In order for the body to make use of dietary lipids, they must first be absorbed from the small intestine. The predominant form of dietary lipid in the human diet is triglyceride. Since these molecules are oils, they are essentially insoluble in the aqueous environment of the intestine. The solubilization (or emulsification) of dietary lipids is accomplished principally in the small intestine by means of the bile acids. Bile acids are synthesized from cholesterol in the liver and then stored in the gallbladder. Following the ingestion of food, bile acids are released and secreted into the gut. Some lipid emulsification occurs in the stomach due to the churning action in this organ which renders some of the lipid accessible to gastric lipase.
The emulsification of dietary fats renders them accessible to various pancreatic lipases in the small intestine. These lipases, pancreatic lipase and pancreatic phospholipase A2 (PLA2) generate free fatty acids and a mixture of mono- and diglycerides from dietary triglycerides. Pancreatic lipase degrades triglyceride at the sn-1 and sn-3 positions sequentially to generate 1,2-diglycerides and 2-acylglycerols. Phospholipids are degraded at the sn-2 position by pancreatic PLA2 releasing a free fatty acid and the lysophospholipid. The products of pancreatic lipases then enter the intestinal epithelial cells via the action of various transporters as well as by simple diffusion. Within the enterocyte the lipids are used for re-synthesis of triglycerides.
Dietary triglyceride and cholesterol, as well as triglyceride and cholesterol synthesized by the liver, are solubilized in lipid-protein complexes. These complexes contain triglyceride lipid droplets and cholesteryl esters surrounded by the polar phospholipids and proteins identified as apolipoproteins. These lipid-protein complexes vary in their content of lipid and protein.
Structure of a chylomicron as a representative structure of a typical lipoprotein particle. Image demonstrates the phospholipid and free cholesterol outer layer with primarily triglycerides and cholesteryl esters internally. Each lipoprotein type, chylomicron, LDL, and HDL, contain apolipoproteins. Apolipoprotein B-48 (apoB-48) is specific for chylomicrons just as apoB-100 is specific for LDL.back to the top
|*HDL2||Intestine, liver (chylomicrons and VLDLs)||1.063-1.125||33-35||5-15||32-43||20-30||5-10||0|
|*HDL3||Intestine, liver (chylomicrons and VLDLs)||1.125-1.21||55-57||3-13||26-46||15-30||2-6||6|
aTriglycerides, bPhospholipids, cCholesteryl esters, dFree cholesterol, eFree fatty acids
*HDL2 (HDL3) and HDL3 (HDL2) are derived from nascent HDL as a result of the acquisition of apoproteins and cholesteryl estersback to the top
Standard fasting blood tests for cholesterol and lipid profiles will include values for total cholesterol, HDL cholesterol (so-called "good" cholesterol), LDL cholesterol (so-called "bad" cholesterol) and triglycerides. Family history and life style, including factors such as blood pressure and whether or not one smokes, affect what would be considered ideal versus non-ideal values for fasting blood lipid profiles. Included here are the values for various lipids that indicate low to high risk for coronary artery disease.
<200mg/dL = desired values
200–239mg/dL = borderline to high risk
240mg/dL and above = high risk
With HDL cholesterol the higher the better.
<40mg/dL for men and <50mg/dL for women = higher risk
40–50mg/dL for men and 50–60mg/dL for woman = normal values
>60mg/dL is associated with some level of protection against heart disease
With LDL cholesterol the lower the better.
<100mg/dL = optimal values
100mg/dL–129mg/dL = optimal to near optimal
130mg/dL–159mg/dL = borderline high risk
160mg/dL–189mg/dL = high risk
190mg/dL and higher = very high risk
With triglycerides the lower the better.
<150mg/dL = normal
150mg/dL–199mg/dL = borderline to high risk
200mg/dL–499mg/dL = high risk
>500mg/dL = very high risk
|Apoprotein - MW (Da)||Gene||Lipoprotein Association||Function and Comments|
|apoA-I - 29,016||APOA1
|Chylomicrons, HDL||major protein of HDL, binds ABCA1 on macrophages, critical anti-oxidant protein of HDL, activates lecithin:cholesterol acyltransferase, LCAT|
|apoA-II - 17,400||APOA2
|Chylomicrons, HDL||primarily in HDL, enhances hepatic lipase activity|
|apoA-IV - 46,000||APOA4
|Chylomicrons and HDL||present in triglyceride rich lipoproteins; synthesized in small intestine, synthesis activated by PYY, acts in central nervous system to inhibit food intake|
|apoB-48 - 241,000||APOB
|Chylomicrons||exclusively found in chylomicrons, derived from apoB-100 gene by RNA editing in intestinal epithelium; lacks the LDL receptor-binding domain of apoB-100|
|apoB-100 - 513,000||APOB
|VLDL, IDL and LDL||major protein of LDL, binds to LDL receptor; one of the longest known proteins in humans|
|apoC-I - 7,600||APOC1
|Chylomicrons, VLDL, IDL and HDL||may also activate LCAT; clustered with APOC2 and APOE genes on chromsome 19|
|apoC-II - 8, 916||APOC2
|Chylomicrons, VLDL, IDL and HDL||activates lipoprotein lipase; clustered with APOC1 and APOE genes on chromsome 19|
|apoC-III - 8,750||APOC3
|Chylomicrons, VLDL, IDL and HDL||inhibits lipoprotein lipase, interferes with hepatic uptake and catabolism of apoB-containing lipoproteins, appears to enhance the catabolism of HDL particles, enhances monocyte adhesion to vascular endothelial cells, activates inflammatory signaling pathways|
|HDL||closely associated with LCAT|
|cholesterol ester transfer protein, CETP||CETP
|HDL||plasma glycoprotein secreted primarily from the liver and is associated with cholesteryl ester transfer from HDLs to LDLs and VLDLs in exchange for triglycerides|
|apoE - 34,000 (at least 3 alleles E2, E3, E4); the apoE2 allele has Cys at amino acids 112 and 158; apoE3 has Cys and Arg at these two positions, respectively; apoE4 has Arg at both positions||APOE
|Chylomicron remnants, VLDL, IDL and HDL||binds to LDL receptor; clustered with APOC1 and APOC2 genes on chromsome 19; apoEε4 allele amplification associated with late-onset Alzheimer's disease|
|apoH - 50,000 (also known as β2-glycoprotein I)||APOH
|negatively charged surfaces||inhibits serotonin release from platelets, alters ADP-mediated platelet aggregation|
|apo(a) - protein ranges in size from 300,000–800,000 as a result of from 2–43 copies of the Kringle-type domain; Kringle domains contain around 80 amino acids which form the domain via three intrachain disulfide bonds||LPA
|LDL||disulfide bonded to apoB-100, forms a complex with LDL identified as lipoprotein(a), Lp(a); strongly resembles plasminogen; may deliver cholesterol to sites of vascular injury, high risk association with premature coronary artery disease and stroke|
Apolipoprotein A-IV (apoA-IV) is synthesized exclusively in the small intestine and the hypothalamus. The apoA-IV gene (gene symbol = APOA4) is located on chromosome 11q23 and is closely linked to the apoA-I and apoC-III genes. The gene is composed of only two exons and encodes a protein of 46 kDa. Utilizing isoelectric focusing it has been determined that two isoforms of apoA-IV, designated A-IV-1 and A-IV-2, can be identified in plasma. Intestinal synthesis of apoA-IV increases in response to ingestion and absorption of fat and it is subsequently incorporated into chylomicrons and delivered to the circulation via the lymphatic system. Systemic apoA-IV has been shown to have effects in the CNS involving the sensation of satiety.
Following the consumption of fat, the intestinal absorption of the lipid content stimulates the synthesis and secretion of apoA-IV. The increased production of apoA-IV by the small intestine in response to lipid absorption is the result of enhanced transcription of the apoA-IV gene in intestinal enterocytes. The precise signal for this increase in intestinal transcription is the formation and secretion of chylomicrons. It has been shown that neither digestion, uptake, or the re-esterification of absorbed monoglycerides and fatty acids to form triglyceride is the inducing signal for apoA-IV transcription. This was conclusively demonstrated in experiments showing that the intestinal absorption of only myristic acid or long-chain fatty acids is sufficient to stimulate the lymphatic transport of both chylomicrons and apoA-IV. However, it is still unclear whether different types of triglyceride (those containing either saturated, monounsaturated, or polyunsaturated fatty acids) are equally effective in stimulating the secretion of apoA-IV. Although it is known that chylomicrons serve as the inducing signal for apoA-IV transcription and secretion, the precise mechanism by which the transcriptional enhancement is effected is currently undetermined. What is known is that an intact vagal innervation from the CNS to the gut is not necessary since vagotomy does not affect intestinal apoA-IV synthesis in response to lipid absorption.
Leptin is a peptide synthesized and secreted by adipocytes whose principle effects result in decreased food intake and increased energy expenditure. The levels of circulating leptin increase in response to the consumption of a high-fat diet and are directly correlated to the amount of fat stored in adipose tissue. The level of apoA-IV transcription has been shown to be reduced within 90 minutes of ingesting a high fat meal and this reduction is a result of increased leptin secretion. Although numerous studies have demonstrated a negative correlation between leptin levels and apoA-IV expression, the mechanism by which this effect is exerted is not fully understood. There are leptin receptors in the gut and, therefore, leptin binding to these receptors could lead to direct effects on intestinal enterocytes. Alternatively, leptin could exert indirect effects on intestinal cells by increasing fatty acid oxidation through the induction of enzymes that shift fuel metabolism to favor β-oxidation of fatty acids. Given that circulating leptin levels increase as an individual becomes more obese it is likely that leptin is involved in the attenuation of the intestinal apoA-IV response to lipid ingestion. Although the initial response to consumption of a high-fat diet is increased plasma apoA-IV levels, this increase disappears over time. This finding makes it tempting to speculate that the autoregulation of apo AIV in response to chronic high fat feeding is related to the elevation of circulating leptin.
