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165.Stannard AK, Riddell DR, Sacre SM, Tagalakis AD, Langer C, von Eckardstein A, Cullen P, Athanasopouos T, Dickson G, Owen JS: Cell-derived apolipoprotein E (apoE) particles inhibit vascular cell adhesion molecule-1 (VCAM) expression in human endothelial cells. J Biol Chem 246: 46011–46016, 2001.

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170.Mistry MJ, Clay MA, Kelly ME, Steiner MA, Harmony JA: Apolipoprotein E restricts interleukin-dependent T-lymphocyte proliferation at the G1A/G1B boundary. Cell Immunol 160: 14–23, 1995.

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172.Swertfeger DK, Hui DY: Apolipoprotein E receptor binding versus heparan sulfate proteoglycan binding in its regulation of smooth muscle cell migration and proliferation. J Biol Chem 276: 25043–25048, 2001.

173.Riddell DR, Vinogradov DV, Stannard AK, Chadwick N, Owen JS: Identification and characterisation of LRP8 (apoER2) in human blood platelets. J Lipid Res 40: 1925–1930, 1999.

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177.Boisvert WA, Spangenberg J, Curtiss LK: Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation in mice. J Clin Invest 96: 1118–1124, 1995.

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phosphatidic acid phosphohydrolase; PPAR, peroxisomal proliferator-activated receptor; SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory elements binding protein; TG, triacylglycerol; TGH, triacylglycerol hydrolase; VLDL, very low-density lipoprotein.

Triacylglycerol Metabolism is Linked to Several Human Diseases

The ability to store neutral lipids plays a critical role in the ability of an organism to withstand fuel deprivation. Triacylglycerol (TG) is the most concentrated form of energy available to biological tissues. It can provide over double the amount of energy by metabolic oxidation than an equal mass of either carbohydrates or proteins [1]. TG is composed of a glycerol backbone to which three fatty acids are attached via ester bonds. Most cells and tissues have the ability to synthesize and store TG to some degree. TG metabolism is of tremendous clinical relevance. Dysregulation of TG lipid synthesis, transport, or storage contributes to the development of disease. Consequently there is tremendous clinical and pharmacological relevance for enzymes that metabolize TG. Obesity alone is associated with type 2 diabetes, hyperlipidemia, cardiovascular disease, stroke, gallbladder disease, and some cancers [2–7]. Excessive TG storage within muscle and pancreatic β-cells correlates with insulin resistance, leading ultimately to diabetes [8, 9]. Excessive hepatic TG secretion contributes to high levels of circulating TG in the blood, which is a risk factor for cardiovascular disease (CVD). Plasma TG levels >150 mg/dL or 1.70 mM in a fasted state are considered elevated [10].

Components and Classification of Lipoproteins

Neutral lipids such as TG and CE are hydrophobic in nature, and hence have very low solubility in aqueous environments including blood. Therefore, in order to be transported throughout the body, where they are required for various cellular functions, hydrophobic lipids are packaged into complexes with specific proteins known as apolipoproteins (apos) as well as partly polar phospholipids. All mature TG-rich lipoproteins consist of a hydrophobic core of nonpolar lipids including cholesteryl esters (CE) and TG surrounded by a phospholipid monolayer. Free or unesterified cholesterol can also exist within the monolayer of phospholipid. Apos are also amphipathic and confer solubility to the lipoprotein particle as well as influence trafficking via interactions with various cell surface receptors, hydrolytic enzymes, and lipidtransferring proteins [11]. Most apos are exchangeable; they can detach from the surface of a lipoprotein then reattach to another. Hence the complement

of apos changes during the lifetime of these particles. The exception to apos that exchange is more hydrophobic and very large apoB, which is integral and remains bound to the particle it is initially assembled with [12, 13].

