# 17.1: Digestion, Mobilization, and Transport of Fats

### Introduction

In this chapter we will discuss the breakdown of fats to produce useful energy for biosynthesis and for ATP production. Most of the available chemical energy stored in fats is in the form of highly reduced fatty acids. Clearly, one form of fatty acid-containing lipids come from our diet, which includes triacylglycerides (TAGs) and membrane lipids. Fatty acids, mostly in the form of TAGs, are moved in the circulation in the form of large lipid-carrying vesicles called lipoproteins. The lipids can be imported into cells for storage and energy use.

Another source of fatty acids comes from those synthesized within cells from the small molecule acetyl-CoA. Fatty acids are synthesized by an enzyme complex call fatty acid synthase. This enzyme is found most prevalently in adipose (fat) tissue and in the liver. In addition it is significantly expressed in the brain, lung and mammary gland.

TAGs, stored in lipid droplets, are found in most cells.. The major tissue for TAG storage is adipose (fat) tissue, whose volume consist mostly of lipid droplet(s). Given the large mass of muscle tissue, there is also a considerable amount of TAGs stored as small lipid droplets in muscle cells. However, skeletal muscle cells don't synthesize fatty acids. They have the genes for fatty acid synthase but do not transcribe it into RNA so no enzyme is made. They can however import them for catabolism.  Muscle TAGs can be oxidized for energy, especially during endurance excise .

TAGs are also stored in the liver in lipid droplets. The liver also assembles lipoproteins, which are released by the liver. Excess TAGs are stored in the liver in various disease including alcoholism and also in nonalcoholic fatty liver disease (NAFLD), which can progress into nonalcoholic steatohepatitis (NASH), a much worse disease.

There are two major forms of triacylglyeride-storing fat tissues, white adipose tissue (WAT) and brown adipose tissue (BAT). The more abundant WAT store triacylglycerides in one large lipid droplet in the cell and release fatty acid in processes control by the hormones insulin and epinephrine. This simple role can mask the fact that adipose tissue is a major player in the endocrine system and is involved in cells signaling and systematic control of metabolism. Adipose tissue releases the key hormone leptin and adipisin, which in analogy to the hormones and signaling agents released by immune cells (cytokines, lymphokines), can be called adipokines. They also secrete other adipokines including tumor necrosis factor α (TNF-α), adiponectin and resistin.

In contrast, BAT are specialized not to store and release fatty acids. but rather to oxidize fatty acids in ways that maximize heat production, preventing hypothermia. The have multiple smaller lipid droplets, displaying a larger surface area for lipolyis, the hydrolytic cleavage of fatty acids from the TAGs. A particular mitochondrial protein, uncoupling protein 1 (UCP1), is expressed in brown but not white adipocytes, allows a "futile" metabolic cycle leading to dissipation of heat instead of ATP synthesis. The relative abundance of white and brown adipocytes is critical in diseases like obesity and type 2 diabetes. BAT tissue is especially important in small animals (and in newborns) for themoregulation. For smaller organisms, the surface area to volume ratio is greater than the ratio for larger animals, allowing more heat loss. The ratio of surface area AS per volume V for a sphere is given by:

$\dfrac{\mathrm{SA}_{\text {sphere }}}{\mathrm{V}_{\text {sphere }}}=\dfrac{4 \pi \mathrm{r}^{2}}{\left(\dfrac{4}{3}\right) \pi \mathrm{r}^{3}}$

Let's assume an average large adipocyte is a sphere of diameter 100 uM. Compare this to a large sphere with a 100 time greater diameter (10,000 uM).  The smaller sphere has a 1/100 of the diameter but a surface area/volume ratio 100 times greater than the large sphere.

An intermediate type of fat tissue consists of "bright" adipocytes. White adipocytes can be coaxed to differentiate to bright and brown cells, which could be a treatment for obesity.

In this chapter section, we will follow the fate of fatty acids from dietary lipids which are cleaved from TAGs, loaded into chylomicrons, lipoproteins assembled in the small intestine, secreted into the circulation and taken up by the liver. The liver can store the incoming fatty acids in TAGs or release them back into the circulation in the form of another lipoprotein, very low density lipoproteins (VLDL). Circulating VLDL can exchange lipids with other circulating lipoproteins. Lipoproteins delivery fatty acids to cells after interaction with the cell surface of target cells and either cleavage of TAGs by cell membrane-associated enzyme lipase, followed by fatty acid uptake, or by endocytosis of lipoproteins into the cells.

