Cholesterol Processing and Lipid Transport Flashcards
- Following formation of HMG-CoA, the remaining steps in cholesterol formation occur in the smooth endoplasmic reticulum.
- The first unique reaction in cholesterol synthesis is catalyzed by HMG-CoA reductase. This enzyme converts HMG-CoA to mevalonate. -HMG-CoA reductase is rate limiting and consequently highly regulated.
- The synthesis of cholesterol is divided into 4 stages.
Stage 1: mevalonate (6-carbon compound) is synthesized from acetyl CoA.
Stage 2: conversion of mevalonate to “active” isoprenoid (5-carbon) units with the aid of several ATP and loss of CO2. Six isoprenoid units condense to form squalene, the linear 30-carbon intermediate.
Stage 3: an enzyme complex, squalene epoxidase/cyclase, cyclizes squalene to yield lanosterol, the parent sterol.
Stage 4: cholesterol forms from lanosterol after 19 further steps, including the loss of 3 methyl groups.
•Key points of this pathway include
1) NADPH is required at all four synthesis stages;
2) ATP is utilized in stage 2; and
3) O2 is necessary for stages 3 and 4.
•NADPH is derived from both the malic enzyme reaction as well as from the oxidative branch of the pentose phosphate pathway.
•Major sites of cholesterol synthesis include liver, intestine, skin, and especially the endocrine glands that produce steroid hormones (i.e, adrenal cortex and gonads).
-Virtually all nucleated cells are capable of cholesterol synthesis.
- The reactions from acetyl CoA to cholesterol occur in the cytoplasm and on the smooth endoplasmic reticulum of the cell.
- Carbons for cholesterol synthesis require formation of acetyl CoA derived primarily from the beta-oxidation of saturated fatty acids.
- These carbons are carried from the mitochondrial matrix to the cytoplasm as citrate via the citrate transporter.
- Citrate is then lysed in the cytoplasm via citrate lyase to produce acetyl CoA and oxaloacetate.
- Citrate lyase requires coenzyme A (CoA) as a cofactor (coenzyme; cosubstrate) and ATP as a source of energy for the reaction.
- The oxaloacetate product is further processed to malate, which in turn is decarboxylated to pyruvate via malic enzyme with the concomitant production of NADPH (and CO2, not shown) that is required for cholesterol synthesis.
Cholesterol synthesis is controlled by regulation of HMG-CoA reductase - Glucagon and Insulin
•Regulation of cholesterol synthesis is exerted near the beginning of the pathway, at the unique HMG-CoA reductase step.
-Both the activity and the amount (i.e., number of molecules) of enzyme are controlled.
•Following food deprivation, the activity of HMG-CoA reductase is decreased acutely through posttranslational phosphorylation of the enzyme.
- This event is triggered by the elevated concentration of glucagon in the circulation.
- This action of glucagon explains the decreased synthesis of cholesterol during fasting.
•Conversely, in the fed state, insulin activates HMG-CoA reductase by causing removal of this phosphate from the enzyme by protein phosphatase.
Cholesterol synthesis is controlled by regulation of HMG-CoA reductase - Cholesterol
- The amount of HMG-CoA reductase (or LDL receptor) can be down regulated acutely by cholesterol, which binds to a sterol-sensing domain in the enzyme leading to activation of its proteolytic degradation.
- A separate chronic mechanism also exists whereby the concentration of HMG-CoA reductase is modulated by cholesterol, the endproduct of the pathway.
-A normal or high concentration of cholesterol represses formation of the HMG-CoA reductase enzyme (or LDL receptor) thereby lowering its amount.
Cholesterol synthesis is controlled by regulation of HMG-CoA reductase - Cholesterol and SREBP
•The gene for HMG-CoA reductase contains a regulatory DNA sequence in its promoter that must bind sterol regulatory element binding protein (SREBP) to be actively transcribed.
-In the absence of SREBP, transcription of the gene does not occur.
