Metabolism of VLDL & LDL

For a review of the metabolism of very-low-density lipoprotein (VLDL) and LDL see references [Herz, 1999; Packard, 1997; Durrington, 1995a; Kane, 1995; Schumaker, 1992].

The liver secretes a triglyceride-rich lipoprotein known as VLDL, which allows the supply of triglycerides to tissues in the fasting state as well as postprandially.
VLDL particles are somewhat smaller than the chylomicrons (diameter 30-75 nm; density <1006 g/L).
Once secreted, VLDL particles undergo exactly the same sequence of changes as chylomicrons: the acquisition of apolipoproteins and the progressive removal of triglycerides from their core by lipoprotein lipase.
In humans, however, some additional transformations are involved in the metabolism of VLDL particles.

Additional transformations in humans
In humans, the liver (unlike the gut) does not esterify cholesterol before it is secreted (this is different in the liver of some other species, such as the rat). Most of the cholesterol released into the circulation from the liver each day is secreted in VLDL as free cholesterol, which is then transferred from VLDL to HDL along a concentration gradient.

What is the role of LCAT?
The cholesterol is esterified in the circulation by the action of LCAT, which esterifies the hydroxyl group in the 3-position of cholesterol to a fatty acyl group. The fatty acyl group is then selectively removed by LCAT from the 2-position of phosphatidylcholine to give lysophosphatidylcholine. The fatty acyl group in this position is generally unsaturated, and the cholesteryl esters thus formed are frequently cholesteryl oleate or cholesteryl linoleate.

Familial LCAT deficiency is a very rare disorder, in which HDL fails to mature and circulating free cholesterol levels increase [Glomset, 1995]. This leads to:

anaemia;
corneal opacities;
proteinuria; and
renal failure.

What is the role of cholesteryl ester transfer protein?
Esterified cholesterol on HDL is transferred back to VLDL, but this cannot take place by simple diffusion because cholesteryl ester is intensely hydrophobic and because the concentration gradient is unfavourable. A transfer protein called cholesteryl ester transfer protein (CETP) is present in plasma, which transports cholesteryl ester from HDL to VLDL [Barter, 2000]. It does this in exchange for triglycerides in VLDL, and in this way also contributes to the removal of core triglycerides from VLDL. The major mechanism for the removal of triglycerides from VLDL is, however, lipolysis catalysed by lipoprotein lipase.

What apolipoproteins are produced by the liver?
Another major difference between VLDL and chylomicrons is that the apo B produced by the liver in humans is apo B100, not apo B48. As in the case of chylomicrons, the quantum of apo B packaged in the VLDL remains tightly associated with the particle until its final catabolism, and its amount does not vary after secretion. Each molecule of VLDL contains one molecule of apo B100. The apo B100 produced in the liver contains the protein sequence necessary to bind to the LDL receptors, whereas that produced by the gut (although derived from the same gene) does not. This is due to a process of 'gene editing', which stops the ribosome translating the messenger RNA before the receptor-binding sequence, producing an apo B with a molecular mass that is 48% of the mass of the version from the liver. Essential for the process by which both apo B48 and apo B100 are packaged with triglyceride in the enterocyte and hepatocyte to form chylomicrons or VLDL, respectively, is microsomal triglyceride transfer protein (MTP), which is defective in abetalipoproteinaemia [Kane, 1995] (see later).

Formation of intermediate-density lipoprotein
The circulating VLDL particles become progressively smaller as their core is removed by lipolysis and surface materials are transferred to HDL. In healthy individuals, most of the VLDL is converted to smaller LDL particles through an intermediary lipoprotein known as intermediate density lipoprotein (IDL). This has a density of 1006-1019 g/L, and possesses apo E (which in the latter respect is similar to chylomicron remnants).

Formation of LDL
The enzyme, hepatic lipase, also participates in the conversion of IDL to LDL. In some species, such as the rat, the liver is largely responsible for the removal of VLDL, and LDL formation is thus bypassed. In humans, LDL particles - which are relatively enriched in cholesterol, but small enough (diameter 18-25 nm; density 1019-1063 g/L) to cross the vascular endothelium and enter the tissue fluid - serve to deliver cholesterol to the tissues. The concentration of LDL particles in the extracellular fluid is probably approximately 10% of that in the plasma.

Why do cells need cholesterol?
Cells require cholesterol for membrane repair and growth and, in the case of specialised tissues, such as the adrenal gland, gonads and skin, as a precursor for steroid hormone and vitamin D synthesis.

