TG-Rich Lipoproteins and LPLs Flashcards
How is dietary fat absorbed?
Most dietary fat is found as TAG, which is a major source of energy (30-40%) for the body. This is absorbed by duodenal enterocytes as monoglycerides and free fatty acids, which are produced by the action of bile salts and pancreatic enzymes in the intestine.
What is the role of chylomicrons?
These provide an alternative source of fats and cholesterol to LDLs, as they eventually reach the bloodstream after secretion into the lymph vessels.
Instead of providing cells with TAG and cholesterol by being endocytosed, the giant particles instead have the TAG within them hydrolysed by lipoprotein lipase enzymes to release FFAs into the bloodstream, where they are taken up by myocytes and adipocytes. Once they have been largely emptied the chylomicron remnants are cleared by the liver.
How are sterols absorbed in the gut lumen and what are their possible pathways after this?
Sterols form mixed micelles in the gut lumen; structures designed to solubilise the dietary and biliary cholesterol by combination with phospholipids and bile acids. Sterols are extracted from these and absorbed by enterocytes at the apical surface using the sterol binding protein NPC1L1.
After absorption by the enterocytes, there are two possible pathways for the sterols that eventually lead to either packaging into chylomicrons or secretion back into the gut lumen for egestion, allowing for control of cholesterol uptake.
What is the net sterol absorption in the gut lumen and what mutation affects this?
Net absorption of dietary cholesterol is around 40%, but the figure is significantly lower for plant-derived sterols, >5% of which are esterified and packaged.
However, genetic conditions characterised by loss of function of the ABCG5/8 transporter can lead to high levels of plant sterols in the plasma, a condition called sitosterolaemia.
What enables re-secretion of sterols back into the gut lumen from enterocytes?
Secretion of the cholesterol is actively accomplished by a heterodimer of G5/G8 ATP-binding Cassette transporters (ABCG5/8).
How are sterols internalised by enterocytes?
This process occurs in enterocytes and hepatocytes, hence the enrichment of Niemann-Pick C1 like 1 (NPC1L1) in the brush-border membrane enterocytes in the proximal jejunum of the small intestine.
NPC1L1 binds into the membrane and concentrates at clathrin coated pits by a cytosolic triskelion binding domain. Once cholesterol has been recruited into this part of the membrane by extraction from mixed micelles by NPC1L1, the area is internalised and taken to an endocytic recycling compartment of the lysosome. During this they are esterified by ACAT2.
How are cholesterol esters sorted in the lysosome?
Cholesterol is extracted from the lysosome, both in this pathway and when LDLs are broken down here, by two proteins within the acidic sac: the membrane bound NPC1 (Niemann-Pick C1) and the soluble NPC2.
Cholesterol first binds into NPC2 through hydrophobic interaction with a cavity in the protein and the cholesterol iso-octyl side chain, exposing the 3’-OH to the lysosome interior. This is the same binding mechanism as is used by NPC1L1.
The cholesterol is then transferred to the NPC1, which instead binds it by its hydroxyl group, exposing the hydrophobic side chain and hence leading to insertion of the cholesterol into the lysosome membrane. From here the cholesterol is trafficked to the ER for processing.
How was the cholesterol uptake pathway discovered, and how is it targeted in therapy?
This pathway was discovered during a screen for ACAT inhibitors, when a form of the compound now called ezetimibe was found to potently and selectively inhibit uptake of dietary and biliary cholesterol in rodents, without interfering with uptake of TAG, fat soluble vitamins or bile acids. NPC1L1 was subsequently identified through mutation screening of proteins with cholesterol binding domains.
Ezetimibe reduces blood cholesterol as much as 15-20%, an effect which is additive with the inhibition of cholesterol synthesis provided by statins. However, its efficacy in reducing clinical events and improving outcomes is less clear. Also a possible increase in cancer is suggested to associate with combined drug use, so prescription remains controversial.
What is the structure of NPC1L1 and how is it targeted by ezetemibe?
The N-terminal domain crystal structure of NPC1L1 (AA 22-265) revealed a fold almost identical to that formed by the homologous NPC1 region, but is larger to allow for a broader sterol substrate specificity. The protein also has a sterol sensing domain (SSD).
The Ezetimibe binding site (amino acids 510-571) is in the first extracellular loop and thus is distinctly separated from that of the cholesterol binding domain.
What mutations are known to occur in NPC1L1?
Various rare mutations have been identified that prevent proper cholesterol internalisation in the intestinal cells, which are generally sorted into two classes depending on whether the mutation leads to incorrect folding and ER associated degradation (ERAD) or if the mutation prevents proper trafficking to the plasma membrane.
How are immature TG-rich lipoproteins produced?
The current model is that of cotranslational deposition into the ER of the ApoB protein, throughout which microsomal triglyceride transfer protein (MTP) adds small amounts of lipids taken from the cytosolic pool of TG (i.e. a lipid droplet) to the nascent chain, moving the TG droplet into the ER lumen for later addition.
This produces only a small lipoprotein; chylomicrons and VLDLs are formed by addition of more TG by an MTP-independent mechanism.
If insufficient lipid is available for ApoB to assume a stable conformation, the poorly lipidated particles are degraded in the cytosol by the proteasome and/or by lumenal proteases.
How are VLDLs secreted from the liver?
In the liver, the small precursors are termed pre-VLDLs. These are trafficked to the Golgi, and are lipidated during transit, meaning that they become VLDL2s by the time they reach the Golgi, where they merge with the tips of the cisternae and transfer through the trans-golgi network.
These can be secreted or further lipidated to form VLDL1 which lead to production of small, dense LDL particles which are particularly susceptible to oxidation and hence strongly pro-atherogenic.
How is the TG-rich lipoprotein production pathway targeted in therapy?
Approved by the FDA in 2012 for FH treatment, this prevents the production/secretion of VLDLs and chylomicrons by binding to MTP and directly preventing it from transferring TG to ApoB.
How are TG-rich lipoproteins harvested?
As large triglyceride-rich particles (chylomicrons and VLDL) flow through blood capillaries they are acted on by lipoprotein lipase (LPL). The enzyme is bound and displayed on the surface of the endothelium.
Energy-rich triglyceride molecules in the core of the CM/ VLDL particles are hydrolysed to produce two fatty acids and a 2-monoglyceride, which the tissues take up, most commonly by heart and skeletal muscle for energy or adipose for storage. Depletion of the TG store produces CM remnants and LDLs.
What is the structure of LPL?
LPL is a head to tail homodimer, so has two N-terminal active sites. Dimerisation enables correct functioning of the catalytic centre – a serine protease triad contained in a hydrophobic groove that is protected from solvent by a lid region.
Where is LPL found?
LPL is found bound to the endothelium on heparin sulphate proteoglycans (HSPGs), transiently interacts with the passing TG-rich lipoproteins. Around 40 LPLs can be attached to each structure, allowing each capture of a CM/VLDL to provide large quantities of TG.
How is LPL binding to TG-rich lipoproteins regulated?
LPL binding requires ApoCII (a small - 8.8kDa, 79AA - apolipoprotein present in both chylomicrons and VLDLs) as well as ApoB, and is inhibited by the presence of ApoCIII, thus lipolysis of CMs and VLDLs can be controlled by regulation of their apolipoprotein content. The binding site of ApoCII to LPL is unknown.
