Cholesterol and Lipoproteins 101

Cholesterol is a type of the sterols.  Sterols are a subgroup of steroids that occur naturally in plants and animals.  Cholesterol is the only sterols in animal cells.  In human body, cholesterol is an essential component of the cell membrane, the precursor for all steroid hormones, vitamin D and bile acids biosynthesis, and plays a crucial role in the structure and function of the central nerve system.  A typical human body contains about 900 mg of cholesterol, of which 200–500 mg is from diet and the rest is synthesized in the liver and the central nerve system (Levy et al. 2007). 

Figure 1. The chemical structures of sterol, cholesterol and a representative of cholesteryl esters, cholesteryl stearate.
Cholesterol exists in free cholesterol when first synthesized in hepatic cells.  In circulating blood, cholesterol exists mainly in the form of cholesteryl esters as a component of lipoproteins.  Cholesteryl esters are compounds forms by an ester bond between a cholesterol molecule and a fatty acid molecule (Fig.1). They are the inactive form and the transportation mode of cholesterol.  Lipoproteins are multi-molecule complexes that transport and deliver cholesterol and other components to the target tissue (Fig.2).

Figure 2. A diagram of a lipoprotein.  See text for details.

Other components of lipoprotein include triglycerides, phospholipids and apolipoproteins. Triglycerides are the main form of energy storage molecules in human and animals.  They are composed of one glycerol and three fatty acids.  Phospholipids are derivatives of glycerides or other fat in which one fatty acid is replaced by a phosphate group, which in turn linked to another organic base such as amino acid or alcohol.  Apolipoproteins are the protein component of lipoprotein.  There are five main types (A, B, C, D, and E) of apolipoproteins in human plasma. Some of the apolipoproteins are further categorized into subtypes (e.g., A-I, -II, and -IV; and C-I, -II, and -III). In general, these apolipoproteins play important roles in lipid metabolism: maintaining the structural integrity of lipoproteins for lipid transportation, serving as cofactors in enzymatic reactions, and acting as ligands for lipoprotein receptors.

The principal plasma lipoproteins are the chylomicrons, VLDL (very low density lipoprotein), LDL (low density lipoprotein) and HDL (high density lipoprotein).  The main difference of these lipoprotein types are the size, density and protein components of the particles.  In the rank of chylomicrons, VLDL, LDL and HDL, the lipoprotein particle sizes are smaller and the densities greater (Table 1).

Table 1. Major types of lipoproteins in circulating blood (adapted from Durrington, 2007).

Chylomicrons are the product of diet fat digestion.  They are produced in absorptive cells of small intestines and released to blood circulation as nascent chylomicrons, which is mainly composed of cholesterol, triglycerides and Apo B48.  In circulation, nascent chylomicrons incorporate apolipoprotein C-II (Apo C2) and apolipoprotein E (Apo E) donated from HDL to form a mature chylomicron (often referred to simply as "chylomicron"). Chylomicrons transport exogenous lipids to liver, adipose, cardiac, and skeletal muscle tissue, where their triglyceride components are unloaded by the activity of lipoprotein lipase.  Once triglycerides are distributed, the chylomicrons returns Apo C2 to the HDL (but keeps Apo E), and becomes chylomicron remnants.  Chylomicron remnants are cleared from circulation mainly by the HSPG/LRP pathway on hepatic cells.  Some portion of the chylomicron remnants can be converted to LDL and cleared by the LDL receptor (also called Apo B100/E receptor) pathway.

VLDL is a class of lipoproteins smaller than chylomicrons.  They are mainly formed in liver cells and serve the function of distributing intracellular triglycerides from liver to other tissues. First triglycerides and Apo B100 form an initial complex.  Then cholesterol and more triglycerides join the complex to form VLDL on the Golgi complex.  The VLDL is then secreted to the circulation.  Once in circulation, more cholesterol is transferred from HDL to VLDL through the action of CEPT (Cholesterol ester transfer protein) while Apo C-II and Apo E are also incorporated into VLDL. Subsequent progressive hydrolysis of VLDL by lipoprotein lipase unloads triglycerides to various tissues during circulation and results in VLDL remnants, also known as IDL (Intermediate Density Lipoprotein). ILDL are eventually converted to LDL in circulation by lipoprotein lipase. 