### Lipoproteins

Before we look at more detail at the individual steps in lipids processing, let's look at the different lipoproteins, the large vesicular structures that allow the transport of fats, very insoluble molecules, in the circulation. Unlike normal liposomes or vesicles that have a lipid bilayer surrounding an interior aqueous compartment, lipoproteins have only a single monolayer of phosphoplipids encapsulating a nonaqueous interior filled with TAGs, cholesterol and cholesterol esters. The protein part of the lipoprotein consists of one or several proteins bound on the outside of the particle. The proteins help solubilize the lipoprotein, confine its size and prevent aggregation of the lipoproteins, which would be a health risk. The structure of a typical lipoprotein is shown in Figure $$\PageIndex{1}$$.

Lipoproteins are classified based on density. The lowest density chylomicrons are the largest with the most lipids (mostly TAGs) in their interior compartment. Very large density lipoproteins (VLDL), intermediate density (IDL), low density (LDL) and high density (HDL) have decreasing size, less encapsulated lipid and increasing density. The relative sizes are shown in Figure $$\PageIndex{2}$$.

Lipoproteins (with the exception of chylomicrons) could be classified as nanoparticles, which typically vary in size from 1-100 nm. Larger lipoproteins as well as chylomicrons form emulsions in the blood, much as milk (also cloudy) is an emulsion of lipid/protein particles. The serum of people with high levels of lipids (hyperlipidemia) can actual look milky white, especially after eating foods rich in TAGs, when levels of chylomicrons are very high. Figure $$\PageIndex{3}$$ shows blood of a patient with hyperlipidemia after addition of EDTA (which binds Ca2+ and prevents clotting) that has settled (without centrifugation). The milky white plasma on top (lower density) most likely has high concentrations of chylomicrons and/or LDL. The lower layer contains mostly red blood cells.

No x-ray structures of lipoproteins are available. However a structure of a nascent HDL particle (3k2s) has been determined by small angle neutron scattering. Figure $$\PageIndex{4}$$ below shows an interactive iCn3D model of it.

The major protein in HDL, a lipoprotein that protects against cardivascular disease, is apolipoproteinA-I (apoA-I).  Figure $$\PageIndex{4}$$ shows that it adopts an antiparallel double superhelix as it wraps around the nascent HDL.  The more hydrophobic surfaces of apo A-I are oriented inward allowing interactions with hydrophobic lipids in the core.  It is probably prototypical for nascent lipoproteins. It will give you an idea of how proteins wrap around the outside of the particle. Mature lipoproteins are most likely spherical in shape. This nascent HDL in the model contains 200 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholines (POPC) molecules, 20 cholesterols and a single copy of apolipoproteinA-I (apoA-I).

Figure $$\PageIndex{5}$$ below shows a cartoon image of VLDL, assembled from lipids synthesized/taken up by and released form the liver, and chylomicrons, assembled from dietary fats and released from enterocytes in the small intestine (size of the lipoproteins is not to scale). Note that VLDL has one copy of Apo B-100 while chylomicrons have one copy of Apo B-48.

All lipoproteins, except HDL, are a member of the Beta-lipoprotein family as they contain an apo-B protein. The liver synthesizes apo B100, which becomes a permanent part of VLDL (i.e it is not exchangeable with other lipoproteins) and its metabolic derivatives so any lipoprotein containing apo B100 arose form the liver. Other proteins on lipoproteins are exchangeable.  In contrast, enterocytes in the small intestine produce apo B-48 (48% the size of apo B100) so this protein marks the lipoproteins (chylomicrons and chylomicron remnants) that were assembled in the small intestine. Apo B100 has over 4500 amino acids and a molecular mass of 555K.  The gene for the intestinal apo B48 is the same as for apo B100 except that it has a premature stop codon which leads to the shorter truncated apo B-48.

The apoproteins bind to specific receptors on cells which may allow uptake of the lipoprotein. For example, the LDL receptors binds to the apo B-100 protein on a region removed from the apo B48 protein of chylomicrons.  It also binds ApoE, which is found mostly predominately on HDL an VLDL but some is present in LDL.  The LDL receptors has also been called the ApoB/ApoE receptor.

Over 90% of the apoB-containing particles in circulation are LDL.  In addition, chylomicrons are present in circulation only after eating.  Some apoproteins can act cofactors and inhibitors for liproprotein processing.

