Alterations in high density lipoprotein (HDL) composition that occur in dyslipidemic states may modulate a number of events involved in cholesterol homeostasis. To elucidate the details of how HDL-core composition can affect the molecular structure of different kinds of HDL particles, the conformation and stability of apoA-I have been investigated in homogeneous recombinant HDL particles (LpA-I) containing palmitoyloleoyl phosphatidylcholine (POPC), triolein (TG), and/or cholesteryl linoleate (CE). In a discoidal particle containing two molecules of apoA-I and 85 molecules of POPC, apoA-I exhibits an ␣-helix content of 70% and a free energy of stability of its ␣-helical segments (⌬G D 0 ) of 2.2 kcal/mol. Inclusion of eight molecules of TG into the complex significantly reduces the ␣-helix content and stability of apoA-I, whereas inclusion of four molecules of CE into the complex has an opposite effect in that the ␣-helix content is significantly reduced and the stability of the remaining ␣-helical structure of apoA-I is increased. Neutral lipids have a different effect on apoA-I conformation in spherical LpA-I particles. In a sonicated-spherical LpA-I particle containing two molecules of apoA-I and 70 molecules of POPC, apoA-I exhibits an ␣-helix content of about 60% and a ⌬G D 0 of 1.2 kcal/mol apoA-I. Inclusion of either 10 molecules of TG or six molecules of CE into such a particle increases both the ␣-helix content and stability of apoA-I. Increasing the CE/TG ratio in LpA-I particles that contain both neutral lipids enhances the stability of the ␣-helical segments. ApoA-I molecules tend to dissociate and cause particle instability when ⌬G D 0 for the lipid-bound ␣-helices is less than that for helices in the lipid-free state. The stabilities of both discoidal and spherical LpA-I particles are relatively low when the only neutral lipid present is TG but the particle stability is enhanced by the presence of CE molecules. Such dissociation of apoA-I molecules from LpA-I particles that have a low CE/TG ratio would be promoted in the hypertriglyceridemic state in vivo. High density lipoproteins (HDL)1 comprise a heterogeneous class of particles that contain apolipoprotein A-I (apoA-I) (LpA-I) or apoA-I and A-II (LpA-I,A-II) as their primary protein constituents (1). The central role that HDL plays in cholesterol metabolism is thought to involve the transport of cholesterol from peripheral tissues to the liver (2). Several studies have suggested that the efficiency of HDL in mediating this flux may be impaired in hypertriglyceridemic patients and that this effect may be related to modifications in HDL composition and size (for review, see Ref.3). Investigations in a variety of laboratories have shown that changes in HDL size and composition can lead to altered interactions between HDL and lecithin:cholesterol acyltransferase (4 -6), cholesteryl ester transfer protein (7-9), and cell surfaces (10, 11). There is evidence that an increased number of small, neutral lipid-poor, HDL particles in hypertriglyceridemic p...

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Hepatic lipase (HL) is a lipolytic enzyme that contributes to the regulation of plasma triglyceride (TG) levels. Elevated TG levels may increase the risk of developing coronary heart disease, and studies suggest that mutations in the HL gene may be associated with elevated TG levels and increased risk of coronary heart disease. Hepatic lipase facilitates the clearance of TG from the very low density lipoprotein (VLDL) pool, and this function is governed by the composition and quality of high density lipoprotein (HDL) particles. In humans, HL is a liver resident enzyme regulated by factors that release it from the liver and activate it in the bloodstream. HDL regulates the release of HL from the liver and HDL structure controls HL transport and activation in the circulation. Alterations in HDL-apolipoprotein composition can perturb HL function by inhibiting the release and activation of the enzyme. HDL structure may therefore affect plasma TG levels and coronary heart disease risk. Triglycerides and Heart DiseaseElevated plasma triglyceride (TG) levels have been viewed as a risk factor for coronary heart disease (CHD) for more than a decade.1,2 Plasma TG levels are regulated by both synthesis and degradation of both very low density lipoprotein (VLDL) and chylomicron particles. The clearance of TG-rich lipoproteins from the circulation is controlled by the actions of lipoprotein lipase (LPL) and hepatic lipase (HL) and by the interlipoprotein exchange of TG by cholesteryl ester transfer protein. Lipoprotein lipase is the predominant TG lipase and is responsible for hydrolyzing TG in chylomicrons and VLDL, whereas HL is both a phospholipase and a TG lipase and plays an important role in HDL metabolism and in the conversion of VLDL to LDL.3 Single nucleotide polymorphisms (SNPs) in the HL gene (LIPC) have been shown to associate with plasma lipid concentrations and increased CHD risk. 4,5 Hepatic lipase deficiency is a result of relatively rare LIPC mutations that give rise to a loss in circulating HL activity (due to impaired secretion or inactive enzyme) and cause an increase in TG-rich HDL and VLDL remnants and increased CHD risk.

