Lipid Metabolism 3

The statins are the major class of medications used to lower elevated serum cholesterol by initially inhibiting the major rate-limiting step of cholesterol synthesis. Which metabolite of the pathway would accumulate under conditions of taking a statin?

Hydroxymethylglutaryl CoA (HMG-CoA)

3 multiple choice options

Cholesterol, and its precursors and products, have a variety of functions within cells. Which one statement correctly describes a function of a cholesterol precursor, cholesterol itself, or a product derived from cholesterol?

Precursors of cholesterol can be converted to vitamin D

3 multiple choice options

A new patient is being evaluated for cardiovascular disease. The values of his lipid panel are a total cholesterol of 400 mg/dL, an HDL of 35 mg/dL, and a triglyceride reading of 200 mg/dL. What would be his calculated LDL cholesterol reading?

325

3 multiple choice options

Which one of the following apolipoproteins acts as a cofactor activator of the enzyme lipoprotein lipase (LPL)?

Apo-CII

3 multiple choice options

Which one of the following sequences places the lipoproteins in the order of most dense to least dense?

HDL/LDL/VLDL/chylomicrons

3 multiple choice options

Which one of the following would you least expect to observe in a patient lacking MTP (microsomal triglyceride transfer protein) after eating a normal diet, in which each meal consisted of 30% fat?

Steatorrhea

3 multiple choice options

Patients with elevated serum LDL levels (>120 mg/dL) are first encouraged to reduce these levels through a combination of diet and exercise. If this fails, they are often prescribed statins. The key for statin treatment reducing circulating cholesterol levels is which of the following?

Upregulation of LDL receptors

3 multiple choice options

A consequence of abetalipoproteinemia is a fatty liver (hepatic steatosis). This occurs due to which one of the following?

Inability to produce VLDL

3 multiple choice options

Hormones are typically synthesized in one type of tissue, often in response to the release of a stimulatory hormone. Which one of the following correctly matches a hormone with its stimulatory hormone, and its site of synthesis?

Cortisol, ACTH, adrenal cortex

3 multiple choice options

Because the steroid muscle cannot be degraded by the human body, excretion of bile salts in stool serves as a major route of removal of steroids from the body. Which one of the following must occur to bile salts in order for bile salts to be excreted in the stool?

Intestinal bacterial deconjugate bile salts

3 multiple choice options

A 52-year-old male visited a cardiologist due to intermittent chest pain. A nuclear stress test revealed blockages in two arteries, indicative of atherosclerotic disease. Serum LDL levels in the patient were 155 mg/dL (for a patient with atherosclerotic disease a value of 100 mg/dL or lower is desirable). In addition to improving nutrition and increasing exercise, the patient was prescribed atorvastatin, which after 3 months had lowered the LDL levels to 120 mg/dL. Combined treatment with an anti-PCSK9 antibody resulted in lowering the LDL levels to 100 mg/dL. The mechanism of drug action in the patient is best represented by which one of the following?

Both lead to upregulation of the LDL receptor.

3 multiple choice options

Acute intermittent porphyria (AIP) is a disorder that interrupts the pathway of heme synthesis. Individuals who are experiencing an onset of symptoms have been demonstrated to express reduced cortisol levels. This could occur due to which one of the following?

Reduced activity of enzymes in the zona fasciculata of the adrenal gland.

3 multiple choice options

Under conditions of low energy within the liver cell, both fatty acid biosynthesis and cholesterol synthesis are inhibited. This bifunctional regulation occurs through the activation of which one of the following?

A protein kinase

3 multiple choice options

The regulation of HMG-CoA reductase is complex and involves transcriptional regulation. The transcription factor involved in this regulation is regulated via intracellular cholesterol levels, and a key regulatory event involving this transcription factor is best described as which one of the following?

Activation of a protease

3 multiple choice options

What is cholesterol?

A lipophilic steroid with a double bond in 5,6-position, an OH group in 3-position (secondary alcohol, S-configuration) and a branched C8-side chain attached at C-17.

Why is cholesterol important?

Important as membrane stabilizer, and as precursor of steroid hormones and the bile acids. The biosynthetic pathway toward cholesterol also branches off into ketone bodies (acetoacetate) and the D-vitamins.

Why can cholesterol be harmful?

Too much is bad. 'Cholesterol' deposition in arteries can cause stroke, cardiovascular diseases, etc.

To what group of lipids does cholesterol belong?

Steroids

What is unique about steroids?

Steroids cannot be broken down. Their metabolism ends with bile acids, which can be excreted through the intestines, but most of them are recycled!

What drugs can be used as cholesterol-lowering agents?

-Statin Drugs (e.g., Lovastatin, Atorvastatin). These are either natural products (fungal metabolites) or synthetic. The all act as HMG-CoA reductase inhibitors.