•A similar mechanism increases the amount of the receptor that recognizes LDL particles to promote their uptake by cells.
- Therefore both the HMG-CoA reductase and LDL receptor genes are regulated in a similar manner.
- These proteins work in tandem to increase the intracellular cholesterol concentration.
- De novo synthesis of cholesterol is enhanced by increased amounts of the reductase enzyme, and uptake of cholesterol into the cell is accelerated by having more receptors to pick up LDL that is laden with cholesterol ester.
- SREBP (sterol regulatory element binding protein) is responsible for signaling the synthesis of HMG-CoA reductase by binding to the DNA promoter region associated with coding for HMGCoA reductase or LDL receptor.
- Under conditions of normal or elevated cell cholesterol, SREBP is trapped in the endoplasmic reticulum (ER) membrane as part of a complex consisting of INSIG, SCAP (SREBP cleavage activating protein) and SREBP.
- Both INSIG and SCAP bind to cholesterol, which facilitates the binding of INSIG and SCAP. Thus SREBP cannot be mobilized and HMG-CoA reductase or LDL receptor transcription remains repressed.
- In this way, a high concentration of cholesterol represses its own synthesis and reduces the number of LDL receptors to decrease uptake of cholesterol ester into the cell.
•When the concentration of cholesterol in the cell becomes too low, INSIG and SCAP dissociate so that the SCAP-SREBP complex can move to the Golgi membrane for insertion.
-In the Golgi, two distinct proteolytic enzymes (S1P and S2P), cleave the SREBP from SCAP to release SREBP into the cytoplasm.
- SREBP then enters the nucleus and binds to the sterol responsive element (SRE) promoter region to induce the transcription of the gene encoding for HMG-CoA reductase or the LDL receptor.
- Consequently the cholesterol concentration increases in the cell to correct the cellular deficit.
Fates of Cholesterol
- Membrane structure
- Precursor of steroid hormones and vitamin D
- Esterification for storage
- Esterification for elimination
- Precursor to bile salts
Esterification for Storage or Elimination
- Esterification is catalyzed either by ACAT (acylCoA:cholesterol acyltransferase) for cell storage or by LCAT (lecithin:cholesterol acyltransferase) for transport by high-density lipoprotein (HDL).
- The ACAT and LCAT reactions provide major routes for storage and eventual elimination of cholesterol, respectively.
Cholesterol Elimination
- Ultimately, cholesterol enters the liver where it can be metabolized for excretion in the bile primarily as bile acids.
- A large proportion of the bile acids excreted from the gall bladder is reabsorbed into the portal circulation, taken up by the liver, and recirculated in the bile.
-In this way bile acids used in lipid absorption are conserved for the most part.
- The bile salts also play an important role in regulating their own formation from cholesterol.
- The hydroxylation of cholesterol to form 7 alpha hydroxycholesterol is the committed, as well as the rate limiting, step in the synthesis of bile acids.
- The reaction is catalyzed by 7 alpha-hydroxylase, which is expressed exclusively in smooth endoplasmic reticulum of liver. This reaction requires oxygen, NADPH and vitamin C (ascorbic acid).
7 alpha hydroxylase
•catalyzes the hydoxylation of cholesterol
cholesterol —> bile acids
- rate limiting step
- 7 alpha-hydroxylase enzyme is regulated by induction or repression.
- The enzyme is induced by cholesterol (feedforward effect) which binds to the liver nuclear receptor, LXR.
- The enzyme is feedback repressed by bile acids (feedback effect) which binds instead to the liver farnesoid receptor (FXR) to repress the 7 alpha-hydroxylase promoter on the gene.
•Hypercholesterolemia may be treated by interrupting the recirculation of bile acids back through the liver.
Removal of Cholesterol by Cells
- Though the detailed processing of HDL will be discussed later in the lecture, it is valuable at this point to understand how non-hepatic cells rid themselves of cholesterol for transport to the liver.
- The liver rids its cells of cholesterol primarily by conversion to bile acids that are excreted.