LDL is also known as the BAD cholesterol since high level of LDL predispose to coronary heart disease.  LDL is mainly from the lipoprotein lipase digestion of chylomicrons and VLDL. It is small enough to cross the vascular endothelium to enter the tissue fluid to supply tissue with cholesterol.  Composition wise, LDL has the highest proportion of cholesterol among all type of lipoproteins, consistent with its major role of delivering cholesterol to various tissues.  LDL delivers cholesterol to cells via two routes: receptor dependent and receptor independent.  The receptor dependent route is regulated by several pathways that will be discussed in detail later.  The receptor independent route is mainly dependent on the extra cellular concentration of LDL.

HDL, also known as the GOOD cholesterol, is the smallest of the lipoprotein particles. They are the densest because they contain the highest proportion of protein to cholesterol. Their most abundant apolipoproteins are Apo A-I and Apo A-II.  HDL is synthesized in liver as disc-shaped complexes of apolipoproteins and phospholipids. These complexes then pick up cholesterol from cells by interaction with the ATP-binding cassette transporter A1 (ABCA1). A plasma enzyme called lecithin-cholesterol acyltransferase (LCAT) converts the free cholesterol into cholesteryl ester, which is then sequestered into the core of the lipoprotein particle, eventually making the newly synthesized HDL spherical. They increase in size as they circulate through the bloodstream and incorporate more cholesterol and phospholipid molecules from cells and other lipoproteins. HDL transports cholesterol from other tissue back to  the liver (also known as reverse cholesterol transport) or to steroidogenic organs such as adrenals, ovary, and testes by direct and indirect pathways.

The metabolism and interactions of these four major types of lipoproteins are shown in Fig.3.

Figure 3. An outline of the major metabolic pathways of the major lipoproteins (adapted from Durrington, 2007).

Cholesterol biosynthesis

Cholesterol biosynthesis is a multi-step process involving at least thirty different enzymatic reactions (Fig. 4). Konrad Bloch and Fyodor Lynen were awarded the Nobel Prize in 1964 for elucidating these reactions. The multistep biosynthesis of cholesterol starts with acetyl CoA, an intermediate metabolite from the breakdown of most energy source from diet.  The rate limiting step of cholesterol biosynthesis is catalyzed by the enzyme HMG-CoA reductase, a target of the statins, the most prescribed cholesterol lowing medicine in the United States.  Another important enzyme in the cholesterol biosynthetic pathway is squalene synthase, also a cholesterol lowing target that is responsible for the production of lanosterol, an intermediate metabolite solely committed to cholesterol synthesis. Cholesterol biosynthesis is regulated by free cholesterol levels in hepatic cells and by the LDL-receptor pathway.

Figure 4.  Major steps of cholesterol biosynthesis.

The major factor that regulates cholesterol biosynthesis is the intracellular concentration of free cholesterol in the cholesterol factory hepatic cells, which is in turn regulated by the level of circulating LDL through the LDL-receptor pathway and other pathways.

Many other factors influence the rate of cholesterol synthesis.  The factors that increase cholesterol synthesis include: cholesterol absorption interfering agent (b-sitosterol), enterohepatic circulation interruption (by biliary fistula, ileal by pass, etc.), hormones (insulin, thyroxine, catecholamines), medicine (phenobarbitone, chelestyramine) and dark.   The factors that decrease cholesterol synthesis include: dietary cholesterol, portacaval shunt, hormones (glucagon, glucocorticoids), medicine (clofibrate, nicotinic acid, stains etc.) and light (Durrington, 2007).