Direct infusion of lipid into the ileum results in increased expression of ileal and jejunal apoA-IV, whereas, infusion of lipid into the duodenum only results in increased jejunal apoA-IV expression. These results strongly suggest that a signal is released by the distal gut during active lipid absorption which is capable of stimulating apoA-IV synthesis in the proximal gut. A strong candidate for this signal is the ileal peptide PYY. To determine if PYY is indeed involved in increased apoA-IV expression experiments were performed in rats involving intravenous injections of physiological doses of PYY. These experiments showed that PYY infusion does indeed result in significant stimulation of jejunal apoA-IV synthesis and lymphatic transport in fasting animals. Further experiments demonstrated that the stimulation of jejunal apoA-IV synthesis by PYY is the result of effects on translation of the mRNA as opposed to increased transcription of the gene since the levels of the mRNA were unaltered but synthesis of the protein was markedly stimulated. Whereas fat absorption-mediated increases in apoA-IV expression do not require vagal innervation, the responses to PYY do involve the vagal nerve.
Only recently was it determined that both the mRNA and apoA-IV protein are present in the hypothalamus, primarily in the ARC. The presence of apoA-IV in the hypothalamus, a site intimately involved in regulating energy homeostasis, suggests that the effects exerted on appetite by apoA-IV may be due to direct hypothalamic synthesis and secretion. Experiments in rodents, aimed at determining the role of apoA-IV in hypothalamic functions, clearly demonstrated a role for this apolipoprotein in feeding behaviors. Blocking apoA-IV actions by central injection of antibodies to the protein results in increased food consumption, even during the light phase when rodents normally do not eat. Additional studies have shown that apoA-IV is involved in inhibiting food intake following the ingestion of fat. Infusion of lymph fluid that contains chylomicrons results in markedly suppressed food intake during the first 30 min of administration. However, it is not the lipid content of the chylomicrons that is responsible for the suppression of food intake since infusion of a mixture of triglycerides and phospholipids does not exert the same effect. If apoA-IV is removed from chylomicrons prior to infusion, via the use of specific antibodies, there is no observed effect on food intake. If apoA-IV itself is infused, the level of suppression of food intake is the same as that seen with infusion of fatty lymph fluid containing chylomicrons.
The plasma levels of apoA-IV in humans adapt in response to prolonged consumption of fat. Chronic consumption of a high-fat diet initially results in significantly elevated plasma apoA-IV levels. However, the increased level disappears over time. Conversely, on a low-fat diet, intestinal apoA-IV gene expression is sensitive to fasting and lipid feeding, being low during fasting and high during lipid absorption. Consumption of a high-fat diet results in a slow and progressive reduction in hypothalamic apoA-IV mRNA over time. The response of hypothalamic apoA-IV gene expression to chronic consumption of a high-fat diet is only partially similar to the response seen in the small intestine. In animals that are chronically fed a high-fat diet there is no observable increase in hypothalamic apoA-IV expression in response to intragastric infusion of lipid following a period of fasting. In contrast, intragastric infusion of lipid into fasted animals that have been consuming normal chow, results in significant stimulation of hypothalamic apoA-IV mRNA levels. These results demonstrate that chronic consumption of a high-fat diet significantly reduces apoA-IV mRNA levels and the response of hypothalamic apoA-IV gene expression to dietary lipids. Therefore, it is highly likely that dysregulation of hypothalamic apoA-IV could contribute to diet-induced obesity.back to the top
Chylomicrons are assembled in the intestinal mucosa as a means to transport dietary cholesterol and triglycerides to the rest of the body. Chylomicrons are, therefore, the molecules formed to mobilize dietary (exogenous) lipids. The predominant lipids of chylomicrons are triglycerides (see Table above). The apolipoproteins that predominate before the chylomicrons enter the circulation include apoB-48 and apoA-I, apoA-II and apoA-IV. ApoB-48 combines only with chylomicrons.
Chylomicrons leave the intestine via the lymphatic system and enter the circulation at the left subclavian vein. In the bloodstream, chylomicrons acquire apoC-II and apoE from plasma HDLs. In the capillaries of adipose tissue and muscle, the fatty acids of chylomicrons are removed from the triglycerides by the action of lipoprotein lipase (LPL), which is found on the surface of the endothelial cells of the capillaries. The apoC-II in the chylomicrons activates LPL in the presence of phospholipid. The free fatty acids are then absorbed by the tissues and the glycerol backbone of the triglycerides is returned, via the blood, to the liver and kidneys. Glycerol is converted to the glycolytic intermediate dihydroxyacetone phosphate (DHAP). During the removal of fatty acids, a substantial portion of phospholipid, apoA and apoC is transferred to HDLs. The loss of apoC-II prevents LPL from further degrading the chylomicron remnants.
Chylomicron remnants, containing primarily cholesteryl esters, apoE and apoB-48, are then delivered to, and taken up by, the liver. The remnant particle must be of a sufficiently small size such that can pass through the fenestrated endothelial cells lining the hepatic sinusoids and enter into the space of Disse. Chylomicron remnants can then be taken up by hepatocytes via interaction with the LDL receptor which requires apoE. In addition, while in the space of Disse chylomicron remnants can accumulate additional apoE that is secreted free into the space. This latter process allows the remnant to be taken up via the chylomicron remnant receptor, which is a member of the LDL receptor-related protein (LRP) family. The recognition of chylomicron remnants by the hepatic remnant receptor also requires apoE. Chylomicron remnants can also remain sequestered in the space of Disse by binding of apoE to heparan sulfate proteoglycans and/or binding of apoB-48 to hepatic lipase. While sequestered, chylomicron remnants may be further metabolized which increases apoE and lysophospholipid content allowing for transfer to LDL receptors or LRP for hepatic uptake.
Detail of the uptake of chylomicron remnants by the liver. Diagram depicts the interaction of the vasculature of hepatic sinusoids with hepatocytes. The space between hepatic sinusoidal endothelium and hepatocytes is called the space of Disse. Chylomicron remnants containing primarily cholesterol esters, apoE, and apoB-48 are rapidly taken up by the liver. The remnants pass through the endothelial lining of the hepatic sinusoid and in the space of Disse interact with specific receptors as well as heparin sulfated proteoglycans (HSPG). Hepatocyte uptake of remnants is initiated by sequestration of the particles on HSPG followed by receptor-mediated endocytosis of the remnants. The receptor-mediated endocytic process may be mediated by LDL receptors (LDLR) and/or LDL receptor-related protein (LRP). The interaction of remnants with HSPG involves apoB-48 and the interaction with LDLR or LRP involves apoE.back to the top
The dietary intake of both fat and carbohydrate, in excess of the needs of the body, leads to their conversion into triglycerides in the liver. These triglycerides are packaged into VLDLs and released into the circulation for delivery to the various tissues (primarily muscle and adipose tissue) for storage or production of energy through oxidation. VLDLs are, therefore, the molecules formed to transport endogenously derived triglycerides to extra-hepatic tissues. In addition to triglycerides, VLDLs contain some cholesterol and cholesteryl esters and the apoproteins, apoB-100 (a single copy), apoC-I, apoC-II, apoC-III and apoE. Like nascent chylomicrons, newly released VLDLs acquire apoCs and apoE from circulating HDLs.
The fatty acid portion of VLDLs is released to adipose tissue and muscle in the same way as for chylomicrons, through the action of lipoprotein lipase. The action of lipoprotein lipase coupled to a loss of certain apoproteins (the apoCs) converts VLDLs to intermediate density lipoproteins (IDLs), also termed VLDL remnants. IDLs contain multiple copies of apoE and a single copy of apoB-100. The presence of the multiple copies of apoE enable these lipoprotein particles to have very high affinity for the LDL receptor on cells such as hepatocytes and adrenal cortex cells. Conversion of VLDL to IDL is also associated with loss of apoCs by transfer back to HDLs. Further loss of triglycerides, as well as transfer of apoE back to HDL converts IDLs to LDLs. The presence of the apoB-100 protein allows LDL to be recognized by the LDL receptor but the lack of apoE makes the affinity much lower than that of IDL.
The liver takes up IDLs after they have interacted with the LDL receptor to form a complex, which is endocytosed by the cell. For LDL receptors in the liver to recognize IDLs requires the presence of apoB-100 and is enhanced in the presence of apoE. The LDL receptor is also sometimes referred to as the apoB-100/apoE receptor. The importance of apoE in cholesterol uptake by LDL receptors has been demonstrated in transgenic mice lacking functional apoE genes. These mice develop severe atherosclerotic lesions at 10 weeks of age.
The cellular requirement for cholesterol as a membrane component is satisfied in one of two ways: either it is synthesized de novo within the cell, or it is supplied from extra-cellular sources, namely, chylomicrons and IDL/LDL. As indicated above, the dietary cholesterol that goes into chylomicrons is supplied to the liver by the interaction of chylomicron remnants with the remnant receptor. In addition, cholesterol synthesized by the liver can be transported to extra-hepatic tissues if packaged in VLDLs. In the circulation VLDLs are converted to IDLs and LDLs through the action of lipoprotein lipase. IDLs and LDLs are the primary plasma carriers of cholesterol for delivery to all tissues.