Removal of Cholesterol from Cells by HDL
- Besides cholesterol ester, HDL molecules contain lecithin, unique proteins and LCAT, for esterifying free cholesterol from cells.
- HDL in the circulation acquires cholesterol by employing LCAT to extract cholesterol from the plasma membranes of peripheral cells.
- This process of cholesterol removal by HDL is facilitated by an ATP-binding cassette protein (ABCA1) transporter called cholesterol efflux regulatory protein (CERP).
- CERP is activated by apolipoprotein A1, which is a protein component of HDL particles. CERP pumps cholesterol across the plasma membrane (i.e., flips the molecules from the cytoplasm to the membrane bilayer) and delivers them to HDL particles as a substrate for the LCAT enzyme found on these particles.
- Once loaded with cholesterol ester, HDL is transported to the liver where it dumps its cholesterol load for potential conversion to bile acids.
-Since HDLs are ultimately cleared by the liver, where cholesterol is converted to bile acids and excreted in bile, this step constitutes “reverse cholesterol transport” from cells to HDL, and is one of the crucial mechanisms whereby the body eliminates cholesterol.
Factors Influencing Cholesterol Balance Inside Cells - Intracellular Decrease
Decrease in the intracellular concentration of free (unesterified) cholesterol is a result of a combination of the following factors.
[a] The esterification of cholesterol by ACAT (acyl CoA:cholesterol acyl transferase) for storage in droplets.
[b] The utilization of cholesterol for the synthesis of other steroids, such as the hormones of the adrenal cortex and gonads, of bile acids in the liver, or of vitamin D in the skin
[c] The release of cholesterol for transport to the liver following:
i) trafficking to the plasma membrane (a process directed by the Golgi)
ii) pumping to the exterior surface by CERP (cholesterol efflux regulatory protein)
iii) transfer to HDL particles (promoted by LCAT)
Factors Influencing Cholesterol Balance Inside Cells - Intracellular Increase
Increase in the intracellular concentration of free cholesterol is a result of a combination of the following factors.
[1] The endocytotic uptake of low-density lipoproteins by the LDL receptor; also uptake of cholesterol-containing lipoproteins by scavenger receptors in macrophages, or by nonreceptor-mediated pathways and diffusion of free cholesterol from cholesterol-rich lipoproteins through the plasma membrane
[2] The synthesis of cholesterol by the pathway described earlier in these notes.
[3] The hydrolysis of cholesterol esters by the enzyme cholesterol esterase.
- The LDL receptor is found on the cell surface.
- The LDL receptor binds the LDL apoprotein B100, and the LDL, with its receptor, is taken up intact via endocytosis.
- The LDL particle is degraded in the lysosome/ late endosome. The protein component is degraded to amino acids, while the cholesterol ester is hydrolyzed by an acid hydrolase.
- The free cholesterol is then translocated by a protein (NPC-1) into the free cholesterol pool in the Golgi apparatus [w].
- This influx of cholesterol via internalization of LDL particles leads to the degradation and repressed synthesis of HMG-CoA reductase to shut down cholesterol synthesis.
- Additionally, the increased concentration of cholesterol represses the formation of new LDL receptors [x].
•Internalized cholesterol also stimulates ACAT activity [y] to enhance cholesterol storage and, via binding to LXR, induces the synthesis of CERP [z] to promote cholesterol efflux.
Therapies for Treating Hypercholesterolemia
- Statins
- Bile Acid Sequestering Resins
- Nicotinic Acid
- Fibrates
- Decreasing Intestinal Cholesterol Absorption
Therapies for Treating Hypercholesterolemia - Statins
- All statins are competitive inhibitors of hydroxyl-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis. T
- hey inhibit the enzyme in the nano (10-9 ) molar range and work by blocking part of the site where HMG-CoA, the substrate, binds thereby limiting access of the substrate to the catalytic (active) site.
- By inhibiting HMG-CoA reductase, statins decrease synthesis of cholesterol by the liver and other tissues.