Lipoproteins are classified based on their density, which is determined by their lipid and protein composition and is inversely proportional to their diameter. Chylomicrons are the largest and least dense class of TG-rich lipoproteins, with a very TG-rich core (75–1200 nm in diameter) [14]. They are synthesized by the small intestine utilizing ingested dietary lipids, and are carried by the lymphatic system to the bloodstream. Very low-density lipoprotein (VLDL) is the second largest lipoprotein (30–100 nm), and is synthesized and secreted by the liver via an intricate secretory pathway [15, 16]. VLDL is also rich in TG and is catabolized in the circulatory system via a complex series of reactions involving lipases and the transfer of lipids and apos among lipoproteins [17] to produce intermediate (IDL) and low-density lipoproteins (LDL; LDL is approximately 18–25 nm in diameter).

The final class is the smallest (25 nm in diameter or smaller), and these particles are known as high-density lipoproteins (HDL). HDL does not carry apoB, but rather contains at least one molecule of apoA-I among other apos [11]. HDL has a relatively CE-rich core. It is important for the removal of excess cholesterol from peripheral (nonhepatic) cells and its transport back to the liver for recycling or excretion as bile. The collection of cholesterol from the periphery and delivery back to the liver is known as the reverse cholesterol transport pathway. The cardioprotective nature of HDL has been shown in numerous epidemiological studies worldwide over the last 50 years. A striking inverse correlation between HDL cholesterol and incidence of CVD has been observed [18–21], strongly suggesting that a major risk factor for CVD development is low-plasma HDL [22, 23]. Although the detailed mechanisms of how cells efflux cholesterol to HDL and how HDL protects against atherosclerosis are not fully elucidated, potential strategies to treat and prevent CVD exist in raising plasma HDL levels [24].

VLDL Assembly and Secretion

The major apo component of chylomicrons, VLDL, and the sole apo of LDL is a single apoB molecule per particle [12, 17]. Several excellent reviews regarding the mechanism of VLDL assembly and secretion have been published [16, 25, 26]. Gene targeting experiments in mice revealed that apoB is the sole apo required for the secretion of a VLDL particle [27]. ApoB is encoded by a single gene, though it occurs in two forms due to mRNA editing by a deaminase that converts codon 2153 from CAA (glutamine) to a stop codon [28, 29]. The larger form, termed apoB100, is the sole form of apoB secreted by the human liver and includes 100% of the mature protein. Human apoB100 has 4536 amino acids and a calculated molecular weight of over 512

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kDa [30, 31]. The shorter form, apoB48, represents the N-terminal 48% of apoB100 with 2152 amino acids and a calculated molecular weight of over 264 kDa [13]. ApoB48 is secreted by human intestine on chylomicrons for transport of dietary lipids. While apoB100 is capable of an interaction with the LDL receptor to mediate cellular uptake via endocytosis, apoB48 lacks the LDL-receptor interacting domain. It is initially counterintuitive that chylomicrons can be up to ten times larger than VLDL and carry more lipids, while at the same time they contain the shorter apoB protein. This means that the C-terminal 52% of apoB100 is not necessary for acquisition of large amounts of lipid during lipoprotein assembly. Both forms of apoB are secreted by the liver of rats and mice [27], making these animals a more challenging model in which to investigate lipoprotein metabolism as it applies to humans.

Triacylglycerol Biosynthesis

The fatty acids present within the TG molecule are provided by dietary lipids or are generated de novo. Variations in the composition and quantity of the diet affect the rate of de novo fatty acid and TG synthesis to satisfy the body’s demands for lipid (or energy) storage [32]. The source of the fatty acids for TG synthesis varies depending upon the tissue [33]. For example, the enterocyte takes up dietary fatty acids and 2-monacylglycerols (MG) and resynthesizes TG from these substrates primarily through the 2-monoacylglycerol (MG) pathway. Hepatocytes take up fatty acids derived from remnant lipoproteins and free fatty acids released from adipocytes. It is generally accepted that the synthesis of TG is controlled primarily by the amount of fatty acid available. Thus, de novo TG synthesis is enhanced only when fatty acid synthesis or uptake exceeds the cellular need for energy and phospholipid synthesis. In hepatocytes, as well as adipocytes, de novo TG synthesis is primarily achieved through the glycerol-3-phosphate pathway (Fig. 7.1).