The exclusive apolipoprotein of LDLs is apoB-100. LDLs are taken up by cells via LDL receptor-mediated endocytosis, as described above for IDL uptake. The uptake of LDLs occurs predominantly in liver (75%), adrenals and adipose tissue. As with IDLs, the interaction of LDLs with LDL receptors requires the presence of apoB-100. The endocytosed membrane vesicles (endosomes) fuse with lysosomes, in which the apoproteins are degraded and the cholesterol esters are hydrolyzed to yield free cholesterol. The cholesterol is then incorporated into the plasma membranes as necessary. Excess intracellular cholesterol is re-esterified by acyl-CoA-cholesterol acyltransferase (ACAT), for intracellular storage. The activity of ACAT is enhanced by the presence of intracellular cholesterol.
Insulin and tri-iodothyronine (T3) increase the binding of LDLs to liver cells, whereas glucocorticoids (e.g., dexamethasone) have the opposite effect. The precise mechanism for these effects is unclear but may be mediated through the regulation of apoB degradation. The effects of insulin and T3 on hepatic LDL binding may explain the hypercholesterolemia and increased risk of atherosclerosis that have been shown to be associated with uncontrolled diabetes or hypothyroidism.
The consumption of alcohol is associated with either a protective or a negative effect on the level of circulating LDL. Low level alcohol consumption, particularly red wines which contain the antioxidant resveratrol, appear to be beneficial with respect to cardiovascular health. Resveratrol consumption is associated with a reduced risk of cardiovascular, cerebrovascular, and peripheral vascular disease. One major effect of resveratrol in the blood is the prevention of oxidation of LDLs, (forming oxLDL). Oxidized LDLs contribute significantly to the development of atherosclerosis. Conversely excess alcohol consumption is associated with the development of fatty liver which in turn impairs the ability of the liver to take up LDL via the LDL receptor resulting in increased LDL in the circulation. Clearly a reduction in alcohol consumption will have a significant impact on overall cardiovascularr and hepatic function.
An abnormal form of LDL, identified as lipoprotein-X (Lp-X), predominates in the circulation of patients suffering from lecithin-cholesterol acyl transferase (LCAT, see HDL discussion for LCAT function) deficiency or cholestatic liver disease. In both cases there is an elevation in the level of circulating free cholesterol and phospholipids.back to the top
HDLs represent a heterogeneous population of lipoproteins in that they exist as functionally distinct particles possessing different sizes, protein content, and lipid composition. One of the major functions of HDLs is to acquire cholesterol from peripheral tissues and transport this cholesterol back to the liver where it can ultimately be excreted following conversion to bile acids. This function is referred to as reverse cholesterol transport (RCT). The role of HDLs in RCT represents the major atheroprotective (prevention of the development of atherosclerotic lesions in the vasculature) function of this class of lipoprotein. In addition to RCT, HDLs exert anti-inflammatory, antioxidant, and vasodilatory effects that together represent addition atheroprotective functions of HDLs. Evidence has also been generated that demonstrates that HDLs possess anti-apoptotic, anti-thrombotic, and anti-infectious properties. With respect to these various atheroprotective functions of HDLs, it is the small dense particles (referred to as HDL3) that are the most beneficial.
HDLs are synthesized de novo in the liver and small intestine, as primarily protein-rich disc-shaped particles. These newly formed HDLs are nearly devoid of any cholesterol and cholesteryl esters. The primary apoproteins of HDLs are apoA-I, apoC-I, apoC-II and apoE. In fact, a major function of HDLs is to act as circulating stores of apoC-I, apoC-II and apoE. ApoA-I is the most abundant protein in HDLs constituting over 70% of the total protein mass. In addition to apoproteins, HDLs carry numerous enzymes that participate in the anti-oxidant activities. These enzymes include glutathione peroxidase 1 (GPx), paraoxonase 1 (PON1) and platelet activating factor acetylhydrolase (PAF-AH, also called lipoprotein-associated phospholipase A2, Lp-PLA2: see below for functions of Lp-PLA2). Two additional functionally important enzymes found associated with HDLs are lecithin:cholesterol acyltransferase (LCAT, see next paragraph) and cholesterol ester transfer protein (CETP, see below and the next section). Another important HDL component is the compound sphingosine-1-phosphate (S1P; details of S1P activities can be found in the Sphingolipids page). Depending upon the HDL subclass characterized, as many as 75 different proteins have been shown to be associated with circulating HDLs.
The primary mechanism by which HDLs acquire peripheral tissue cholesterol is via an interaction with monocyte-derived macrophages in the subendothelial spaces of the tissues. Macrophages bind nascent HDLs, that contain primarily apoA-I, through interaction with the ATP-binding cassette transport protein A1 (ABCA1). The transfer of cholesterol from macrophages via the action of ABCA1, involves apoA-I and results in the formation of nascent discoidal lipoprotein particles termed pre-β HDLs. The free cholesterol transferred in this way is esterified by HDL-associated LCAT. LCAT is synthesized in the liver and so named because it transfers a fatty acid from the C-2 position of lecithin to the C-3-OH of cholesterol, generating a cholesteryl ester and lysolecithin. The activity of LCAT requires interaction with apoA-I, which is found on the surface of HDLs. The cholesteryl esters formed via LCAT activity are internalized into the hydrophobic core of the pre-β HDL particle. As pre-β HDL particles enlarge with progressive uptake of cholesterol they become larger and spherical generating the HDL2 and HDL3 particles as indicated above. The importance of ABCA1 in reverse cholesterol transport is evident in individuals harboring defects in ABCA1 gene. These individuals suffer from a disorder called Tangier disease which is characterized by two clinical hallmarks; enlarged lipid-laden tonsils and low serum HDL.
Detail of the interactions between HDL and LDL within the vasculature. As indicated in the text HDL begins as protein-rich discoidal structures, composed primarily of apoA-I, produced by the liver and intestines. Within the vasculature apoA-I interacts with the ATP-binding cassette transporter, ABCA1 (such as is diagrammed for interaction with macrophages) and extracts cholesterol from cells. Through the action of LCAT the apoA-I-associated cholesterol is esterified forming cholesterol esters. This process results in the generation of HDL3 particles. As the HDL3 particles continue through the circulation they pick up more cholesterol and through the action of LCAT, generate more cholesterol esters. As HDL migrates through the vasculature there is an interaction between them and IDL and LDL. This interaction occurs through the action of CETP which exchanges the cholesterol esters in the HDL for triglycerides from LDL. This interaction results in the conversion of HDL3 particles to HDL2. The differences between these two types of HDL particle are detailed in the Table at the start of this page. HDL can also remove cholesterol from cells via interaction with the ATP-binding cassette transporter ABCG1. Approximately 20% of HDL uptake of cellular cholesterol occurs via ABCG1. HDL is then removed from the circulation by the liver through binding of the HDL to hepatic scavanger receptor SR-B1. Cholesterol ester-rich IDL and LDL can return to the liver and be taken up through interaction with the LDL receptor (LDLR). Within the vasculature the generation of ROS results in oxidation of lipid components of LDL generating oxidized LDL (oxLDL) which is taken up by macrophages via the scavenger receptor, FAT/CD36.
HDLs also acquire cholesterol by extracting it from cell surface membranes. This process has the effect of lowering the level of intracellular cholesterol, since the cholesterol stored within cells as cholesteryl esters will be mobilized to replace the cholesterol removed from the plasma membrane. The transfer of cholesterol from peripheral tissue cells to HDLs in this way involves the action of the ATP-binding cassette protein G1 (ABCG1). Approximately 20% of HDL uptake of peripheral tissue cholesterol occurs via the ABCG1-mediated pathway.
Cholesterol-rich HDLs return to the liver, where they bind to a receptor that is a member of the scavenger receptor family, specifically the scavenger receptor BI: SR-BI (see below). When HDL binds to SR-BI it is not internalized as is the case for LDLs following their binding to the LDL receptor but the cholesteryl esters of HDLs are taken up by the hepatocytes through caveolae while the HDL and SR-BI remain on the plasma membrane. Caveolae (Latin for little caves) are specialized "lipid rafts" present in flask-shaped indentations in the plasma membranes of many cells types that perform a number of signaling functions.
HDL particles exhibit complex, and sometimes contradictory rolls in vascular biology. Depending upon the vascular context, as well as the make-up of HDL particle, these lipoproteins can serve antiatherogenic or proatherogenic functions. In the absence of systemic inflammation many of the enzymes and apolipoproteins associated with HDLs play important roles in reducing the amount of oxidized lipid to which peripheral tissues are exposed. Some of these important proteins are apoA-I, PON1, GPx (an important anti-oxidant enzyme), and PAF-AH (see section below for the discussion of this important activity). However, when an individual has an ongoing systemic inflammatory state, these anti-oxidant proteins can be dissociated from the HDL or become inactivated resulting in the increased generation of oxidized and peroxidized lipids which are proatherogenic. Atherosclerotic plaques also produce myeloperoxidase which chemically modifies HDL-associated apoA-I rendering it less capable of interacting with cell surfaces such as macrophages. This latter effect results in a reduced capacity for removal of cholesterol from lipid-laden macrophages (foam cells) leaving the foam cells in a more pro-inflammatory state.