Cholesterol biosynthesis regulation by intracellular cholesterol concentration

Cholesterol biosynthesis is regulated by the intracellular concentration of free cholesterol in the hepatic cells.  The cellular cholesterol sensor is an escort protein called SREBP cleavage-activating protein (SCAP).  At lower cellular cholesterol levels, SCAP binds to the sterol regulatory element-binding protein (SREBP), which contains an N-terminal membrane domain and a C-terminal regulatory domain.  The SCAP-SREBP complex then moves to the Golgi, where two specific proteases (site-1 and site-2 proteases) cleave the SREBP enabling the C-terminal regulatory domain to enter the nucleus. There it activates the transcription of the genes coding for the LDL receptor and for the key enzyme in cholesterol biosynthesis, HMG-CoA reductase. This in turn stimulates the rate of cholesterol uptake and synthesis. Conversely, when cellular cholesterol levels are higher, the SCAP fails to activate the transcription factor and uptake and synthesis of cholesterol are not enhanced (Figure 5).  Further regulation of cholesterol biosynthesis may be exerted by certain oxysterols, which can suppress the activation of SREBP by binding to an oxysterol-sensing protein in the endoplasmic reticulum or by direct effects on the biosynthetic and transport enzymes (Brown & Goldstein, 1997; 1999).

Figure 5.  The SREBP pathway in cholesterol regulation.

Cholesterol biosynthesis and the LDL-receptor pathway

The LDL receptor pathway is the major route by which the LDL lipoproteins are cleared from blood circulation.  The discovery of this pathway earned the Nobel Prize in Physiology or Medicine to Joseph Goldstein and Michael Brown in 1985 (Goldstein & Brown, 2009). The LDL receptor is also known as Apo B100/E receptors. They are cell surface proteins that are capable of binding Apo B100 or Apo E proteins in vitro.  In vivo, they usually bind to the Apo B100-containing lipoproteins, in particular LDL, because 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 internalized within the cell, where it undergoes lysosomal degradation; the Apo B of the complex is hydrolyzed to its constituent amino acids and the cholesteryl ester is hydrolyzed to free cholesterol. The LDL receptor is recycled back to the cell surface (Fig. 6).

Figure 6.  The LDL pathway in cholesterol regulation (Adopted from Goldstein & Brown, 2009. Arterioscler Thromb Vasc Biol. 29: 431–438).

When the LDL level in blood is high, the LDL receptor pathway is actively clearing the LDL and turns it into free intracellular cholesterol, resulting in the inhibition of cholesterol synthesis by suppressing the gene expression of HMG-CoA reductase and LDL receptors.  Conversely, when the LDL level in blood circulation is low, the LDL receptor pathway is less active and the suppression of HMG-CoA reductase and LDL receptors gene expression is lifted and cholesterol biosynthesis is activated.

The HSPG/LRP pathway

The HSPG/LRP pathway is also called the chylomicron remnant receptor pathway or Apo E receptor pathway in literature.  This pathway involves two cell surface receptors LRP (LDL receptor-related protein) and HSPG (heparin sulfate proteoglycans) to function in hepatic clearance of remnant lipoproteins. Initially, Apo E-containing lipoproteins bind to cell surface HSPG. The lipoproteins become enriched in Apo E, with the HSPG apparently serving as a reservoir for Apo E. The Apo E-enriched proteins are then transferred to the LRP, and the LRP either initiates uptake or, more likely, forms a complex with HSPG that is subsequently taken up by the cells (Mahley et al, 1999).  Not all the Apo E containing lipoproteins can bind to LRP. Apolipoproteins CI and CII inhibit the binding. Therefore VLDL, which contains a higher proportion of Apo CI and Apo CII, cannot be taken up by the LRP pathway. LRP is certainly not dedicated to lipoprotein clearance.  Its main function includes clearance of alpha2-macroglobulin from the circulation.