How does LDL enter cells?
LDL is able to enter cells by two routes making a major contribution to its catabolism.

The first route is via the LDL receptor, which is regulated according to the cholesterol requirement of each individual cell.
The second route is non-receptor mediated and depends almost entirely on the extracellular concentrations of LDL.

The LDL receptor route
The LDL receptor, although capable of binding apo E-containing lipoproteins, in practice, usually binds to the apo B100-containing lipoproteins, in particular LDL [Goldstein, 1995]. This is due to the fact that LDL is the most widely distributed and abundant of the apo B100-containing and apo E-containing lipoproteins. After binding, the LDL-receptor complex is internalised within the cell, where it undergoes lysosomal degradation; the apo B of the complex is hydrolysed to its constituent amino acids and the cholesteryl ester is hydrolysed to free cholesterol.

How is cellular cholesterol content regulated?
The release of the free cholesterol is the signal by which the cellular cholesterol content is precisely regulated by three co-ordinated reactions.

First, the rate-limiting enzyme for cholesterol biosynthesis (3-hydroxy-3-methylglutaryl CoA [HMG-CoA] reductase) is repressed, effectively centralising cholesteryl biosynthesis to organs, such as the liver and gut.
Second, the synthesis of the LDL receptor itself is suppressed.
Third, acyl-CoA:cholesterol O-acyltransferase is activated, so that any cholesterol that is surplus to immediate requirements can be converted to cholesteryl ester, which, because of its hydrophobic nature, forms droplets within the cytoplasm and is, thus, conveniently stored.

What is the effect of lysosomal release of free cholesterol?
The effect of the lysosomal release of free cholesterol on the expression of the LDL receptor contrasts with its effect on the hepatic LRP, which is not subject to any similar down-regulatory process. Free cholesterol, released by lysosomal digestion of cholesteryl ester-rich, apo E-containing lipoproteins entering the hepatocyte via the LRP, does not influence expression of this receptor mechanism. Defective LDL uptake by the LDL receptor is the basis of familial hypercholesterolaemia (see later).

The non-receptor-mediated route
The other quantitatively important mechanism by which LDL-cholesterol (LDL-C) may enter cells is by a non-receptor-mediated pathway: LDL binds to cell membranes at sites other than those at which LDL receptors are located, and some of it passes through the membrane by pinocytosis. HDL is able to compete with LDL for this type of cell-membrane association. The absence of a receptor means that the 'binding' is of low affinity and thus, at low concentrations, LDL entry by this route may have little significance. Unlike receptor-mediated entry, however, non-receptor-mediated LDL uptake is not saturable, but continues to increase with increasing extracellular LDL concentrations. When LDL levels are relatively high, entry of cholesterol into the cells by this route may thus assume greater quantitative importance than that via the LDL receptor, which will be both saturated and down-regulated. This appears to be the situation in the typical western adult consuming a high-fat diet, whose LDL-C is high compared with the level in most animal species, and in whom only about one-third of LDL is catabolised by receptors and two-thirds by non-receptor-mediated pathways. In hypercholesterolaemia, even more is catabolised via the non-receptor pathway (four-fifths in patients heterozygous for familial hypercholesterolaemia, virtually all in homozygotes, see later).

Other receptors
LDL may also be removed from the circulation by a number of receptors other than the classical LDL receptor. It is likely that these are responsible for the catabolism of only relatively minor amounts of LDL, but some of these receptors present on the macrophage have excited considerable interest because they may have a central role in atherogenesis. They include the scavenger receptors and oxidised LDL receptors, which permit the uptake of oxidised LDL by macrophages [Witztum, 1991]. In addition, the macrophage can take up the beta-VLDL present in type III hyperlipoproteinaemia [Mahley, 1995] (see later) by a receptor-mediated route, although the precise nature of the receptors involved (which could include a VLDL receptor) is currently unclear. Uptake at both these macrophage receptors is so rapid in vitro that foam cells resembling those in arterial fatty streaks and atheromatous lesions are formed. In contrast, uptake of unmodified LDL by the macrophage via the LDL receptor is too slow for the formation of foam cells to occur. Oxidation of LDL may occur in vivo and is of potential relevance to atherogenesis.