Reverse cholesterol transport can also involve the transfer of cholesterol esters from HDLs to VLDLs and LDLs. This transfer requires the activity of the plasma glycoprotein cholesterol ester transfer protein (CETP). The transfer of cholesteryl esters from HDLs to VLDLs via CETP activity also involves an exchange of triglycerides from the VLDLs to the HDLs. VLDLs are eventually converted to LDLs and the cholesterol acquired from HDLs can be returned to the liver via the interaction of LDL with the hepatic LDL receptor. This action of HDL CETP has the added effect of allowing the excess cellular cholesterol to be returned to the liver through the LDL receptor. However, some of the LDL is oxidized in the periphery (generating oxLDL) where it can participate in atherogenesis. Additionally, when HDL particles become enriched with triglycerides they are better targets for the action of hepatic lipase. As hepatic lipase acts on the triglyceride-rich HDLs they become progressively smaller and unstable which results in the release of apoA-I. The loss of apoA-I renders the HDL particle unable to participate in reverse cholesterol transport. Blocking the activity of CETP keeps HDL particles less triglyceride-enriched while also reducing cholesterol transfer to VLDLs resulting in reduced circulating levels of proatherogenic oxLDL. This latter observation suggests that CETP inhibition may be a viable therapeutic approach for elevating the circulating levels of HDLs. This is discussed in the section below.back to the top
Using a range of both in vitro and in vivo assays it has been possible to quantify the anti- and pro-inflammatory properties, as well as the anti-oxidant functions of HDLs. Cell-free assays have been used to measure the ability of HDLs to prevent the formation of oxidized phospholipids in LDLs as well as to determine the ability of HDLs to degrade oxidized phospholipids that are already formed. In cell culture assays HDLs have been shown to inhibit monocyte chemotaxis in response to oxidized LDL or to prevent the upregulation of cell adhesion molecules on endothelial cells. Both of these latter effects are strongly anti-inflammatory since monocytes need to migrate to a site of inflammation via a chemotactic gradient and then adhere to the endothelium at the site of injury or inflammatory event. The role of HDLs in promoting cholesterol efflux from cells, especially from macrophages, (the process of reverse cholesterol transport) reduces the activation of inflammatory responses in these cells. The analysis of HDL functions in oxidative and inflammatory events has identified the role of various apolipoproteins associated with HDLs in these processes which are outlined in the following sections.
Apolipoprotein A-I: Numerous lines of evidence demonstrate that apoA-I is a major anti-atherogenic and anti-oxidant factor in HDL due to its critical role in the HDL-mediated process of reverse cholesterol transport. In addition to reverse cholesterol transport, apoA-I can remove oxidized phospholipids from oxidized LDLs (oxLDLs) and from cells. Specific methionine residues (Met112 and Met148) of apoA-I have been shown to directly reduce cholesterol ester hydroperoxides and phosphatidylcholine hydroperoxides.
Apolipoprotein A-II: Experiments in transgenic mice have demonstrated that human apoA-II-enriched HDLs served to protect VLDLs from oxidation more efficiently than HDLs from control animals. The human apoA-II-enriched HDLs support highly effective reverse cholesterol transport from macrophages. Although there is a demonstrated benefit of apoA-II in reverse cholesterol transport and in reduced LDL oxidation, these transgenic mice exhibited increased displacement of PON1 and PAF-AH from HDLs. The displacement of these two beneficial HDL-associated proteins (see below) likely explains the increased atherosclerosis seen in dyslipidemic mice that overexpress either human or murine apoA-II. However, recent clinical studies in human patients show that the higher the plasma apoA-II concentration the lower is the risk of developing coronary artery disease (CAD).
Apolipoprotein A-IV: Apolipoprotein A-IV has multiple activities related to lipid and lipoprotein metabolism as well as the control of feeding behaviors (see earlier section related to this protein). ApoA-IV participates in reverse cholesterol transport by promoting cholesterol efflux as well as through by activation of LCAT. ApoA-IV has also been shown to have anti-oxidant, anti-inflammatory and anti-atherosclerotic actions. ApoA-IV is secreted only by the small intestine in humans (although it is expressed in the hypothalamus) and its synthesis in the gut is stimulated by active lipid absorption. Intestinal apoA-IV synthesis is enhanced by peptide tyrosine-tyrosine (PYY) secreted from the ileum. Intestinal apoA-IV, present in the circulation following ingestion of fat, as well as hypothalamic apoA-IV is an anorexigenic peptide which mediates, in part, the appetite suppressing effects of a lipid-rich meal.
Apolipoprotein E: The anti-atherosclerotic activity associated with apoE is well known. This beneficial effect of apoE is due primarily to its role in the process of receptor-mediated uptake of LDLs by the liver. Although apoE-mediated hepatic uptake of LDLs results in a reduction in hypercholesterolemia, apoE has also been shown to inhibit atherosclerosis without any significant effect on hypercholesterolaemia. In addition, different apoE alleles have demonstrated activities. For example apoE2 stimulates endothelial nitric oxide (NO) release and has anti-inflammatory activities, whereas, apoE4 is pro-inflammatory.
Paraoxonases 1 and 3: Paraoxonases are a family of enzymes that hydrolyze organophosphates. Paraoxonase 1 (PON1) is synthesized in the liver and is carried in the serum by HDL. PON1 possesses anti-oxidant properties, in particular it prevents the oxidation of LDLs. Evidence suggests that the direct anti-oxidant effect of HDLs, on LDL oxidation, is mediated by PON1. PON1 has been shown to enhance cholesterol efflux from macrophages by promoting HDL binding mediated by ABCA1, which in turn results in a reduction of pro-inflammatory signaling. This anti-inflammatory action of PON1 serves an anti-atherosclerotic function of the protein. That PON1 is indeed important in preventing atherosclerosis has been demonstrated in mice deficient in the protein. Atherosclerotic lesions that develop in these mice when fed a high-fat diet are twice the size that develop in similarly fed control mice. In human clinical studies, a higher level of PON1 activity is associated with a lower incidence of major cardiovascular events. Other pathological conditions in humans that are associated with oxidative stress, such as rheumatoid arthritis and Alzheimer disease, are frequently associated with reduced activity of PON1.
PON3, which is another HDL-associated paraoxonase, has also been shown to prevent the oxidation of LDL. Transgenic mice expressing human PON3 have been shown to be protected from the development of atherosclerosis, without any significant changes in plasma lipoprotein cholesterol, triglyceride or glucose levels.
Platelet-activating factor acetylhydrolase (PAF-AH): There are two major forms of PAF-AH, cytosolic and plasma lipoprotein-associated. The plasma form of PAF-AH circulates bound to HDLs. Given that PAF-AH is a member of the PLA2 family and that it also circulates bound to lipoprotein it is more commonly referred to as the lipoprotein-associated PLA2 (Lp-PLA2 section below). Experimental data suggests that Lp-PLA2, rather than PON1, is the major HDL-associated hydrolase that is responsible for the hydrolysis of oxidized phospholipids. Lipoproteins that are isolated from transgenic mice expressing human Lp-PLA2 are more resistant to oxidative stress. In addition, these mice have been shown to have reduced levels of foam cell (lipid-rich macrophages) formation and enhanced rates of cholesterol efflux from macrophages. In experimental atherosclerosis models, gene transfer of LP-PLA2 inhibits atherosclerotic lesion formation in apoE-deficient mice. In humans, Lp-PLA2 deficiency is associated with increases in cardiovascular disease, while conversely circulating levels of Lp-PLA2 serve as an independent marker of the risk for developing coronary artery disease.
Glutathione peroxidase 1: Glutathione peroxidase 1 (GPx1) functions primarily to reduce hydrogen peroxide to water, but it has been shown to also reduce lipid hydroperoxides to corresponding hydroxides effectively detoxifying these types of abnormally modified lipids. Numerous human clinical studies indicated that GPx1 provides a protective role against the development of atherosclerosis. These effects of GPx1 have also been shown in mice deficient in apoE where concomitant loss of the peroxidase results in increased rates of atherosclerotic plaque formation. The role of GPx1 in the protection from development of atherosclerosis is most pronounced under conditions of significant oxidative stress.
Sphingosine-1-phosphate (S1P): S1P is a bioactive lysophospholipid involved in a number of physiologically important pathways. For more detailed information of S1P activities visit the Sphingolipids page. Within the blood, HDLs are known to be the most prominent carriers of S1P. Indeed, many of the biological effects of HDL are mediated, in part, via S1P binding to its cell surface receptors. Effects of HDL on endothelial cells, such as migration, proliferation, and angiogenesis, are mediated, in part, by S1P associated with HDLs. HDL-associated S1P inhibits pro-inflammatory responses, such as the generation of reactive oxygen species, activation of NAD(P)H oxidase and the production of monocyte chemoattractant protein-1. While the HDL-associated forms of S1P exhibit these anti-inflammatory effects, free plasma S1P can activate inflammatory events dependent upon the receptor sub-type to which it binds.back to the top
Numerous epidemiological and clinical studies over the past 10 years have demonstrated a direct correlation between the circulating levels of HDL cholesterol (most often abbreviated HDL-c) and a reduction in the potential for atherosclerosis and coronary heart disease (CHD). Individuals with levels of HDL above 50mg/dL are several time less likely to experience CHD than individuals with levels below 40mg/dL. In addition, clinical studies in which apoA-I, (the predominant protein component of HDL-c) or reconstituted HDLs are infused into patients, raises circulating HDL levels and reduces the incidence of CHD. Thus, there is precedence for therapies aimed at raising HDL levels in the treatment and prevention of atherosclerosis and CHD. Unfortunately current therapies only modestly elevate HDL levels. Both the statins and the fibrates have only been shown to increase HDL levels between 5%–20% and niacin is poorly tolerated in many patients. Therefore, alternative strategies aimed at increasing HDL levels are being tested.