The effect of apolipoprotein A-II (apoA-II) on the structure and stability of HDL has been investigated in reconstituted HDL particles. Purified human apoA-II was incorporated into sonicated, spherical LpA-I particles containing apoA-I, phospholipids, and various amounts of triacylglycerol (TG), diacylglycerol (DG), and/or free cholesterol. Although the addition of PC to apoA-I reduces the thermodynamic stability (free energy of denaturation) of its ␣ -helices, PC has the opposite effect on apoA-II and significantly increases its helical stability. Similarly, substitution of apoA-I with various amounts of apoA-II significantly increases the thermodynamic stability of the particle ␣ -helical structure. ApoA-II also increases the size and net negative charge of the lipoprotein particles. ApoA-II directly affects apoA-I conformation and increases the immunoreactivity of epitopes in the N and C termini of apoA-I but decreases the exposure of central domains in the molecule (residues 98-186). ApoA-II appears to increase HL association with HDL and inhibits lipid hydrolysis. ApoA-II mildly inhibits PC hydrolysis in TG-enriched particles but significantly inhibits DG hydrolysis in DG-rich LpA-I. In addition, apoA-II enhances the ability of reconstituted LpA-I particles to inhibit VLDL-TG hydrolysis by HL. Therefore, apoA-II affects both the structure and the dynamic behavior of HDL particles and selectively modifies lipid metabolism.

A unique class of lipid-poor high-density lipoprotein, pre-beta1 HDL, has been identified and shown to have distinct functional characteristics associated with intravascular cholesterol transport. In this study we have characterized the structure/function properties of poorly lipidated HDL particles and the factors that mediate their conversion into multimolecular lipoprotein particles. Studies were undertaken with homogeneous recombinant HDL particles (LpA-I) containing apolipoprotein (apo) A-I and various amounts of palmitoyloleoylphosphatidylcholine (PC) and cholesterol. Complexation of apoA-I with small amounts of PC and cholesterol results in the formation of discrete lipoprotein structures that have a hydrated diameter of about 6 nm but contain only one molecule of apoA-I (Lp1A-I). While the molecular charge and alpha-helix content of apoA-I are unaffected by lipidation, the thermodynamic stability of the protein is reduced significantly (from 2.4 to 0.9 kcal/mol of apoA-I). Evaluation of apoA-I conformation by competitive radioimmunoassay with monoclonal antibodies shows that addition of small amounts of PC and cholesterol to apoA-I significantly increases the immunoreactivity of a number of domains over the entire molecule. Increasing the ratio of PC:apoA-I to 10:1 in the Lp1A-I complex is associated with increases in the alpha-helix content and stability of apoA-I. However, incorporation of 10-15 mol of PC destabilizes the Lp1A-I complex and promotes the formation of more thermodynamically stable (1.8 kcal/mol of apoA-I) bimolecular structures (Lp2A-I) that are approximately 8 nm in diameter. The formation of an Lp2A-I particle is associated with an increased immunoreactivity of most of the epitopes studied, with the exception of one central domain (residues 98-121), which becomes significantly less exposed. This structural change parallels a significant increase in the net negative charge on the complex. Characterization of the ability of these lipoproteins to act as substrates for lecithin:cholesterol acyltransferase (LCAT) shows that unstable Lp1A-I complexes stimulate a higher rate of cholesterol esterification by LCAT than the small but more stable Lp2A-I particles (Vmax values are 5.8 and 0.3 nmol of free cholesterol esterified/h, respectively). The ability of LCAT to interact with lipid-poor apoA-I suggests that LCAT does not need to bind to the lipid interface on an HDL particle but that LCAT may directly interact with apoA-I. The data suggests that lipid-poor HDL particles may be metabolically reactive particles because they are thermodynamically unstable.