What is the role of cholesterol transport in atherosclerosis?
It should be recognised, however, that because of the largely unexplained high levels of LDL in humans, the removal and transport back to the liver of excess cholesterol delivered to the tissues (particularly the arterial wall) may be critical for atherosclerosis. The process involves uptake of HDL, and some aspects of it must be mentioned here because the pathway for reverse cholesterol transport (RCT; the transport of cholesterol from peripheral tissues back to the liver) interacts with the pathway producing LDL from VLDL.

How is excess cholesterol removed?
Some two-thirds of excess cholesterol arriving on HDL from the tissues can be removed from this HDL during its passage through the liver. Some cholesterol is, however, transferred to VLDL by CETP and can thus contribute to LDL-C. CETP can also remove cholesteryl ester from LDL in exchange for triglyceride, which is then removed from the LDL by hepatic lipase, giving rise to a small dense LDL.

Small dense LDL
Small dense LDL is taken up poorly by the LDL receptor and is more susceptible to oxidative modification than more buoyant LDL [Chait, 1993]. It may thus be particularly atherogenic [Lamarche, 1997]. Both the removal of cholesteryl ester from HDL and the formation of small dense LDL are increased when VLDL levels are high (hypertriglyceridaemia). This gives rise to the so-called atherogenic profile [Austin, 1990], in which low levels of HDL-cholesterol (HDL-C) and increased concentrations of small dense LDL occur in the presence of even quite moderate hypertriglyceridaemia. Because of the relative paucity of cholesterol in this type of LDL, serum cholesterol levels are unaffected by its presence and can be normal or even low. Also, because triglyceride catabolism, mediated by lipoprotein lipase, is often decreased in hypertriglyceridaemia, the rate at which the components required for the formation of HDL are released from VLDL and chylomicrons is diminished, further contributing to low HDL levels.

Lipoprotein (a)
Lipoprotein (a) [Lp(a)] was first identified as a result of blood transfusion reactions occurring due to genetic variation in its antigenicity. The precise location of Lp(a) in LDL and in the more buoyant form of HDL (HDL₂; see Figure 2) also varies from individual to individual, as does its serum concentration. Lp(a) may be undetectable in some people and present at concentrations equalling those of LDL in others. The protein moiety of Lp(a), like that of LDL, contains apo B100, but, in addition, apo(a) is also present.

Apolipoprotein (a)
Apo(a) is a member of the plasminogen supergene family, part of which is homologous to the plasminogen protein sequence. Its kringle 4 domain is repeated many times, and the number of these repeats (determined at a genetic locus adjacent to the plasminogen gene) determines the molecular mass of apo(a). Individuals expressing polymorphisms with fewer kringle 4 repeats have the highest serum concentrations of Lp(a) [Hobbs, 1999; Durrington, 1995b].

What are the functions of Lp(a) in the disease process?

High levels of Lp(a) are associated with the risk of coronary heart disease (CHD) in individuals of European origin [Sweetnam, 2000; Craig, 1998], particularly when serum cholesterol levels are also raised and when there is a family history of premature CHD.
Unlike plasminogen, Lp(a) does not possess fibrinolytic activity, because of a modification of its activation site. It has, therefore, been suggested that Lp(a) may interfere with thrombolysis.
Furthermore, because Lp(a) binds to a wide variety of cells and connective tissue matrices, it is retained in the arterial wall longer than LDL and is thus more likely to undergo oxidative modification and macrophage uptake, leading to atheroma.

Incidence of CHD
The incidence of CHD varies enormously in different parts of the world. Those countries with a northern European culture (and in particular diet) have the highest incidence, while places, such as China, Japan and rural Africa, have the lowest incidence (Mediterranean countries are intermediate). There are, of course, many differences between these countries, but the one that relates most closely to the incidence of CHD is the median cholesterol of the middle-aged male population [Simons, 1986]. Of considerable interest is the fact that, in a country such as Japan, where the average serum cholesterol is low, other coronary risk factors do not seem to operate. Thus, CHD is comparatively uncommon in Japan, even in cigarette smokers and in people with diabetes and hypertension. Cardiovascular diseases cause more than 235,000 deaths in the UK each year and extensive research has shown that if LDL is lowered, then the risk of developing CHD or having a secondary CHD event or stroke is reduced.

What is the relationship between serum cholesterol and incidence of CHD?
Within populations there is an exponential relationship between serum cholesterol and the incidence of CHD (see Figure 4) [Law, 1994; Anderson, 1990]. This depends on the LDL-C, which comprises some 70-80% of total cholesterol in men and a little less in women.

Figure 4. Relationship between serum cholesterol and the incidence of CHD