Lipoproteins are used by cells to transfer hydrophobic lipid molecules such as cholesterol esters and TG through the bloodstream. Their classificiation is based on the associated apolipoproteins and the content of cholesterol, TGs and phospholipids. The combined protein and lipid content define the buoyant density of the particle and separate the lipoproteins into chylomicrons (CM), CM remnants, very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL), LDL and HDL. Lipoprotein particles consist of a hydrophobic lipid core consisting of cholesteryl esters and TGs, surrounded by a hydrophilic membrane of phospholipids, free cholesterol and apolipoproteins. (Hegele 2009).
CMs are formed in enterocytes in the small intestine. They transport dietary TGs and cholesterol to peripheral tissues. CM remnants are formed after lipoprotein lipase (LPL)-mediated lipolysis and are taken up by the liver via the LDLR and low-density lipoprotein related protein 1 (LRP-1). (Hussain 2014). VLDL particles are synthesized by the liver and consist mainly of TGs and cholesterol and contain a single ApoB100 molecule. Similiar to CMs their TGs are hydrolyzed by LPL in peripheral tissues to release fatty acids (FAs). This release of TGs leads to the formation of VLDL remnants or IDL. These can be removed from the circulation via binding of ApoE to hepatic LDLR or LRP1 but while clearance of CM remnants is efficient, most of the TGs in IDL are further hydrolyzed by hepatic lipase (HL). Also, the exchangeable apolipoproteins of IDL are transferred from the IDL to other lipoproteins leading to the formation of cholesterol ester (CE)-rich LDL particles.
(Nakajima et al. 2011)
HDL is formed via multiple steps after synthesis of ApoAI by the liver and intestine. It then accumulates cholesterol and phospholipids (PLs) mediated by ABCA1 to eventually form mature HDL. Cholesterol in HDL particles are esterified to CEs by lecithin:cholesterol acyltransferase (LCAT). (Dominiczak and Caslake
Three main pathways are responsible for the production and transport of lipids within the body; the exogenous pathway, the endogenous pathway, and the pathway of reverse cholesterol transport.
2.4.2 Exogenous pathway
The exogenous pathway mediates the efficient transfer of dietary FAs to muscle and adipose tissue for use in energy metabolism and storage. Dietary TGs are emulsified by bile and hydrolyzed by pancreatic lipase. Released FAs are transported from the intestinal lumen to enterocytes where they are re-esterified by monoacylgycerol acyltransferase (MGAT) and diacylglycerolacyltransferase (DGAT) to form TGs.
Nascent CMs are assembled from TGs, cholesteryl esters and ApoB48 with the help of microsomal triglyceride transfer protein (MTP). They aquire Apo-AIV in the lipidation step and Apo-A1 and Apo-AII when processed in the Golgi. After being transported into the lacteals which are small lymphatic vessels, they enter the circulation from the thoracic duct where they further aquire Apo-CII, Apo-CIII and Apo-E from other lipoproteins such as HDL. (Hussain 2014).
LPL expressed from muscle and adipose tissue, and transported to the luminal surface of capillaries, then hydrolyzes the TGs carried in the CMs to form FAs which are then taken up by the muscle cells and adipocytes with the help of fatty-acid transport proteins (FATPs) and CD36 (Olivecrona 2016). The metabolism of CMs leads to a decrease in their size due to the loss of TGs, and formation of CM remnants.
Subsequently, these are efficiently cleared by hepatic LDLR, through receptor binding of the ApoE on the surface of the CM remnants. CM remnants are also taken up by VLDLR and LRP-1 which are able to bind both ApoB and ApoE. The size and composition of the CM particle is determined by the amount and type of fat ingested and absorbed by the intestine. Larger particles are produced when more fat is absorbed.
2.4.3 Endogenous pathway
The endogenous pathway refers to the synthesis and subsequent metabolism of VLDL to ultimately form LDL particles which are taken up by the liver via LDLR.
TGs and CEs in the liver are transferred by MTP to newly synthesized ApoB-100 to form VLDL. The rate-limiting step in this process is the availability of TGs which determines whether the synthesized ApoB is secreted or degraded. (Cohen and Fisher 2013). VLDL particles are transported to the peripheral tissues where, similar to CM, LPL hydrolyzes the TGs to release FAs to be taken up by the peripheral tissues. The formed IDL particle (VLDL remnant) is enriched in CE and acquires ApoE from HDL particles. Once the particle is small enough, it passess through fenestrae in the liver endothelium to the space of Disse where it is further modified before taken up by hepatic LDLR, LRP-1 and heparin sulfate proteoglycans (HSPG).
Even though ApoE is recognized by hepatic LDLR, the uptake of IDL is not as efficient as uptake of CM remnants, and only about 50 % of IDL is cleared. The
remaining TGs in IDL are hydrolysed by hepatic lipase (HL) and exchangeable apolipoproteins are transferred from IDL to other lipoproteins, leading to the formation of LDL. (Cohen and Fisher 2013).
Figure 2. The exogenous and endogenous pathways of lipoprotein metabolism. CMs are formed from dietary lipids, transported via the thoracic duct to the circulation and peripheral tissues where lipolysis by LPL hydrolyzes the TGs to release FFAs to be taken up by muscle and adipose tissue. Remnants are cleared via hepatic LDLR and LRP-1. VLDL is secreted by the liver to peripheral tissues where lipolysis by LPL similiar to CMs happens. The formed IDL is either taken up by the LDLR in the liver or further hydrolysed by HL to form LDL. Picture modified from (Lusis, Fogelman, and Fonarow 2004).