The Glycerol-3-Phosphate Pathway of TG Biosynthesis

The glycerol-3-phosphate pathway is the main route of TG biosynthesis in nonenteric tissues. Briefly, it begins with the acylation of glycerol-3-phosphate by glycerol-3-phosphate acyltransferase (GPAT) at the sn-1 position to form 1-acylglycerol-3-phosphate. Subsequently, the acylation of 1-acylglycerol-3- phosphate at the sn-2 position by 1-acylglycerol-3-phosphate acyltransferase (AGPAT) (also referred to as lysophosphatidic acid acyltransferase) synthesizes phosphatidic acid (PA). The hydrolysis of PA by phosphatidate phosphohydrolase (PAPH) generates sn-1,2-diacylglycerol (DG). DG is then acylated to form TG via the action of diacylglycerol acyltransferase (DGAT). The utilization of molecular cloning and gene targeting techniques has elucidated many new insights regarding these enzymes and their regulation. The

storage

secretory droplet pathway

FIGURE 7.1. The glycerol phosphate pathway of triacylglycerol synthesis. Glycerol-3- phosphate is acylated to form 1-acyl-glycerol-3-phosphate by glycerol-3-phosphate acyltransferase (GPAT). A subsequent acylation is accomplished by 1-acyl-glycerol-3- phosphate acyltransferase (AGPAT), resulting in phosphatidic acid. Hydrolysis of the sn-3-phosphate through the action of phosphatidic acid phosphohydrolase (PAPH) produces diacylglycerol. Diacylglycerol may be converted to triacylglycerol by diacylglycerol acyltransferase (DGAT) or diacylglycerol transacylase (DGTA). Alternately, it can be diverted towards phospholipid biosynthesis. In liver, triacylglycerol is stored in droplets or secreted on VLDL.

regulation of TG synthesis and its breakdown determines the quantity of TG stored within a cell.

Glycerol-3-Phosphate Acyltransferase

GPAT represents the committed step for the synthesis of all glycerolipids. Two isoforms for this enzyme exist. A mitochondrial GPAT has been cloned

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and demonstrated to be tightly regulated [34]. A microsomal GPAT activity also exists, though a mammalian cDNA has not been cloned and its activity appears to be largely unregulated [34]. The murine mitochondrial GPAT cDNA is primarily expressed in liver, adipose, and muscle tissues [35].

The nutritional and hormonal regulation of the murine mitochondrial GPAT is well characterized and occurs primarily at the transcriptional level. Posttranslational reduction of mitochondrial GPAT activity has been reported to occur upon stimulation of adenosine monophosphate-activated kinase, a sensor of cellular fuel deprivation [36].

GPAT activity has strong links to obesity. For example, Zucker rats are a commonly used model for obesity as well as insulin resistance. These animals exhibit enhanced GPAT activity in both hepatic and adipose tissues [37]. The regulation of mitochondrial GPAT is consistent with a function in reducing TG synthesis and increasing oxidation when nutrients are limiting, and conversely to direct fatty acids toward TG synthesis and away from oxidation when energy is in excess [38]. The localization of this GPAT activity on the cytosolic surface of mitochondria also suggests a role in preventing fatty acids (more accurately fatty acyl-CoAs) from entering this organelle and undergoing β-oxi- dation under conditions of metabolic excess by converting it to 1-acylglycerol- 3-phosphate and thus directing the fatty acid towards TG synthesis (Fig. 7.1).