Cholesterol ester transfer protein (CETP) is plasma glycoprotein secreted primarily from the liver and plays a critical role in HDL metabolism by facilitating the exchange of cholesteryl esters (CE) from HDL for triglycerides (TG) in apoB containing lipoproteins, such as LDL and VLDL. The activity of CETP directly lowers the cholesterol levels of HDLs and enhances HDL catabolism by providing HDLs with the TG substrate of hepatic lipase. Thus, CETP plays a critical role in the regulation of circulating levels of HDL, LDL, and apoA-I. It has also been shown that in mice naturally lacking CETP most of their cholesterol is found in HDL and these mice are relatively resistant to atherosclerosis. The potential for the therapeutic use of CETP inhibitors in humans was first suggested when it was discovered in 1985 that a small population of Japanese had an inborn error in the CETP gene leading to hyperalphalipoproteinemia and very high HDL levels. To date three CETP inhibitors have been used in clinical trials. These compounds are anacetrapib, torcetrapib, and dalcetrapib. Although torcetrapib is a potent inhibitor of CETP, its' use has been discontinued due to increased negative cardiovascular events and death rates in test subjects. Treatment with dalcetrapib results in increases in HDL (19–37%) and a modest decrease (≈6%) in LDL levels. Treatment with anacetrapib results in a significant increase in both HDL (≈130%) and LDL (≈40%). Anacetrapib is currently in phase III clinical studies.
As described in the section below on therapeutic intervention in hyperlipidemias/hypercholesterolemias, the fibrates (e.g. fenofibrate) are a class of drugs that has been shown to result in small increases in HDL levels. The fibrates function by activation of the peroxisome proliferator-activated receptor-α (PPARα) class of transcription co-activators. However, the level of HDL increase with the current PPARα agonists is minimal at best primarily due to lack of specificity for PPARα. Therefore, current research is focused on subtype-specific PPARα agonists that have increased potency. One compound currently being tested, GFT505, is a selective PPARα agonist with a potency 100-fold greater than fenofibrate.
The liver X receptors (LXRα and LXRβ) are transcription co-activators that are involved in the regulation of lipid metabolism and have also been associated with regulation of inflammation. LXR agonists have been shown to inhibit the progression of atherosclerosis in mouse models of the disorder. Although the precise mechanism by which these LXR agonists effect a reduction in the progression of atherosclerosis is not clear, it is known that the genes encoding ABCA1 and ABCG1 contain LXR-binding sites. In fact, LXR agonists up-regulate the expression of both ABCA1 and ABCG1 in macrophages which leads to increased reverse cholesterol transport. Less cholesterol in macrophages leads to a reduced inflammatory activity of the macrophage which in turn likely contributes to the reduced atherosclerosis. However, there is a limitation to the utility of LXR agonists as shown by the first generation synthetic LXR ligands which activate both LXRs and lead to marked increases in hepatic lipogenesis and plasma triglyceride levels. These effects are due to the role of LXRs in activation of hepatic SREBP-1c and the resultant activation of each of its target genes as described above. Although it could be theoretically possible to enhance the reverse cholesterol effects of LXRs without targeting hepatic lipogenesis with the use of LXRβ-specific ligands since most of the hepatic responses are due to activation of LXRα, this will be a difficult challenge as the ligand binding pocket in both isoforms has been shown to be nearly identical. In addition, there are species-specific differences in overall LXR responses that need to be carefully considered meaning the use of animal models that more closely resemble humans in their metabolic pathways.back to the top
LDLs are the principal plasma carriers of cholesterol delivering cholesterol from the liver (via hepatic synthesis of VLDLs) to peripheral tissues, primarily the adrenals and adipose tissue. LDLs also return cholesterol to the liver. The cellular uptake of cholesterol from LDLs occurs following the interaction of LDLs with the LDL receptor (also called the apoB-100/apoE receptor). The sole apoprotein present in LDLs is apoB-100, which is required for interaction with the LDL receptor.
The LDL receptor is a polypeptide of 839 amino acids that spans the plasma membrane. An extracellular domain is responsible for apoB-100/apoE binding. The intracellular domain is responsible for the clustering of LDL receptors into regions of the plasma membrane termed coated pits. Once LDL binds the receptor, the complexes are rapidly internalized (endocytosed). ATP-dependent proton pumps lower the pH in the endosomes, which results in dissociation of the LDL from the receptor. The portion of the endosomal membranes harboring the receptor are then recycled to the plasma membrane and the LDL-containing endosomes fuse with lysosomes. Acid hydrolases of the lysosomes degrade the apoproteins and release free fatty acids and cholesterol. As indicated above, the free cholesterol is either incorporated into plasma membranes or esterified (by ACAT) and stored within the cell.
The level of intracellular cholesterol is regulated through cholesterol-induced suppression of LDL receptor synthesis and cholesterol-induced inhibition of cholesterol synthesis. The increased level of intracellular cholesterol that results from LDL uptake has the additional effect of activating ACAT, thereby allowing the storage of excess cholesterol within cells. However, the effect of cholesterol-induced suppression of LDL receptor synthesis is a decrease in the rate at which LDLs and IDLs are removed from the serum. This can lead to excess circulating levels of cholesterol and cholesteryl esters when the dietary intake of fat and cholesterol exceeds the needs of the body. The excess cholesterol tends to be deposited in the skin, tendons and (more gravely) within the arteries, leading to atherosclerosis.
The LDL receptor-related protein family represents a group of structurally related transmembrane proteins involved in a diverse range of biological activities including lipid metabolism, nutrient transport, protection against atherosclerosis, as well as numerous developmental processes. The LDL receptor (LDLR) described above represents the founding member of this family of proteins. The LRPs include LRP1, LRP1b, LRP2 (also called megalin), LRP4 (also called MEGF7 for multiple epidermal growth factor-like domains protein 7), LRP5/6, LRP8 (also called apolipoprotein E receptor 2), the VLDL receptor (VLDLR), and LR11/SorLA1 (LDL receptor relative with 11 ligand binding repeats/sorting protein related receptor containing LDLR class A repeats).
LRP1 is also known as CD91 or α2-macroglobulin receptor. This receptor is expressed in numerous tissues and is known to be involved in diverse activities that include lipoprotein transport, modulation of platelet derived growth factor receptor-β (PDGFRβ) signaling, regulation of cell-surface protease activity, and the control of cellular entry of bacteria and viruses. Regulation of PDFGRβ activity mediates the protective effects of LRP1 in development of atherosclerosis. LRP1 is synthesized as a 600kDa precursor that is proteolytically processed into a 85kDa transmembrane protein and a 515kDa extracellular protein. The extracellular protein non-covalently associates with the transmembrane protein. LRP1 has been shown to bind more than 40 different ligands that include lipoproteins, extracellular matrix proteins, cytokines and growth factors, protease and protease inhibitor complexes, and viruses. This diverse array of ligands clearly demonstrates that LRP1 is involved in numerous biological and physiological processes.
LRP2 was originally identified as an autoantigen in a rat model of autoimmune kidney disease called Heymann nephritis. LRP2 is expressed in numerous tissues and is found in the apical surfaces of epithelial borders as well as intracellularly in endosomes. In the proximal convoluted tubule of the kidney LRP2 is involved in the reabsorption of numerous molecules. LRP2 binds lipoproteins, hormones, vitamins, vitamin-binding proteins, proteases and, protease inhibitor complexes.
The LRP5/6 proteins serve as co-receptors in Wnt signaling (see the Wnts, TGFs, and BMPs page for more details).
The founding member of the scavenger receptor family was identified in studies that were attempting to determine the mechanism by which LDL accumulated in macrophages in atherosclerotic plaques. Macrophages ingest a variety of negatively charged macromolecules that includes modified LDLs such as oxidized LDLs (oxLDLs). These studies led to the characterization of two types of macrophage scavenger receptors identified as type I and type II. Subsequent research determined that the scavenger receptor family consists of several families that are identified as class A receptors, class B receptors, mucin-like receptors, and endothelial receptors. After binding ligand the scavenger receptors can either be internalized, similar to the process of internalization of LDL receptors, or they can remain on the cell surface and transfer lipid into the cell through caveolae or they can mediate adhesion.
The class A receptors include the type I and II macrophage scavenger receptors as well as an additional macrophage receptor called MARCO (macrophage receptor with collagenous structure).