Lipoproteins and Cardiovascular Risk

High concentrations of LDL are associated with increased cardiovasclar risk.  Chylomicron levels, given their transient and lower concentration levels, do not pose a health risk unless the enzyme required to removed fatty acids from them, lipoprotein lipase, is missing or defective, or if another apoprotein component, apo CII, which mediates the interaction with lipoprotein lipase, is missing.  LDL-C (a term used to describe the total cholesterol in LDL partricles which is routinely measured in clinical labs) can be lowed by a health diet centered around plant food). Drugs like statins, which decrease endogenous cholesterol synthesis, also remarkable lower LDL levels and decrease cardiovascular risk.

However, another protein, lipoprotein, also called Lp(a) or LP little a is an independent cardiovascular risk factor. It's blood concentration are regulated by genetics and not by diet. These particles contain, in addition to apo B100, apo (a), a protein that has a very unique repeating structure (up to 40 times) called a kringle, which is also found in some proteins involved in the blood coagulation system.  People whose genes encode apo (a) with fewest number of  kringles express lots of that protein and their Lp(a) particles are smaller. This confers a greater cardiovascular risk compared to those expressing proteins with a large number of kringle domains. Figure $$\PageIndex{6}$$ below (from Amgen) shows models of Lp(a) with different numbers of repeating kringle (kinked) domains.

From a biochemical perspective, it is interesting to explore the differences in apolipoprotein binding to a single-leaflet encapsulated lipid nanoparticles compared to the interacction of peripheral and integral membrane proteins with intact bilayers (which we studied in Chapter 12.1).  As mentioned above, the more nonpolar surfaces of apo AI in HDL are oriented inward toward the nonpolar lipid core. Presumably, apo B proteins in chylomicrons and LDL also wrap around the entire lipid surface.

The major organizing scafflolding protein of HDL is apoA-I (see iCn3D model above), It presumably plays a role similar to apoB in chylomicrons and LDL, but it is exchangeable. (Note: ApoA-I is also found in chylomicrons.) It is also a cofactor for the enyzme lecithin:cholesterol acyl transferase (LCAT), which effectively converts free cholesterol in the single bilayer into esterified cholesterol esters within HDL. In its apo-form, it also interacts with the cell surface transporter ATP-binding cassette A1 (ABCA1), which plays a role in the assembly of HDLparticles.  HDL also has apo C and apo E proteins, all of which are exchangeable.

It has been difficult to determine the structure of either the full free or bound apoproteins, probably because they present hydrophobic surfaces that promote self-association and aggregation. Apolipoproteins in the A, C and E class have repeating amphiphilic helices which presumably imbed to some degree in the lipid particles.  In addition, the proteins have significant disorder and can adopt many bound conformations.  Hence it's very difficult to predict the 3D structure of a membrane- or lipoprotein-bound protein using structural prediction programs such as RoseTTAFold.  It's not as simple as simply inputting the linear sequence of a peptide or protein and producing a helical wheel diagram like we showed in Chapter x.xx.

The exchangeable apoliproteins have similar genetic sequences (four exons and three introns), as well as similar amino acids sequences.  They have 11-mer amino acid tandem repeat and some (A-I, A-IV) have 22-mer tandem repeats.  These repeats form amphiphilic helices as determined by sequence analysis. The first amino acid in the amphiphilic helix often has a positive charged amino acid and a negative one is often found in the middle. Proline, a helix breaker, is often, but not always found between the helices.

Figure $$\PageIndex{7}$$ belows shows the primary sequence of apoA-I.  An 11-mer repeat is shown in yellow highlight.  The other highlighted stretches (different colors) are 22-mer repeats.  Note that the repeats are not of identical sequences but rather of sequences that can form amphiphilic helices (i.e. secondary structure repeats).

Figure $$\PageIndex{7}$$.belows shows the primary sequence of apoA-I.  An 11-mer repeat is shown in yellow highlight.  The other highlighted stretches (different colors) are 22-mer repeats.  Note that the repeats are not of identical sequences but rather of sequences that can form amphiphilic helices (i.e. secondary structure repeats).

The bottom part of Figure $$\PageIndex{7}$$shows a helical wheel projection (using Heliquest) of the red-highlighted 22-mer repeat.  The arrow shows the hydrophobic moment with the arrow head pointing to the more nonpolar face.  The particular amphiphilic helix shown may or may not facilitate binding of the bound conformation of the protein.