- This action in liver decreases the availability of cholesterol for the synthesis of very low-density lipoproteins (VLDL) particles that eventually become LDL particles.
- Additionally, slowing the rate of cholesterol synthesis lowers the concentration of intracellular cholesterol that leads to an increase in the number of LDL receptors and hence in the uptake of LDL from the blood.
-Hence the major overall effect of statins is to lower the plasma concentration of LDL and hence of circulating cholesterol.
- An additional benefit of statins is to raise the plasma concentration of HDL (‘good cholesterol’) by increasing the production of apolipoprotein A1, that activates CERP.
- Statins provide significant benefits to a broad range of patients at risk for coronary heart disease and this benefit in some seems independent of the circulating LDL concentration in the patient.
- Despite the undeniable benefits of statins, myopathies seem to be an issue for all or most statins and can even lead to a fatal rhabdomyolysis.
-Myopathies are monitored by testing for changes in the blood concentration of the skeletal muscle isoform of creatine kinase.
•Additionally physicians are cautioned to be aware of possible medication interactions between statins and other lipid lowering medications such as niacin and fibrates.
General Lipoprotein Composition
- Lipoprotein particles undergo continuous metabolic processing/remodeling, so they have variable properties and compositions, but the main lipid components are triacylglycerols, cholesterol esters and phospholipids.
- Other lipids carried by lipoproteins include free cholesterol, fat-soluble vitamins and trace amounts of free fatty acids.
Unlike most membranes, the one around a lipoprotein is a monolayer. The outer surface is coated with apoproteins and the core is comprised of cholesterol ester and triacylglycerol in proportions that vary with each type of lipoprotein
VLDL
- VLDL, a triacylglycerol-rich lipoprotein, is produced and secreted by the liver.
- The triacylglycerol is formed from free fatty acids that are either mobilized from adipose cells or synthesized in liver from acetyl CoA derived from nonlipid precursors, primarily from glucose.
- A major function of this lipoprotein is to transport endogenously synthesized triacylglycerol from the liver to extrahepatic tissues.
- VLDL secreted into the circulation also contains apolipoprotein B100 that is synthesized in liver.
- The primary role of apo B100 in this process is to aid in the proper assembly and export of nascent VLDL.
- The only protein contained in the nascent form of VLDL is apo B100.
- Chylomicrons are assembled using apo B48 in a similar manner.
- Apo B48 is a truncated version of apo B100 lacking 52% of the linear structure.
- The additional portion of apo B100 accounts for its ability to bind to the LDL receptor.
- Release of VLDL by liver is augmented by any condition that results in increased flux of free fatty acids to the liver in the absence of their conversion to ketone bodies.
- Obesity, increased caloric intake, chronic ingestion of ethanol and estrogens stimulate release of VLDL and are important factors in hypertriglyceridemia.
- The second major function of VLDL is to provide liver-generated cholesterol esters for LDL that carries the cholesterol to peripheral cells, and back to the liver.
- After secretion from the liver, VLDL matures by acquiring apolipoproteins CII and E in the plasma from HDL particles.
-Liver cells synthesize and export the apo CII and E proteins to be picked up by HDL particles.
- Mature VLDL converts to IDL and then LDL particles following hydrolysis of triacylglycerol to three fatty acids and the glycerol backbone. T
- Triacylglycerols in VLDL (also chylomicrons) undergo lipolysis catalyzed by lipoprotein lipase (LPL).
- LPL lies on endothelial cell surface of capillary walls and hydrolyzes triacylglycerols to release fatty acids taken up and used primarily by adipose and muscle cells.
- LPL is primarily associated with heart, skeletal muscle, adipose and mammary cells. In muscle, released fatty acids are oxidized for energy.
- In adipose cells fatty acids are re-esterified for storage as triacylglycerols.
- LPL is activated by apolipoprotein CII, bound to phospholipids on the VLDL and chylomicrons.
- In the fed state, insulin increases activity of LPL while in starvation the activity declines.