We have devised a combined in vivo, ex vivo, and in vitro approach to elucidate the mechanism(s) responsible for the hypoalphalipoproteinemia in heterozygous carriers of a naturally occurring apolipoprotein A-I (apoA-I) variant (Leu 159 to Arg) known as apoA-I Finland (apoA-I FIN ). Adenovirus-mediated expression of apoA-I FIN decreased apoA-I and high density lipoprotein cholesterol concentrations in both wild-type C57BL/6J mice and in apoA-I-deficient mice expressing native human apoA-I (hapoA-I). Interestingly, apoA-I FIN was degraded in the plasma, and the extent of proteolysis correlated with the most significant reductions in murine apoA-I concentrations. ApoA-I FIN had impaired activation of lecithin:cholesterol acyltransferase in vitro compared with hapoA-I, but in a mixed lipoprotein preparation consisting of both hapoA-I and apoA-I FIN there was only a moderate reduction in the activation of this enzyme. Importantly, secretion of apoA-I was also decreased from primary apoA-I-deficient hepatocytes when hapoA-I was co-expressed with apoA-I FIN following infection with recombinant adenoviruses, a condition that mimics secretion in heterozygotes. Thus, this is the first demonstration of an apoA-I point mutation that decreases LCAT activation, impairs hepatocyte secretion of apoA-I, and makes apoA-I susceptible to proteolysis leading to dominantly inherited hypoalphalipoproteinemia. Plasma concentrations of high density lipoprotein (HDL)1 cholesterol (HDL-C) are inversely correlated with the risk of developing coronary heart disease (1). However, the complex and often poorly understood etiology for variations in HDL-C concentrations within the general population has made the therapeutic control of HDL levels an elusive target to date. This is attributed to the intricate nature of HDL metabolism that involves many components including the major HDL structural protein apolipoprotein A-I (apoA-I) and multiple factors required for cholesteryl ester (CE) formation, lipolysis, lipid transfer, cellular lipid efflux, and cell surface interactions (reviewed in Refs. 2-4). Nascent HDLs that are derived from the liver and intestine are poorly lipidated (2, 5) and must acquire additional lipids for their maturation into the more stable ␣-migrating HDLs in plasma. Defective clearance of triglyceride-rich lipoproteins (TRL) is recognized as a major determinant of HDL-C concentrations. Recessive mutations in lipoprotein lipase and its major activator protein apolipoprotein C-II, which result in impaired hydrolysis of TRL, also contribute to low HDL-C concentrations. Also the efficient conversion of free cholesterol (FC) to CE on HDL by lecithin:cholesterol acyltransferase (LCAT) is necessary for HDL maturation and depends on apoA-I as its physiological activator (reviewed in Refs. 2-4). Recent work has also highlighted the importance of both the ATP-binding cassette transporter A1 (ABCA1) protein and phospholipid transfer protein (PLTP) in maintaining normal HDL-C concentrations. PLTP-deficient mice have HDL-C levels that...

The production of cholesteryl ester (CE) by lecithin: cholesterol acyl transferase (LCAT) is elevated significantly in hyperlipidemic subjects at high risk for coronary artery disease. To elucidate the molecular events involved, the relationship between LCAT activation and apolipoprotein (apo) A-I charge and structure in high density lipoproteins (HDL) has been studied in both native HDL and homogeneous recombinant HDL (Lp2A-I) particles containing apoA-I, palmitoyloleoyl phosphatidylcholine and cholesterol. Increasing the cholesterol content of discoidal Lp2A-I from 4 to 26 molecules/particle raises the maximum rate of cholesterol esterification by LCAT (Vmax) from 3.1 to 9.2 nmol CE/h/unit of LCAT and increases the apparent Km from 0.5 to 3.5 microM cholesterol. Similarly, increasing the cholesterol content in triolein core-containing Lp2A-I (4-18 molecules/particle) and in native HDL3 (12-21 molecules/particle) also significantly increases the Vmax for LCAT (2.8-7.7 and 0.5-3.6 nmol CE/h, respectively) and raises the Km values (7.6-36.9 and 7.3-8.5 microM cholesterol, respectively). In contrast, changes in the cholesterol content of native and recombinant HDL have no significant effect on the apparent Km values when expressed in terms of the concentration of either apoA-I or palmitoyloleoyl phosphatidylcholine. This appears to indicate that interfacial cholesterol content has no effect on the binding affinity of LCAT to different LpA-I particles but directly affects catalysis by modulating the interaction of cholesterol molecules with the active site of LCAT. Increasing the cholesterol content of the different HDL particles progressively increases the particle net negative charge, and these changes in apoA-I charge are strongly correlated with both the Vmax and apparent Km values for LCAT. This suggests that the conformation and charge of apoA-I play a central role in LCAT activation and that these parameters are influenced by the amount of cholesterol in the surface of HDL particles.

Hyperglycemia is associated with abnormal plasma lipoprotein metabolism and with an elevation in circulating nucleotide levels. We evaluated how extracellular nucleotides may act to perturb hepatic lipoprotein secretion. Adenosine diphosphate (ADP) (>10 µM) acts like a proteasomal inhibitor to stimulate apoB100 secretion and inhibit apoA-I secretion from human liver cells at 4 h and 24 h. ADP blocks apoA-I secretion by stimulating autophagy. The nucleotide increases cellular levels of the autophagosome marker, LC3-II, and increases co-localization of LC3 with apoA-I in punctate autophagosomes. ADP affects autophagy and apoA-I secretion through P2Y13. Overexpression of P2Y13 increases cellular LC3-II levels by ∼50% and blocks induction of apoA-I secretion. Conversely, a siRNA-induced reduction in P2Y13 protein expression of 50% causes a similar reduction in cellular LC3-II levels and a 3-fold stimulation in apoA-I secretion. P2Y13 gene silencing blocks the effects of ADP on autophagy and apoA-I secretion. A reduction in P2Y13 expression suppresses ERK1/2 phosphorylation, increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, and blocks the inhibition of Akt phosphorylation by TNFα and ADP. Conversely, increasing P2Y13 expression significantly inhibits insulin-induced phosphorylation of insulin receptor (IR-β) and Akt, similar to that observed after treatment with ADP. Nucleotides therefore act through P2Y13, ERK1/2 and insulin receptor signaling to stimulate autophagy and affect hepatic lipoprotein secretion.