It follows that the relative areas of the hydrophilic and hydrophobic faces in the amphipathic helixes influence the lipid-associating properties of the exchangeable apolipoproteins. Another factor that might influence the lipid binding ability of exchangeable apolipoproteins and which has not been studied in detail so far is the arrangement of tandem repeating amphipathic helixes with respect to one another.

Actual amphiphilic helices would bind to the membrane in a parallel fashion with the nonpolar face anchoring the protein to the lipid surface.  Other experimental techniques are used to determine how a peptide or protein than can form amphiphilic helices actually interact with the lipid surface. These  include site-directed mutagenesis studies coupled with spectroscopic (CD, fluorescence) and binding assay methods (using with liposomes).

The properties of an membrane-bound amphiphilic helices is affected by the exact size and distribution of the polar/charged and nonpolar side chains.  On binding, they sense or cause membrane curvature, interact with specific lipid and stabilize specific membrane conformations (such as spherical for lipoproteins).  Figure $$\PageIndex{8}$$ below shown how different proteins with amphiphilic membranes interact with membrane surfaces.

Figure $$\PageIndex{8}$$: Interactions of amphiphilic helices with membranes

Manuel Giménez-Andrés et al. Biomolecules. 2018 Sep; 8(3): 45.  doi: 10.3390/biom8030045.   Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

A key point to note is the large conformational changes that occur as the protein or parts of it goes from the free, more disordered state, to the bound state with lipid-associating amphiphilic helices.  The following proteins are depicted in the figure.

(a) The peroxisomal membrane protein Pex11 amphiphilic helix causes a distortion of the membrane;

(b)  ARF1 is a small G protein in which only the GTP form localizes and binds through an amphiphilic helix to the membrane;

(c) The ALPS motif of the golgin GMAP-210 binds to only highly curved vesicles;

(d) The yeast transcriptional repressor Opi1 binds to endoplasmic reticulum (ER) membrane in part through an amphiphilic helix;

(e) The heat shock protein Hsp12 has a long amphiphilc helix which helps stabilize the membrane;

(f) The extremely long amphiphilic helix of perilipin 4 coats lipid droplets and stabilizes even if there is a lack of phospholipids.

### Dietary uptake and release into the circulation

Now how are the lipid nanoparticles assembled? We'll start with dietary lipids in the form of TAGs, glycerophospholipids and cholesterol ester. The figure below show key steps which are described in Figure $$\PageIndex{9}$$ below

Here are some key steps depicted in the figure:

• hydrolysis (lipolysis) of TAGs by pancreatic lipase, cholesterol esters (CE) by cholesterol esterase, and glyerophospholipids (GP)by phospholipase A2 in the lumen of the intestine. These enyzmes interact at the interface of the lipid substrates and aqueous surroundings;
• the resulting products,which include free fatty acids (FA), 2-monoglycerol (MG), free cholesterol (FC) and lyso-glycerolphospholipids (lyso-GP), aggregate with the help of bile salts to form emulsions (like oil drops in water), which can be taken up by diffusion or possibly endocytosis when present in high amounts. Alternatively, membrane transporters (like FABPs and others proteins) can move them into the cell by facilitated diffusion;
• cytoplasmic transporters like fatty acid binding proteins move the lipolysis product to the ER where free fatty acids are reesterfied. The enzymes involved include mono- and diacylglycerol acyltransferases (MGAT, DGAT) and sterol O-acyltransferase 2, also known as acyl-coenzyme A:cholesterol acyltransferase (ACAT-2). Multiple enzymes are involved in the resynthesis of glycerophospholipids);
• Apo B-48 is synthesized by ribosomes bound to the ER and interacts with a heterodimer of microsomal triglyceride transfer protein large subunit two (MTP) and protein disulfide isomerase (PDI). This facilitates folding of apo B48 and loading of lipids using MTP into pre-chylomicrons;
• pre-chylomicron vesicles move to the Golgi with the help of Sar1b, a small G-protein (and GTPase) where the particle assembles to the full chylomicron, which is released from the cells as the mature large lipid nanoparticle.

An intriguing feature of lipases is that they work at the interface between the aqueous and nonaqueous (in this case lipid nanoparticle) environments. Let's briefly consider the mechanism of hydrolysis of a TAGs by equine pancreatic mechanism. This enzyme utilizes the same mechanism we have seen earlier for the hydrolysis of a peptide bond by serine proteases. A catalytic triad of Asp 176, His 263 and Ser 152 as a nucleophilic catalyst is shown in the partial reaction displayed in Figure $$\PageIndex{10}$$.