The class B receptors include CD36 and scavenger receptor class B type I (SR-BI). The CD36 receptor is also known as fatty acid translocase (FAT; thus often designated FAT/CD36) and it is one of the receptors responsible for the cellular uptake of fatty acids as well as for the uptake of oxidized LDL (oxLDL) by macrophages. FAT/CD36 and SR-BI are closely related multi-ligand receptors and are most recognized for their roles in lipid and lipoprotein metabolism. The role of these receptors in platelet function has recently been the focus of numerous studies. Several of the identified ligands for FAT/CD36 include the gut hormone ghrelin, phospatidylserine (PS), β-amyloid, serum amyloid A, bacterial lipopeptides, and specific forms of oxidized phospholipids (oxPLs) either associated with LDLs (referred to a oxLDL) or free that contain an oxidized polyunsaturated fatty acid at the sn-2 position. These latter oxPLs are referred to as oxPCCD36 because they are predominantly phosphatidylcholine PLs and they bind FAT/CD36. The endothelial receptors that bind oxLDL and are called the LOX-1 receptors. LOX-1 is a member of the C-type lectin superfamily of carbohydrate recognition proteins. The receptor is also called the oxidized LDL receptor 1 (OLR1) and as such the LOX-1 protein is encoded by the OLR1 gene. The mucin-like receptors include CD68/macrosialin and the fruit fly scavenger receptor; dSR-CI.
The SR-BI protein has been shown to be the endogenous receptor for HDLs in the liver. Additionally, the HDL-SR-BI interaction in the adrenal glands is the mechanism for the delivery of cholesterol to the steroid hormone synthesizing cells of this tissue. HDLs first bind to SR-BI and then the cholesteryl esters present in the HDLs are transferred to the membrane for uptake via caveolae. The importance of the fact that the HDL-SR-BI complex remains at the cell surface is evident from the observation that this ligand-receptor interaction is also involved in the removal of cholesterol from cells by HDLs in the process of reverse cholesterol transport.back to the top
Platelet activating factor (PAF) is a lipid compound of the plasmalogen family of phospholipids (ether-linked glycerophospholipid) that is involved in numerous proinflammatory activities. Inactivation of PAF was originally ascribed to an activity called PAF-acetylhydrolase (PAF-AH). Subsequent to its initial characterization, PAF-AH was shown to be a member of a large family of enzymes that all hydrolyze the sn-2 position of glycerophospholipids. This family of enzymes is the PLA2 family. A detailed discussion of the PLA2 family of enzymes can be found on the Bioactive Lipids page. There are two major forms PAF-AH, one that is cytosolic and one that is secreted and found in the plasma. The plasma form of PAF-AH circulates bound to lipoproteins. Given that PAF-AH is a member of the PLA2 family and that it also circulates bound to lipoprotein it is more commonly referred to as the lipoprotein-associated PLA2 (Lp-PLA2). Lp-PLA2 is found in the plasma bound primarily to LDLs but is also found associated with HDLs and lipoprotein(a) [Lp(a)]. Of clinical significance is the fact that Lp-PLA2 has been implicated in atherosclerosis and cardiovascular disease but its precise role in these pathophysiological processes is not completely understood.
The human Lp-PLA2 protein is encoded by the PLA2G7 gene and is composed of 441 amino acids following cleavage of the signal peptide. The protein contains two sites of N-glycosylation. The enzymatic activity of Lp-PLA2 is specific for short chain acyl groups (up to 9 methylene groups) at the sn-2 position of phospholipids. When PAF is the substrate for Lp-PLA2 the products are lyso-PAF and acetate. When phospholipids of the phosphatidylcholine (PC) family are oxidized by free radical activity (referred to as oxPL) they can be a substrate for Lp-PLA2 even if the unsaturated fatty acid at the sn-2 position is longer than 9 carbon atoms. The ability of Lp-PLA2 to recognize oxPL as substrates is due to the presence of aldehydic or carboxlic moieties at the omega (ω) end of the sn-2 peroxidized fatty acyl residues. The products of Lp-PLA2 activity on oxPL are oxidized free fatty acids (oxFFA) and lyso-PC. Numerous types of oxPL have been identified in oxidized LDL (oxLDL) particles and many of them exhibit biological activity and exert key effects in atherogenesis. Lp-PLA2 can also hydrolyze long chain fatty acyl phospholipid hydroperoxides, phospholipids containing isoprostanes esterified at the sn-2 position and other lipid esters such as short-chain diglycerides, triglycerides, and acetylated alkanols. In addition to its hydrolytic activity Lp-PLA2 exhibits transacetylase activity. The transacetylase function transfers acetate and short-chain fatty acids from PAF to ether- and ester-linked lysophospholipids. The transacetylase function is evident when Lp-PLA2 is associated with LDL.
In humans with normal levels of circulating lipids and no detectable Lp(a), essentially all of the Lp-PLA2 in the plasma is bound to LDL. The interaction of Lp-PLA2 with LDL occurs through apolipoprotein B-100 (apoB-100). When plasma levels of Lp(a) rise in excess of 30mg/dL there is an enrichment in the association of Lp-PLA2 with this abnormal lipoprotein particle. When expressed as enzyme mass, Lp(a) carries 1.5–2 times more Lp-PLA2 than does LDL. As in its association with LDL, Lp-PLA2 interacts with apoB-100 in Lp(a) particles. Abnormalities in lipoprotein metabolism, such as those resulting in Lp(a) production, significantly affect the plasma levels of Lp-PLA2. For example in familial hypercholesterolemia the level of LDL-Lp-PLA2 activity increases in parallel with the severity of the hypercholesterolemia. The level of plasma Lp-PLA2 can be positively affected by low-calorie diets associated with weight loss or after drug treatment with the various classes of hypolipidemic drugs discussed below in Pharmacologic Intervention. In the context of atherosclerosis and cardiovascular disease numerous epidemiological studies have shown that increased levels of plasma Lp-PLA2 approximately double the risk for primary and secondary cardiovascular events. In fact it is suggested that measurement of Lp-PLA2 levels is useful as a cardiovascular risk marker independent of and additive to traditional risk factors. However, whether Lp-PLA2 is a novel biomarker or is causal in the development of atherosclerotic diseases remains controversial. This is because there are both anti- or proatherogenic activities associated with Lp-PLA2. The antiatherogenic functions of Lp-PLA2 are attributed to its role in hydrolyzing and inactivating the powerful proinflammatory lipid, PAF. Additionally, by hydrolyzing oxPLs Lp-PLA2 effectively lowers the circulating levels of this class of inflammatory mediators. On the other hand the proatherogenic and proinflammatory actions associated with Lp-PLA2 are in fact due to its hydrolysis of oxPLs. The hydrolysis of oxPLs releases both lyso-PC and oxFFA both of which have been shown to have proatherogenic effects.back to the top
Fortunately, few individuals carry the inherited defects in lipoprotein metabolism that lead to hyper- or hypolipoproteinemias (see Tables below for brief descriptions). Persons suffering from diabetes mellitus, hypothyroidism and kidney disease often exhibit abnormal lipoprotein metabolism as a result of secondary effects of their disorders. For example, because lipoprotein lipase (LPL) synthesis is regulated by insulin, LPL deficiencies leading to Type I hyperlipoproteinemia may occur as a secondary outcome of diabetes mellitus. Additionally, insulin and thyroid hormones positively affect hepatic LDL-receptor interactions; therefore, the hypercholesterolemia and increased risk of atherosclerosis associated with uncontrolled diabetes or hypothyroidism is likely due to decreased hepatic LDL uptake and metabolism.
Of the many disorders of lipoprotein metabolism, familial hypercholesterolemia (FH) may be the most prevalent in the general population. Heterozygosity at the FH locus occurs in 1:500 individuals, whereas, homozygosity is observed in 1:1,000,000 individuals. FH is an inherited disorder comprising four different classes of mutation in the LDL receptor gene. The class 1 defect (the most common) results in a complete loss of receptor synthesis. The class 2 defect results in the synthesis of a receptor protein that is not properly processed in the Golgi apparatus and therefore is not transported to the plasma membrane. The class 3 defect results in an LDL receptor that is incapable of binding LDLs. The class 4 defect results in receptors that bind LDLs but do not cluster in coated pits and are, therefore, not internalized.
FH sufferers may be either heterozygous or homologous for a particular mutation in the receptor gene. Homozygotes exhibit grossly elevated serum cholesterol (primarily in LDLs). The elevated levels of LDLs result in their phagocytosis by macrophages. These lipid-laden phagocytic cells tend to deposit within the skin and tendons, leading to xanthomas. A greater complication results from cholesterol deposition within the arteries, leading to atherosclerosis, the major contributing factor of nearly all cardiovascular diseases.back to the top
Lipoprotein(a) [Lp(a)] was originally described as a new serum lipoprotein particle by Kare Berg in 1963. Lp(a) is composed of a common LDL nucleus linked to a molecule of apolipoprotein(a) [apo(a); encoded by the LPA gene] by disulfide bonds between a cysteine residue in a Kringle-IV (KIV) type 9 domain in apo(a) and a cysteine residue in apolipoprotein B-100 (apoB-100). When attached to apoB-100 the apo(a) protein surrounds the LDL molecule. Synthesis of Lp(a) occurs in the liver. The half-life of Lp(a) in the circulation is approximately 3–4 days. Although Lp(a) was described over 40 years ago its precise physiological function remains unclear. However, numerous epidemiological studies have demonstrated that elevated plasma levels of Lp(a) are a significant risk factor for the development of atherosclerotic disease.
The Kringle domains of apo(a) exhibit 75%-85% similarity to the KIV domains of plasminogen. The Kringle domain is a highly glycosylated domain found in numerous proteins and is so-called because the three dimensional structure resembles a looped Danish pastry. Each Kringle domain is composed of approximately 80 amino acid residues and the structure is stabilized by three internal disulfide bonds. There are 10 distinct sub-classes of KIV domain in apo(a) designated KIV1 through KIV10. The apo(a) KIV1 and KIV3 through KIV10 domains are present as single-copy domains. The KIV2 domain is present in a variable number of repeated copies (from 2–43) and constitutes the molecular basis for the highly variable size of Lp(a) in different individuals. Apo(a) also contains a Kringle V (KV) domain that resembles the catalytic domain of plasminogen. Indeed, the apo(a) gene located on chromosome 6q26 is a member of the plasminogen superfamily and given the similarities between apo(a) and plasminogen it has been hypothesized that apo(a) influences the processes of hemostasis.
