboxylation reaction.

PI:These molecules 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 atphysiological pH. These molecules exist in membranes with various levels of phosphate esterified to thehydroxyls of the inositol. Molecules with phosphorylated inositol are termed polyphosphoinositides. Thepolyphosphoinositides are important intracellular transducers of signals emanating from the plasma membrane.

The synthesis of PI involves CDP-activated 1,2-diacylglycerol condensation with myo-inositol. PI subsequentlyundergoes a series of phosphorylations of the hydroxyls of inositol leading to the production ofpolyphosphoinositides. One polyphosphoinositide (phosphatidylinositol 4,5-bisphosphate, PIP2) is a critically

important membrane phospholipid involved in the transmission of signals for cell growth and differentiation fromoutside the cell to inside.

PG:Phosphatidylglycerols exhibit a net charge of –1 at physiological pH. These molecules are found in highconcentration in mitochondrial membranes and as components of pulmonary surfactant. Phosphatidylglycerolalso is a precursor for the synthesis of cardiolipin. PG is synthesized from CDP-diacylglycerol and glycerol-3-phosphate. The vital role of PG is to serve as the precursor for the synthesis of diphosphatidylglycerols (DPGs).

DPG:These molecules are very acidic, exhibiting a net charge of –2 at physiological pH. They are foundprimarily in the inner mitochondrial membrane and also as components of pulmonary surfactant. One importantclass of diphosphatidylglycerols is the cardiolipins. These molecules are synthesized by the condensation ofCDP-diacylglycerol with PG.

The fatty acid distribution at the C–1 and C–2 positions of glycerol within phospholipids is continually in flux, owingto phospholipid degradation and the continuous phospholipid remodeling that occurs while these molecules are inmembranes. Phospholipid degradation results from the action of phospholipases. There are various phospholipasesthat 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 notthe same acyl group present in the phospholipid when it resides within a membrane. The remodeling of acyl groups inphospholipids is the result of the action of phospholipase A1 (PLA1) and phospholipase A2 (PLA2).

Sites of action of the phospholipases A1, A2, C and D.

The products of these phospholipases are called lysophospholipids and can be substrates for acyl transferasesutilizing different acyl-CoA groups. Lysophospholipids can also accept acyl groups from other phospholipids in anexchange reaction catalyzed by lysolecithin:lecithin acyltransferase (LLAT).

PLA2 is also an important enzyme, whose activity is responsible for the release of arachidonic acid from the C–2

position of membrane phospholipids. The released arachidonate is then a substrate for the synthesis of theeicosanoids. In fact there is not just a single PLA2 enzyme. At least 30 enzymes have been identified with PLA2

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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 ofeicosanoid 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 plateletactivating 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 withlipoprotein 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 etherphospholipids. Three major classes of plasmalogens have been identified: choline, ethanolamine and serineplasmalogens. Ethanolamine plasmalogen is prevalent in myelin. Choline plasmalogen is abundant in cardiac tissue.

One particular 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, acuteinflammatory reactions and anaphylactic shock. PAF is synthesized in response to the formation of antigen-IgEcomplexes on the surfaces of basophils, neutrophils, eosinophils, macrophages and monocytes. The synthesis andrelease of PAF from cells leads to platelet aggregation and the release of serotonin from platelets. PAF also producesresponses in liver, heart, smooth muscle, and uterine and lung tissues.

Platelet activating factor

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Last modified: July 28, 2013