Figure $$\PageIndex{10}$$:

An acyl-Ser intermediate forms in step B (above), after the collapse of a oxyanion intermediate in step A, to form the product diacylglyericde. In the second half of the reaction (not shown completely), water, in a hydrolysis reaction, cleaves the acyl-Ser intermediate to reform the active enzyme as it release the free fatty acid, R3CO2H. Other lipases also employ the same catalytic triad.

Figure $$\PageIndex{11}$$ below shows an expanded diagram showing the flow and fate of lipoproteins.

Chylomicrons interact with lipoprotein lipase (LPL), which also uses a Asp-His-Ser catalytic triad, to cleave fatty acid esters, which allows delivery of free fatty acids to adipose cell.  The adipocytes can also undergo de novo fatty acid synthesis.  Fatty acids (FA) can also be produced by lipase-mediated lipolysis of stored TGs.  Any of these free fatty acids (FA) in the adipocyte have two fates.  They can be reesterified to glycerol to form TAGs (TG in the figure), or be exported from the cell and bind to a plasma carrier protein and transported to the liver, where it can be taken up by a variety of membrane proteins importers shown in the figure in yellow boxes.  There, as in adipose cells, they can be reesterified to form TAG stores, which can then be packaged into VLDL particles for export.  The fatty acid delivered (or synthesized) could also be used for energy production through the citric acid cycle and oxidative phosphorylation.

VLDL in circulation can undergo lipolyis by lipoprotein lipase to produce fatty acids for uptake in "extrahepatic" tissue (bottom right of diagram).  As fats are removed from VLDL, its density increase as it forms IDL and LDL, which could be considered VLDL "remnants".  VLDL is very enriched in TAGs, but after metabolic processing, the resulting LDL is depleted in TAGs and enriched in cholesterol/cholesterol esters.  LDL  (not shown in the above figure) can be taken up (endocytosed) by liver and other cells after binding to LDL receptors, which recognize apo-B100 and and other apoproteins.  The allows delivery of predominately cholesterol and cholesterol esters to tissues.

How do adipocytes and hepatocytes determine if free fatty acids should be esterifed for storage or released for energy use by other tissue?  We'll discuss that in a subsequent section but the short answer is that in healthy fasting and exercise states, hormones (glucagon, epinephrine) will activate lipolysis in liver and adipose cells, while in the fed state, insulin will promote storage of fatty acids as triacyglycerides.

Adipose cells don't assemble and release lipoproteins.  Instead they release free fatty acids in the circulation which are carried by albumin, the major serum/plasma protein in the blood. The iCn3D Figure $$\PageIndex{12}$$ below shows an interactive iCn3D model  of the complex of human serum albumin (HSA) binding seven 20:4Δ5,8,11,14 - arachidonic acids (1gnj ).

Figure $$\PageIndex{12}$$ Human Serum Albumin/Arachidonic Acid complex (1gnj).  Click the figure for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/icn3d/share.html?WL7BxK4B23m2p3Z19 (Copyright; author via source)

Given the multiple binding sites for fatty acids in albumin, it should come as no surprise that albumin also binds a host of small drugs, including medicinal drugs and toxins such as warfarin (blood thinner), diazepam, ibuprofen, indomethacin, and amantadine, appear to bind preferentially at two major drug binding sites.  This binding is probably helpful in delivering drug through the circulation but potentially not useful if they aren't delivered to appropriate target tissue.

Remember  when we discuss the structure of micelles which are spherical assemblies of single chain amphiphiles, which act as detergents.  Oil from your clothes can enter the nonpolar interior of the detergent micelle and effectively solubilize the nonpolar molecule in the micelle, which are effectively nanoparticles with a diameter of 5-15 nm.  You should hence not be surprised to discover that lipoproteins can also carry fat-soluble vitamins, steroid-like endocrine-disrupting substances and drugs.

## Lipoprotein lipase

The enyzme that breaks now TAGs in circulating chylomicrons and VLDL is lipoprotein lipase (LPL). It is a soluble protein secreted by  adipocyte and muscle cells, but is made by many cell types.  Most is found outside of cells.  It works at the luminal side of blood vessel endothelial cells and is recruited to that membrane surface by binding to the glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) as well as the proteoglycan heparan sulfate at the cell surface.