Apo(a) proteins exhibit a variability in size due to a polymorphism caused by a variable number of the KIV repeats. To date at least seven different isoforms of Lp(a) have been characterized based upon electrophoretic mobilities. These different isoforms are designated F, B, and S1 through S5. The different isoforms are grouped into low molecular weight (LMW) and high molecular weight (HMW) isoforms determined by the number of KIV repeats in the apo(a) protein found in the Lp(a). The level of Lp(a) found in healthy individuals depends upon whether their plasma contains the LMW or HMW isoforms. Individuals with the LMW isoforms have high plasma Lp(a) concentration while those with the HMW isoforms have low concentrations.
When in the circulation Lp(a) particles can be affected by oxidative modification similar to that of the other plasma lipoprotein particles. Lp(a) and oxidized Lp(a) [oxLp(a)] particles interact with macrophages via scavenger receptor uptake leading to cholesterol accumulation and foam cell formation. Indeed, oxLp(a) are phagocytosed more rapidly than other lipoprotein particles and therefore accumulate in the subendothelial space at high levels. This process can lead to progression of atherogenesis, thus accounting for the direct correlation between the plasma level of Lp(a) and coronary artery disease. In addition to oxidation of Lp(a) leading to increased foam cell production, glycation of the particle also may contribute to atherogenesis. In fact, there is a strong correlation in the level of glycated Lp(a) and the severity of hyperglycemia observed in poorly controlled type 2 diabetes.
Although the precise physiology of Lp(a) is poorly understood, as indicated above, there is a strong correlation between plasma concentration of Lp(a) and atherogenic events that lead to coronary artery disease. For a discussion of the processes of blood coagulation and the role of plasminogen visit the Blood Coagulation page. Because of the high degree of similarity between apo(a) and plasminogen it is suggested that Lp(a) may contribute to the thrombotic aspects of ischemic heart disease. Lp(a) has been shown to competitively inhibit the binding of plasminogen to its receptor on endothelial cells as well as to its binding sites on fibrinogen and fibrin. This interference of plasminogen binding leads to reduced surface-dependent activation of plasminogen to plasmin. The normal function of plasmin is to degrade the fibrin clot that forms as a result of vessel injury. Therefore, high plasma concentrations of Lp(a) may represent a source of antifibrinolytic activity. Of significance to the potential for atherogenesis, the antifibrinolytic potential of Lp(a) particles is related to their size. The LMW isoforms of Lp(a) have been shown to have a higher fibrin-binding capacity than the HMW isoforms. Lp(a) also interferes with other aspects of the normal processes of coagulation in addition to its effects on plasminogen function. Lp(a) stimulates the production of plasminogen activator inhibitor-1 (PAI-1) leading to a reduced ability of t-PA to activate the process of clot dissolution. Increased production of PAI-1 also leads to enhanced proinflammatory events via activation of monocyte adhesion to the vessel wall. Lp(a) has also been shown to modulate platelet activation interfering with the interaction of platelets with exposed collagen fibers in the injured vessel wall. In addition to the role of Lp(a) in inhibiting plasminogen binding, Lp(a) has been shown to inhibit the release of tissue plasminogen activator (t-PA) from endothelial cells. With reduced release of the enzyme (t-PA) that converts plasminogen to plasmin and interference with plasminogen binding to fibrin clots Lp(a) can exert a significant negative effect on the ability to dissolve blood clots.
Role of Lp(a) in promotion of atherogenesis. Lp(a) represents a circulating abnormal variant of LDL. The formation of Lp(a) occurs when apolipoprotein a [apo(a)] forms a disulfide bonded complex with the apoB-100 component of LDL. The apo(a) component of Lp(a) particles promotes the process of atherogenesis, in part, due to its ability to interfere with the normal events of hemostasis. This interference results from apo(a) binding to plasminogen binding sites preventing plaminogen and t-PA from interacting. If t-PA cannot cleave plasminogen to plasmin then fibrin clots cannot be dissolved. Lp(a) also interferes with plamin binding sites on the fibrin clot which also inteferes with the process of clot dissolution all of which leads to enhanced atherogenesis. The green arrows indicated enhanced activity such as the ability of Lp(a) to increase the production and activity of PAI-1. Red T-lines represent inhibitory processes.
In addition to the interactions with plasminogen, leading to enhanced atherogenesis, Lp(a) has been shown to stimulate smooth muscle cell (SMC) growth. This effect of Lp(a) is exerted via an inactivation of transforming growth factor-β (TGF-β). Activated TGF-β inhibits the proliferation and migration of SMC, thus the inhibition of this regulatory effect of TGF-β leads to accelerated blood vessel stenosis with concomitant enhancement of the atherogenic process. oxLp(a) has also been shown to inhibit nitric oxide-dependent vasodilation which will tend to exacerbate the atherogenic process in hypertensive patients.
Lp(a) also interferes with other aspects of the normal processes of coagulation in addition to its effects on plasminogen function. Lp(a) stimulates the production of plasminogen activator inhibitor-1 (PAI-1) leading to a reduced ability of t-PA to activate the process of clot dissolution. Increased production of PAI-1 also leads to enhanced proinflammatory events via activation of monocyte adhesion to the vessel wall. Lp(a) has also been shown to modulate platelet activation by interfering with the interaction of platelets with exposed collagen fibers in the injured vessel wall. All of the observed effects of Lp(a) on hemostasis result in the persistence of clots which is a significant contributor to atherogenesis and increases the potential for abnormal thrombotic episodes.back to the top
(familial LPL deficiency, familial hyperchylomicronemia)
|(a) deficiency of LPL;
(b) production of abnormal LPL;
(c) apoC-II deficiency
|slow chylomicron clearance, reduced LDL and HDL levels; treated by low fat/complex carbohydrate diet; no increased risk of coronary artery disease|
|Familial hypercholesterolemia, FH
Type IIA hyperlipoproteinemia
|5 classes of LDL receptor defect||reduced LDL clearance leads to hypercholesterolemia, resulting in atherosclerosis and coronary artery disease|
(familial dysbetalipoproteinemia, remnant removal disease, broad beta disease, apolipoprotein E deficiency)
|hepatic remnant clearance impaired due to apoE abnormality; patients only express the apoE2 isoform that interacts poorly with the apoE receptor||causes xanthomas, hypercholesterolemia and atherosclerosis in peripheral and coronary arteries due to elevated levels of chylomicrons and VLDLs|
|elevated production of VLDL associated with glucose intolerance and hyperinsulinemia||frequently associated with type-II non-insulin dependent diabetes mellitus, obesity, alcoholism or administration of progestational hormones; elevated cholesterol as a result of increased VLDLs|
|Type V familial||elevated chylomicrons and VLDLs due to unknown cause||hypertriglycerideemia and hypercholesterolemia with decreased LDLs and HDLs|
Type II hyperlipoproteinemia
|increased level of HDLs||a rare condition that is beneficial for health and longevity|
|increased LDL production and delayed clearance of triglycerides and fatty acids||strongly associated with increased risk of coronary artery disease|
|Familial ligand-defective apoB||2 different mutations: Gln for Arg (amino acid 3500) or Cys for Arg (amino acid 3531); both lead to reduced affinity of LDL for LDL receptor||dramatic increase in LDL levels; no affect on HDL, VLDL or plasma triglyceride levels; significant cause of hypercholesterolemia and premature coronary artery disease|
|Familial LCAT deficiency
|absence of LCAT leads to inability of HDLs to take up cholesterol
(reverse cholesterol transport)
|decreased levels of plasma cholesteryl esters and lysolecithin; abnormal LDLs (Lp-X) and VLDLs; diffuse corneal opacities, target cell hemolytic anemia, and proteinuria with renal failure|
(cholesteryl ester storage disease)
|defect in lysosomal cholesteryl ester hydrolase; affects metabolism of LDLs||reduced LDL clearance leads to hypercholesterolemia, resulting in atherosclerosis and coronary artery disease|
|heparin-releasable hepatic triglyceride lipase deficiency||deficiency of the lipase leads to accumulation of triglyceride-rich HDLs and VLDL remnants (IDLs)||causes xanthomas and coronary artery disease|
(acanthocytosis, Bassen-Kornzweig syndrome)
|no chylomicrons, VLDLs or LDLs due to defect in apoB expression||rare defect; intestine and liver accumulate, malabsorption of fat, retinitis pigmentosa, ataxic neuropathic disease, erythrocytes have thorny appearance|
|Familial hypobetalipoproteinemia||at least 20 different apoB gene mutations identified, LDL concentrations 10-20% of normal, VLDL slightly lower, HDL normal||mild or no pathological changes|
|Tangier disease||reduced HDL concentrations, no effect on chylomicron or VLDL production||tendency to hypertriglycerideemia; some elevation in VLDLs; hypertrophic tonsils with orange appearance|
Drug treatment to lower plasma lipoproteins and/or cholesterol is primarily aimed at reducing the risk of atherosclerosis and subsequent coronary artery disease that exists in patients with elevated circulating lipids. Drug therapy usually is considered as an option only if non-pharmacologic interventions (altered diet and exercise) have failed to lower plasma lipids.