2.4.4 Low-density lipoprotein receptor
LDLR is a transmembrane protein and belongs to the low-density lipoprotein receptor gene family. It is encoded by the ldlr gene on chromosome 19p13.1-13.3 (Francke, Brown, and Goldstein 1984). Mutations in the ldlr gene lead to familial hypercholesterolemia as discussed in chapter 2.3. LDLR is expressed ubiquitously
homology region, a region containing O-linked sugars, a transmembrane domain and a C-terminal cytosolic domain (Südhof et al. 1985). The ligand binding domain consists of seven cysteine-rich repeats called LDLR class A (LA) repeats. This domain is crucial for binding of both LDL via ApoB and VLDL via ApoE. The EGF-precursor homology domain consists of three EGF-like repeats. Deletion of this domain does not affect VLDL binding but instead prevents the acid-dependent dissociation of ligand in endosomes. The function of the O-linked glycan region does not affect ligand binding, endocytosis or degradation, and its clear role remains to be elucidated. The hydrophobic 24 amino acid transmembrane domain anchors the LDLR in the lipid membrane while the cytosolic domain is responsible for recruitment to clathrin coated pits, endocytosis and intracellular transport of the ligand-receptor complex.
The LDLR pathway is shown in Figure 3b. Synthesis of the LDLR polypeptide from mRNA takes place on the ribosomes of the rough endoplasmic reticulum where also initial glycosylation of the polypeptide takes place (Goldstein, Hobbs, and Brown 2001). It is then transported to the Golgi complex where glycosylation is completed. The receptor is then transported to the cell surface where the receptors accumulate in clathrin-coated pits (Anderson, Brown, and Goldstein 1977; Anderson, Goldstein, and Brown 1976). LDLR binds LDL in the circulation, after which the ligand-receptor complex is internalized in coated endocytic vesicles. In the cell, uncoating of the vesicle takes place and the ligand-receptor complex is transported to the endosomes where the acidic environment facilitates the release of the ligand.
The receptor is rapidly recycled back to the cell surface, and the LDL is degraded in lysosomes by acid hydrolases and proteases to release unesterified cholesterol which is used in the production of steroid-homones, cell-membranes and bile-acids (Brown, Anderson, and Goldstein 1983).
2.4.5 Metabolism of Low-density lipoproteins
Plasma LDL concentration is determined by the rate of production and clearance of LDL which is partly determined by the number of LDL receptors in the liver. The production of LDL from IDL is partially dependent on LDLR activity. The liver accounts for about 70% of the clearance of LDL with the rest being taken up by extra-hepatic tissues (Goldstein, Hobbs, and Brown 2001). The most important regulator of LDLR expression in the liver is the cholesterol content of hepatocytes via sterol regulatory element binding proteins (SREPBs) (Horton, Goldstein, and Brown 2002).
These are transcription factors that mediate the expression of LDLR and other key genes involved in cholesterol and fatty acid metabolism. When there is a sufficient amount of cholesterol in the cell, SREBPs reside in the endoplasmic reticulum as inactive forms. They are activated by a decrease in intracellular cholesterol, whereby they are transported from the ER to the golgi where proteases cleave SREBPs into active transcription factors which then translocate to the nucleus to stimulate the transcription of LDLR and other genes. (Horton, Goldstein, and Brown 2002). In addition, LDLR transcription is induced by oxidized sterols which activate LXR, a
nuclear hormone receptor that is also a transcription factor. (Zhang et al. 2012).
Furthermore, LDLR activity is regulated by PCSK9 which targets the receptor for degradation. Loss-of-function mutations in PCSK9 lead to increased LDLR activity and decreased LDL levels. This has led to the development a novel group of drugs, PSCK9 inhibitors, to treat severe hypercholesterolemia. (Bergeron et al. 2015).
LDLR-independent pathway is the sole means of clearance of LDL from the plasma of hoFH patients with null-alleles. LDL can be taken up by a non-specific mechanism resembling phagocytosis. However, this is inefficient and only accounts for a minor part of the clearance of LDL from the plasma.
Figure 3. Structure and domains of LDL receptor (a) and metabolism of LDL and functional consequences of LDLR mutations (b). LDLR is synthesized at the endoplasmic reticulum (ER), transported to the Golgi where it is further processed. Mature LDLR is transported to the plasma membrane where it binds LDL particles via ApoB. The LDLR/LDL complex is endocytosed from clathrin-coated pits. In the cell, the LDL is targeted to lysosomal degradation whereas the receptor is recycled to the cell surface for multiple rounds of receptor-mediated endocytosis. LDLR mutations are classified into five functional classes based on the phenotypic behaviour of the mutant protein: Class I mutations affect the synthesis of the receptor (null-alleles) (1); class II: defective transport to Golgi or plasma membrane (2); class III binding-deficient (3); class IV impaired endocytosis (4) and class V recycling deficient (5).
Modified from (Benito-Vicente et al. 2018)
2.4.6 Reverse cholesterol transport
Reverse-cholesterol transport (RCT) is the process by which cholesterol is transported via HDL, from the periphery and lesions to the liver for excretion. It involves the efflux of free cholesterol (FC) from macrophages by ABCA1 to lipid-poor Apo-A1 secreted from the liver and intestinal enterocytes to form discoidal nascent HDL (nHDL) which contains FC, PL and ApoAI. Then the enzyme LCAT catalyzes the conversion of FC to CE to eventually form the spherical, mature HDL particles. (Cohen and Fisher 2013). The liver takes up FC and CE from the mature HDL particle via SR-BI leaving behind a remnant HDL and lipid-free ApoAI that returns for another cycle of RCT (Cohen and Fisher 2013). However, based on current evidence this traditional model of RCT has been suggested to be a minor mechanism and a ‘revised’ model has been proposed where LCAT plays a minor role in RCT.
This newer model suggests that most HDL-FC and nHDL-FC transfer to the liver rapidly independent of LCAT activity. (Gillard et al. 2018).