What is so interesting is that GPIHBP1 is only synthesized by endothelial cells. When lipoprotein lipase is secreted from cells, it binds to extracellular matrix heparan sulfate but dissociates on cleavage of heparan sulfate by heparanases. GPIHBP1 is highly acidic with an intrinsically disordered N-terminal domain containing a sulfated tyrosine and is highly enriched in glutamates and aspartates, which are often sequential in the sequence.  Here is the single letter sequence for amino acids 25-50 of the human version of GPIHBP1:  EEEEEDEDHGPDDYDEEDEDEVEEEE.  This sequence would have similar electrostatic and binding properties to the highly negatively charged heparan sulfate to which it also binds.

LPL aslo binds Ca2+ which stabilize the active dimeric form of the protein.  Its enzymatic activity is activated by apoC-II. Like pancreatic lipase, it employs a Ser-132, Asp-156, and His-241 triad in its hydrolytic action on TAGs/

Figure $$\PageIndex{13}$$l below show an interactive iCn3D model of LPL in complex with GPIHBP1, shown in brown (6E7K).  The calcium ion is shown (grey spacefill) as well as the catalytic triad (labeled, sticks, CPK colors). The highly negatively charged stretch of amino acids in GPIHBP1 was not present in the crystal structure.

The calcium ion is shown (grey spacefill) as well as the catalytic triad (labeled, sticks, atom colors). The HPIHBP1 protein is shown in brown.  The highly negatively charged stretch of amino acids in GPIHBP1 was not present in the crystal structure.

Kumari, A.; Kristensen, K.K.; Ploug, M.;Winther, A.-M.L. The Importance of Lipoprotein Lipase Regulation in Atherosclerosis. Biomedicines 2021, 9, 782. https://

### LDL:  Receptor and Uptake

Liproteins are taken up into cells through receptor-mediated processes. Let's focus generically on the LDL receptor, the major carrier of cholesterol, given its role in cardiovascular disease.  It is found in the cell membranes in most tissues.  It is has many domain repeats, as illustrated in the Figure $$\PageIndex{14}$$ below calculated by SMART.

Figure $$\PageIndex{14$$: Domain strucuture of the LDL receptor

They include the N-terminal region cysteine-rich LDLa domains, which bind LDL, epidermal growth factor domains, LY (or LDLb) domains, and a transmembrane domain (blue rectangle).

Figure $$\PageIndex{15}$$ belows shows an interactive iCn3D model of the extracellular domain of the LDL receptor (1n7d).

Figure $$\PageIndex{15}$$:  Click the figure for a popup model or use this external link: https://structure.ncbi.nlm.nih.gov/i...aT7M6ti5iDAbj8 (Copyright; author via source)

Four tandem LY (LDLb) domains are shown in cyan, LDLa domains are shown in magenta and the EGF domain is shown in dark orange. Glycans are shown in symbolic nomenclature for glycans. Zoom into the structure to see the two disulfide bonds in each LDLa domain as well as the Ca2+ ions that stabilize the domains.

LDL binds binds its receptor at a broad binding interface with multiple LDLa domains.  This may account for the fact the lipid nanparticles with apo B100 or apo E can bind to it. The binding triggers a series of signaling events that leads to internalization  by endocytosis of the receptor in pits coated with the protein chlathrin.  These eventually fuse with lysosomes where they are degraded and cholesterol delivered to the cell.  The steps are described in Figure $$\PageIndex{16}$$ below.

Figure $$\PageIndex{16}$$:

Gilles Lambert et al. J. Lipid Research, 53, 2515-2524 (2012) DOI:https://doi.org/10.1194/jlr.R026658.  Creative Commons Attribution (CC BY 4.0) |

The LDL receptor survives lysosomal degradation and along with newly made receptors is delivered to the plasma membrane in a continuous fashion.  A key protein, proprotein convertase subtilisin kexin type 9 (PCSK9), a serine protease secreted by the liver, promotes enyzmatic degradation of the receptor and prevents its recycling to the membrane.  It also binds to VLDLR and apolipoprotein E receptors and promotes their degradation as well. Its action reduces LDL clearance from the blood, increasing cardiovascular risk, so inhibitors of its action might be potent drugs to decrease circulating LDL.

The LDL receptor is just one member in the LDL receptor family.  Other members of the family are illustrated in Figure $$\PageIndex{17}$$ below.