Alirocumab (Praluent®), Evolcumab (Repatha®): These drugs are the newest type of anti-hypercholesterolemia drugs recently approved by the FDA for use in the US. Both drugs are injectible antibodies that block the function of proprotein convertase subtilisin/kexin type 9, PCSK9. PCSK9 is serine protease of the subtilisin-like proprotein convertase 2 family. A major function of PCSK9 is the endosomal degradation of the LDL receptor (LDLR), thereby reducing the recyling of the LDLR to the plasma membrane. This effect of PCSK9 leads to a reduced ability of the liver to remove IDL and LDL from the blood contributing to the potential for hypercholesterolemia. The potential for the pharmaceutical benefits of the interference in the activity PCSK9 was recognized by a confluence of several studies. Patients with a specific form of familial hypercholesterolemia not due to mutations in the LDLR gene were shown to have severe hypercholesterolemia due to mutations in the PCSK9 gene resulting in hyperactivity of the enzyme. In addition, it was found that in certain individuals with low serum LDL levels there was an association with the inheritance of nonsense mutations in the PCSK9 gene which result in loss of PCSK9 activity. Hypercholesterolemic patients taking another cholesterol-lowering drug while simultaneously utilizing either of these new PCSK9 inhibitors saw further reductions in serum LDL levels of betweeen 55% and 77%.
Atorvastatin (Lipotor®), Simvastatin (Zocor®), Lovastatin (Mevacor®): These drugs are fungal HMG-CoA reductase (HMGR) inhibitors and are members of the family of drugs referred to as the statins. The net result of treatment is an increased cellular uptake of LDLs, since the intracellular synthesis of cholesterol is inhibited and cells are therefore dependent on extracellular sources of cholesterol. However, since mevalonate (the product of the HMG-CoA reductase reaction) is required for the synthesis of other important isoprenoid compounds besides cholesterol, long-term treatments carry some risk of toxicity. A component of the natural cholesterol lowering supplement, red yeast rice, is in fact a statin-like compound.
The statins have become recognized as a class of drugs capable of more pharmacologic benefits than just lowering blood cholesterol levels via their actions on HMGR. Part of the cardiac benefit of the statins relates to their ability to regulate the production of S-nitrosylated COX-2. COX-2 is an inducible enzyme involved in the synthesis of the prostaglandins and thromboxanes as well as the lipoxins and resolvins. The latter two classes of compounds are anti-inflammatory lipids discussed in the Aspirin page. Evidence has shown that statins activate inducible nitric oxide synthase (iNOS) leading to nitrosylation of COX-2. The S-nitrosylated COX-2 enzyme produces the lipid compound 15R-hydroxyeicosatetraenoic acid (15R-HETE) which is then converted via the action of 5-lipoxygenase (5-LOX) to the epimeric lipoxin, 15-epi-LXA4. This latter compound is the same as the aspirin-triggered lipoxin (ATL) that results from the aspirin-induced acetylation of COX-2. Therefore, part of the beneficial effects of the statins are exerted via the actions of the lipoxin family of anti-inflammatory lipids.
Additional anti-inflammatory actions of the statins results from a reduction in the prenylation of numerous pro-inflammatory modulators. Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to acceptor proteins. The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus sequence at the C-terminus of prenylated proteins has been identified and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except alanine) and X is the C-terminal amino acid. In addition to numerous prenylated proteins that contain the CAAX consensus, prenylation is known to occur on proteins of the RAB family of RAS-related G-proteins. There are at least 60 proteins in this family that are prenylated at either a CC or CXC element in their C-termini. The RAB family of proteins are involved in signaling pathways that control intracellular membrane trafficking. The prenylation of proteins allows them to be anchored to cell membranes. In addition to cell membrane attachment, prenylation is known to be important for protein-protein interactions. Thus, inhibition of this post-translational modification by the statins interferes with the important functions of many signaling proteins which is manifest by inhibition of inflammatory responses.
Some of the effects on immune function that have been attributed to the statins are attenuation of autoimmune disease, inhibition of T-cell proliferation, inhibition of inflammatory co-stimulatory molecule expression, decreases in leukocyte infiltration, and promotion of a shift in cytokine profiles of helper T-cell types from Th1 to Th2. Th1 cells are involved in cell-mediated immunity processes, whereas, Th2 cells are involved in humoral immunity process. The cytokines produced by Th2 cells include IL-4, IL-5, IL-10 and IL-13 and these trigger B cells to switch to IgE production and to activate eosinophils.
Nicotinic acid (Niacor® and Niaspan®): Nicotinic acid reduces the plasma levels of both VLDLs and LDLs by inhibiting hepatic VLDL secretion, as well as suppressing the flux of FFA release from adipose tissue by inhibiting lipolysis. In addition, nicotinic administration strongly increases the circulating levels of HDLs. Patient compliance with nicotinic acid administration is sometimes compromised because of the unpleasant side-effect of flushing (strong cutaneous vasodilation). Recent evidence has shown that nicotinic acid binds to and activates the G-protein coupled receptor identified as GPR109A (also called HM74A or PUMA-G). For more detailed information on the normal biological function of GPR109A go to the Bioactive Lipids page. The identity of a receptor to which nicotinic acid binds allows for the development of new drug therapies that activate the same receptor but that may lack the negative side-effect of flushing associated with nicotinic acid. Because of its ability to cause large reductions in circulating levels of cholesterol, nicotinic acid is used to treat Type II, III, IV and V hyperlipoproteinemias.
Signaling events initiated in response to β-hydroxybutyrate or nicotinic acid binding to GPR109A on adipocytes or macrophages. During periods of fasting, hepatic ketone synthesis increases and the released β-butyrate binds to GPR109A on adipocytes triggering activation of the receptor-associated Gi-type G-protein which then inhibits the activity of adenylate cyclase (AC). Inhibition of AC leads to reduced HSL-mediated release of fatty acids from diacylglycerides. Nicotinic acid binding to GPR109A on adipocytes also leads to reduced fatty acid release. The reduced release of adipose tissue fatty acids leads to decreased synthesis and release of VLDL by the liver. It is this effect of nicotinic acid that contributes to the antidyslipidemic action of this drug. The GPR109A receptor on macrophages is also activated by nicotinic acid but this effect contributes to the undesired side-effets of nicotinic acid therapy. Within macrophages, GPR109A activation results in increased activation of PLA2 leading to increased arachidonic acid delivery to COX and increased production of the pro-inflammatory eicosanoids PGE2 and PGD2. The release of these eicosanoids causes increased cutaneous vasodilation resulting in the typical flushing and burning pain response to nicotinic acid therapy.
Gemfibrozil (Lopid®), Fenofibrate (TriCor®): These compounds (called fibrates) are derivatives of fibric acid and although used clinically since the 1930's were only recently discovered to exert some of their lipid-lowering effects via the activation of peroxisome proliferation. Specifically, the fibrates were found to be activators of the peroxisome proliferator-activated receptor-α (PPAR-α) class of proteins that are classified as co-activators. The naturally occurring ligands for PPAR-α are leukotriene B4 (LTB4, see the Lipid Synthesis page), unsaturated fatty acids and oxidized components of VLDLs and LDLs. The PPARs interact with another receptor family called the retinoid X receptors (RXRs) that bind 9-cis-retinoic acid. Activation of PPARs results in modulation of the expression of genes involved in lipid metabolism. In addition the PPARs modulate carbohydrate metabolism and adipose tissue differentiation. Fibrates result in the activation of PPAR-α in liver and muscle. In the liver this leads to increased β-oxidation of fatty acids, thereby decreasing the liver's secretion of triglyceride- and cholesterol-rich VLDLs, as well as increased clearance of chylomicron remnants, increased levels of HDLs and increased lipoprotein lipase activity which in turn promotes rapid VLDL turnover.
Cholestyramine or colestipol (resins): These compounds are nonabsorbable resins that bind bile acids which are then not reabsorbed by the liver but excreted. The drop in hepatic reabsorption of bile acids releases a feedback inhibitory mechanism that had been inhibiting bile acid synthesis. As a result, a greater amount of cholesterol is converted to bile acids to maintain a steady level in circulation. Additionally, the synthesis of LDL receptors increases to allow increased cholesterol uptake for bile acid synthesis, and the overall effect is a reduction in plasma cholesterol. This treatment is ineffective in homozygous FH patients, since they are completely deficient in LDL receptors.
Ezetimibe: This drug is sold under the trade names Zetia® or Ezetrol® and is also combined with the statin drug simvastatin and sold as Vytorin® or Inegy®. Ezetimibe functions to reduce intestinal absorption of cholesterol, thus effecting a reduction in circulating cholesterol. The drug functions by inhibiting the intestinal brush border transporter involved in absorption of cholesterol. This transporter is known as Niemann-Pick type C1-like 1 (NPC1L1). NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 is presumed to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake is regulated by cellular cholesterol content. In addition to the cholesterol lowering effects that result from inhibition of NPC1L1, its inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis. Ezetimibe is usually prescribed for patients who cannot tolerate a statin drug or a high dose statin regimen. There is some controversy as to the efficacy of ezetimibe at lowering serum cholesterol and reducing the production of fatty plaques on arterial walls. The combination drug of ezetimibe and simvastatin has shown efficacy equal to or slightly greater than atorvastatin (Lipitor®) alone at reducing circulating cholesterol levels.back to the top