Acylglycerol-3-Phosphate Acyltransferase

This enzyme is also referred to as lysophosphatidic acid acyltransferase [34]. Several AGPAT cDNA sequences have been identified. Human and mouse cDNAs of 2 and 1.8 kb, respectively that encode proteins with AGPAT activity were identified and designated AGPAT-1 [39, 40]. 3T3-L1 cells are a fibroblastic preadipocyte cell line that can be differentiated into cells with an adipocyte phenotype. AGPAT-1 overexpression in 3T3-L1 adipocytes inhibited fatty acid release while promoting its uptake and TG storage [41]. Expression of AGPAT-1 mRNA has been observed in all tissues examined, with highest expression in skeletal muscle [39, 40]. AGPAT-2 mRNA is expressed at highest levels in the liver, heart, and adipose tissues, and much less in other tissues [42, 43]. The protein encoded by the AGPAT-2 cDNA shares 48% homology with AGPAT-1 and is expressed at twofold higher levels than AGPAT-1 in the adipose tissue, similar levels in liver and AGPAT-1 is expressed at 1.8-fold greater levels than AGPAT-2 in skeletal muscle [44]. AGPAT-1 and -2 were localized to the endoplasmic reticulum (ER) membrane [45, 46]. Several individuals with congenital generalized lipodystrophy were discovered to have a variety of different mutations in the AGPAT-2 gene that led to the production of a dysfunctional protein [44]. These observations suggest that AGPAT-2 is necessary for TG synthesis in adipocytes. Three additional AGPAT cDNAs have been designated AGPAT-3, -4, and -5, respectively. The corresponding mRNAs are barely detectable in liver, adipose, and skeletal muscle and do not yet have a clearly identified function [44].

Phosphatidic Acid Phosphohydrolase

Two forms of phosphatidic acid phosphohydrolase (PAPH) activity exist, PAPH-1 and PAPH-2 (also called lipid phosphate phosphohydrolase). Several cDNAs for the PAPH-2 family or lipid phosphate phosphohydrolases have been cloned. These enzymes appear to play a role in signal transduction pathways by generating the lipid second messenger, DG [47]. PAPH-1 activity exists in both cytosol and microsomal fractions. It has been proposed that long-chain fatty acids stimulate the translocation of PAPH-1 from the cytosol to the ER membrane where PA is hydrolyzed to DG [48, 49]. A cDNA encoding an enzyme with PAPH-1 activity has not been cloned, but this activity is considered as the rate-limiting step for de novo TG synthesis. There is a strong correlation between the activity of membrane-associated PAPH-1 and the rate of TG synthesis [50]. PAPH-1 activity is subjected to dietaryand hormone-induced changes [50]. PAPH-1 activity was increased 2–3-fold by glucagon, cyclic adenosine monophosphate, and dexamethasone in primary rat hepatocytes [51–53]. It was hypothesized that enhanced PAPH-1 activity enables the liver to increase its ability to synthesize TG during the period of maximum feeding.

Diacylglycerol Acyltransferase

DGAT catalyzes the final step in TG synthesis. Two DGAT cDNAs have been cloned. DGAT-1 is ubiquitously expressed in human and murine tissues, with the highest levels of expression in the small intestine, colon, testis, thymus, heart, and skeletal muscle [54]. The mammalian DGAT-2 cDNA is unrelated to the mammalian DGAT-1 cDNA and was cloned based on its homology with a fungal DGAT cDNA [55]. Both proteins have been localized to microsomal membranes. DGAT-2 is highly expressed in liver and adipose, which correlates with a high capacity to synthesize TG in these tissues. Analysis of the predicted amino acid sequence from the DGAT-2 cDNA suggests the presence of a single transmembrane segment and a domain with homology to the glycerol-binding domain of GPAT and AGPAT. The glycerol-binding domain of DGAT-2 is predicted to be within the ER lumen where the synthesis of TG would be ideally linked to the site of VLDL assembly. There is an experimental evidence for the existence of overt (cytosolic) and latent (lumenal) DGAT activities in microsomes [56]. Hence, different acyltransferases (or transacylases), acting in different subcellular compartments could be involved in the synthesis of TG, and the extent of their respective activities could channel TG preferentially for storage within a cytosolic storage pool or towards the secretory pathway via assembly into lipoproteins. Whether the DGAT-1 or -2 enzyme is responsible for directing TG towards VLDL versus storage has been investigated.

Recently, adenovirus constructs were used to overexpress DGAT-1 or DGAT-2 in mice [57]. Intriguingly the data showed that DGAT-1 overexpression led to increased VLDL, while DGAT-2 overexpression led to TG