Figure $$\PageIndex{17}$$:  LDL Receptor Family

Adapted from Theresa Pohlkamp et alFront. Mol. Neurosci., 01 March 2017 | https://doi.org/10.3389/fnmol.2017.00054.   Creative Commons Attribution License (CC BY)

These include the LDL receptor (abbreviated Ldlr in Figure $$\PageIndex{17}$$, as well as the VLDL receptor (Vldlr), apolipoprotein E2 receptor (Apoe2) and LDL receptor related proteins (Lrp)1-4.  These also have a NPxY-motif (asterisk in the cytoplasmic domain) and a YWTD/β-propeller domain.  Given the similarity in domain structure for the LDL family of receptors, the conformational flexibility of the apolipoproteins (at least free in solution), and similar structures for the exchangeable apolipoproteins, it shouldn't be surprising the the LDL receptor would interact with different classes of lipoproteins, albeit with different affinities.

As mentioned previously, apoE is found most abundantly on HDL and on VLDL/chylomicrons and their remnants.  It serve as a ligand which bind to members of the LDL receptors family (remember that LDL generally binds  the LDL receptor through apo B100).

Apo E and Alzheimer

Apoliprotein E has three major variants (alleles) named ε2, ε3, and ε4 (also called ApoE2, 3 and 4).  ApoE3 is most prevalent.  ApoE4 is found in only 15% of people but more than 50% with Alzheimers Disease (AD), so its a risk factor for this disease.  AD affects the brain, which also contains up to 30% of the cholesterol in the body, so aberrations to cholesterol transport and uptake in the brain are not unexpected in neurodegenerative diseases like AD.

ApoE is secreted by brain microglia (immune) cells and astrocytes (specialized glial cells).  It assembles lipids into lipoproteins (HDL-like) and becomes the major vehicle for binding to and import into neurons in a process initiated by the apoE receptor.  The major apoE receptor for clearance of lipoproteins in the brain is sortilin (SorLA in Figure $$\PageIndex{17}$$).

AD is characterized by the accumulation of a toxic amyloid prion protein called aymeloid beta (Aβ).  It is derived from selective but abnormal proteolysis of the neural integral membrane protein amyloid precursor protein (APP).  Aβ aggregates to form insoluble neurotoxic extracellular Aβ amyloid plaques.  The process in normal and diseased cells is shown in Figure $$\PageIndex{18}$$ below.

Figure    $$\PageIndex{18}$$ Processing of amyloid precursor protein (APP).

Emma Ristori et al. Front. Physiol., 27 August 2020 | https://doi.org/10.3389/fphys.2020.01056.   Creative Commons Attribution License (CC BY).

The figure show normal (left) and aberrant processing of APP and the family of proteases (secretases) involved.

While the LDLR doesn't appear to bind to APP or influences it proteolytic processing, it does bind Aβ.  LRP1 is much bigger than LDLR, binds a multitude of ligands and can be cleaved the same enzymes as APP.   Its expressed in the liver and especially in the brain and can regulate the removal of Aβ.

Immune cells in the brain, called microglial, remove Aβ plaques (which are extracellular) by phagocytosis.  ApoE4 increases the inflammatory response (as measured by cytokine release) of the microglia (a good thing if the responses prevents infection or rids Aβ plaques), but also inhibits their ability to phagocytose the Aβ plaques and their metabolic activity.

#### Scavenger Receptors

Patients with homozygous familial hypercholesterolemia (FH) have very high levels of LDL derived from defects in LDLreceptor  binding and update.  Patients display fatty acids streaks under vessel endothelial cells which morph into calcified plaques and lesions filled with fat.  Monocytes/macrophages, which migrate to sites of vascular injury, take up LDL and eventually differentiate inton foam cells filled with lipid.  Somehow, they have receptors that can bind and internalize LDL when the "normal" LDL receptor can't.  Brown and Goldstein found that a specific chemical modification of LDL, acetylation, was necessary for rapid uptake of "modified" LDL into macrophage receptors.  These receptors are now called scavenger receptors (SRs).

There is a large family of scavenger receptors.  It consist of classes A-J proteeins that share functional but not sequence homology.  They are found on macrophages and endothelium.  They bind to and help remove "damage" signals including damage-associated molecular patterns (DAMPs) and chemical species chemically modified by reactive oxygen species. The ligands are often polyanions, end-stage glycans and extracellular matrix proteins. One such example is oxidized-LDL (produced in vivo or by chemical oxidative modification with malondialdehye), which binds to the same scavenger receptor,  SR-A1, also called Macrophage scavenger receptor type I, as acetylated-LDL.  Figure $$\PageIndex{19}$$ below shows the domain structure of the SR family.