The non-receptor-mediated pathway

In the non-receptor-mediated pathway, LDL binds to cell membranes at sites other than LDL receptors. Some of the LDL passes through the membrane by endocytosis. The absence of a receptor means that the 'binding' is of low affinity and not saturable.  Therefore, at low concentrations, LDL entry by this route may have little significance. When extracellular LDL levels are relatively high, entry of cholesterol into the cells by this route may assume greater proportion than the LDL receptor pathway which will be both saturated and down-regulated. For a typical western adult consuming a high-fat diet, about two-thirds of LDL is catabolised by non-receptor-mediated pathways and only one-third by receptors.

Other receptor-mediated pathways in cholesterol metabolism

The β-VLDL receptor is located on the surface of macrophages.  It is responsible for taking up β-VLDL from circulation.  β-VLDL is mainly composed of chylomicron remnant and IDL.  In normal metabolism, the chylomicron remnant is cleared by the HSPG/LRP pathway and IDL is converted to LDL and cleared by the LDL receptor pathway.  In pathological conditions, the clearance of β-VLDL is impaired and the concentration of β-VLDL in circulation is increased.  The macrophages then take up the β-VLDL via the β-VLDL receptor pathway.  Accumulation of β-VLDL turns macrophages into foam cells, a major component of atheromatous plaques.

The VLDL receptor is located on the cell surface of heart, skeletal muscle and adipose tissues.  These tissues general have high activity of lipoprotein lipase and high requirement for triglycerides for either energy supply or energy storage. VLDL receptors belong to the LDL receptor superfamily. Its physiologic function is supposed to assist the uptake of the small Apo E-rick lipoprotein particles formed by lipoprotein lipase hydrolysis of VLDL at the capillary endothelium.

The scavenger receptors, including the acetyl-LDL receptor and oxidized-LDL receptor, are a group of receptors that located on macrophage surface and that are responsive for cleaning (scavenging) waste macromolecules having a negative charge as well as modified LDL. However, the receptor SR-B1 (scavenger receptor class B1) present on the liver cells’ plasma membranes mediates most of the liver’s uptake of cholesteryl esters from HDL in the absence of uptake of apolipoproteins. The overall process by which HDL removes cholesterol from extra hepatic tissues and returns it to the liver is called reverse cholesterol transport.

Cholesterol clearance

Cholesterol is not readily biodegradable so does not serve as an energy source for human. Only the liver possesses the enzymes to degrade significant amounts of cholesterol via pathways that do not lead to energy production.  HDL is responsible for removing the extra cholesterol from various tissues. Some two-thirds of excess cholesterol arriving on HDL from the tissues can be removed from this HDL during its passage through the liver. For example, some cholesterol is transferred to VLDL by CETP and can thus contribute to the formation of LDL. The rest are transferred back to the liver for catabolism by conversion to oxysterols and bile acids. The latter are exported into the intestines to aid digestion and leading to some loss as fecal material. Approximately 90% of cholesterol catabolism occurs via bile acids. The total fecal excretion of bile salts balances hepatic synthesis and represents a major catabolic path in cholesterol metabolism (Cowen & Campbell, 1977).

Personal cholesterol management

The cholesterol level in a person can be controlled by dietary choice, lifestyle and cholesterol lowing medicines.  But each individual would response differently to those interventions due to a different genetic background.  To date, more than 1100 mutation of about two dozen of genes (including  key enzymes, receptors and transporters in cholesterol biosynthesis and transfer) are  implicated in the differential response to dietary or medicinal intervention (Masson et al., 2003; Charlton-Menys & Durrington, 2007).  One particular gene Apo E (Apolipoprotein E) represents the best understood in terms of its polymorphism and association with metabolic regulation of cholesterol (see Apo E and cholesterol management review).  Information based on the genetic background, the cholesterol profile and lifestyle would help tremendously in developing a robust and personalized cholesterol management diet regime.

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