Figure $$\PageIndex{19}$$:  Domain Structure of the Scavenger Receptor Family

SR-A1/MSR1 not only binds acetylated and oxidized LDL but also β-amyloid (), heat shock proteins (), and PAMPs from some bacteria and viruses.

It's very difficult, given the ever increasing amount of "omic" data (genomic, proteomic, lipidomics, interactomics, metabolics), for readers and authors alike, to conceptualize all of the possible combinations of interactions among biological molecules.  For visual learners and perhaps everyone else, its extremely useful to portray information on structures and interactions visually.  An example using STRING, a database of known and predicted protein interactions, for the domain structure and protein:protein interactions of SR-A1/MSR1,is shown in Figure $$\PageIndex{20}$$ below.

Figure $$\PageIndex{20}$$: Visualization of Domain Sructure and Interacitn Map of Macrophage Scavenger Receptor 1

Note the interactions with your now favorite apolipoproteins, apoB, apoE and apoA1. The right hand side of the figure also shows interactions with collagen alpha-2(IV) chain  (COL4A2), which is found in the extracellular matrix.

### HDL metabolism: The Good Cholesterol

High levels of LDL (and Lp(a)) pose a cardiovascular risk.  In contrast, high levels of HDL and apo A-I are cardioprotective. HDL is involved in "reverse" cholesterol transport as it is taken up by the liver and sent to the intestines for elimination from the body.

We have shown earlier in Figure 2 that HDL exists as many variants, reflecting the assembly and remodeling of HDL by enzymes and lipid transfer proteins.  Figure $$\PageIndex{21}$$ below shows the lifecyle of HDL.

Figure $$\PageIndex{21}$$:

Arnold von Eckardstein. Tachometer for Reverse Cholesterol Transport? August 2012. Journal of the American Heart Association 1(4):e003723.  DOI: 10.1161/JAHA.112.003723.  License CC BY 2.5/.  Open Access article under the terms of theCreative Commons Attribution Noncommercial License

Secreted apo A-1 accretes lipids in the circulation through transport and delivery of phospholipids and cholesterol from cell membranes by the ATP-binding cassette transporters (ABC) A1 and G1. Another protein, the scavenger receptor BI (SR-BI), a polytopic integral membrane protein, is also involved.  It acts as a receptor for a variety of "lipid" ligands including phospholipids, cholesterol esters and phosphatidylserine (an outer membrane marker for cell apoptosis) as well as lipoproteins such as HDL.

Other proteins are involved as well in both assembly but especially in remodeling HDL.  The lipolytic enyzmes lecithin-cholesterol acyltransferase (LCAT) removes a fatty acid from phospholipids and adds it to free cholesterol in the HDL to form cholesterol esters.  Two major lipid transfer proteins, phospholipid transfer protein (PLTP)  and cholesterol ester transfer protein (CETP), move lipids between HDLs and other lipoproteins. Cholesterol ester transport protein is made and secreted from the liver. It appears to exchange cholesterol esters from HDL for return of TAGs from VLDL.  Other enzymes (lipoprotein lipase and hepatic lipase are also involved in forming free fatty acids.

In the final step, HDL can deliver cholesterol ester and cholesterol to the liver through binding to liver scavenger receptor BI (SR-BI) mediated by apo A-I. The protein protein is expressed by the liver, adrenal gland, endothelial cells, macrophages and many other tissues.  It appears that the HDL is not primarily taken up the liver by endocystosis.  In contrast, LDL is taken up by endocytosis mediated by the LDL receptor. SR-BI facilitates transfer of cholesterol esters from bound HDL to the liver cell.

HDL and cardiovascular risk

Unlike with the widespread use of statins, which reduce LDL-C concentrations and clinically reduce cardiovascular disease risk, drugs (fibrates, niacin, inhibitors of cholesterol ester transferprotein - CETP), which raise HDL levels, don't seem to lead to significant decreases in cardiovascular risk.  The cholesterol delivered in excess to macrophages after update into endothelial cells can lead to the formation of foam cells under the endothelial layer, which are proinflammatory and convert in time to cholesterol plaques. In contrast, HDL-C does appear to have this direct atherogenic effect.