- Bile acid sequestrants (e.g., Cholestyramine).

- Cholesterol uptake inhibitors (ezetimibe)

How do statin drugs work?

Statin drugs contain a structural element that is an analogue of the product of the HMG-CoA reductase reaction, and this act as competitive inhibitors of HMG-CoA reductase, which catalyzes the rate-limiting step of cholesterol biosynthesis, thereby enhancing the LDL uptake from the blood by the liver.

How do bile acid sequestrants work?

The bile acid sequestrants are polymers that can bind and remove bile acids from the intestines, thereby hindering their return to the liver. The liver reacts through increased uptake of blood lipoproteins.

How do cholesterol uptake inhibitors work?

The uptake inhibitor ezetimibe inhibits the intestinal absorption of cholesterol and related steroids.

What are 2 examples of natural product statins?

lovastatin and pravastatin

What are 2 examples of synthetic statins?

cerivastatin and atorvastatin

What side effects (short term/long term) can be expected from statin drugs and why?

- Statin Drugs (e.g., Lovastatin, Atorvastatin) also inhibit the synthesis of vitamin D3 and of ubiquinone. Both need to be added as supplement!

- Muscular atrophy (also pain) are frequently observed as side effects of statin drugs, and long term dementia has been observed.

- This is because muscular and brain cholesterol and ubiquinone synthesis is also inhibited by statins, the latter especially by statins able to cross the blood-brain barrier. The inhibition has a damaging effect of cell membranes of muscle and brain cells.

What is the purpose of the tricarboxylate transport system?

To transport 'acetyl-CoA' in form of citrate from the mitochondrial matrix to the cytosol; where it is needed for fatty acid biosynthesis and cholesterol biosynthesis.

Which is the transported species in the tricarboxylate transport system?

Citrate, the product of the first TCA cycle enzyme (citrate synthase), is transported.

Which enzymes are involved in the tricarboxylate transport system, and what are their substrates and products?

In the cytosol, citrate is cleaved into acetyl-CoA and oxaloacetate by ATP-citrate lyase, an ATP consuming enzyme. ADP and Pi are the products of the energy-delivering reaction. Oxaloacetate is returned to the mitochondrial matrix in the form of malate, pyruvate, or aspartate.

How can the biosynthesis of cholesterol be characterized (division in phases, most important enzymes and intermediates)?

- Three phases: (i) acetyl-CoA to mevalonic acid, (ii) mevalonate to squalene, (iii) conversion of squalene into the steroid lanosterol and on to cholesterol (19 follow-up steps)

- Important intermediates are HMG-CoA, mevalonate, the isoprenes IPP and DMAPP, the terpenes geranyl PP, farnesyl PP and squalene, and squalene epoxide

- Important enzymes are HMG-CoA reductase, prenyl transferase(head to tail condensation!), squalene synthase (head to head condensation!), squalene epoxidase (P450 oxygenase!) and squalene oxidocyclase (cascade cyclization!)

What cofactor is needed in the squalene epoxidase reaction?

NADPH

Which other biosynthetic pathways share enzymes with the cholesterol biosynthesis and why is this important?

- Ubiquinone (co-enzyme Q10), important for the ETC!

- Vitamin D3 (cholecalciferol), important for Ca2+ management incl. bone breakdown/synthesis and for the immune system!

- Ketone bodies, important as fuel in starvation states

- The synthesis of ubiquinone and vitamin D3 require HMG-CoA reductase as a key enzyme. Thus, HMG-CoA reductase inhibitors also inhibit the synthesis of these important metabolites. Supplementation is necessary!

How is HMG-CoA reductase regulated?

- Feedback regulation by cholesterol and bile acids. This affects the transcription of genes encoding HMG-CoA reductase.

- Hormonal control (insulin, glucagon). Glucagon inactivates HMG-CoA reductase through phosphorylation by a phosphorylated kinase. Insulin can stop that process indirectly through dephosphorylation of that kinase by a phosphatase, which inactivates the kinase. Insulin can also directly dephosphorylate the kinase with a phosphatase.

What is LCAT?

- LCAT = lecithin:cholesterol acyl transferase

- Transfers a fatty acid from lecithin (phosphatidylcholine, a phospholipid) to the 3-OH group of cholesterol. Activated by apoA1 from HDL.

What is ACAT (in context with cholesterol synthesis)?

- ACAT (cholesterol metabolism): Acyl:cholesterol acyl transferase (in liver cells)

- Esterifies cholesterol with a fatty acid at 3-position (for storage)

What are lipoproteins? (names, function, fate, desirable concentration)

- Transport vehicles for lipids in the blood

- Chylomicrons: for dietary lipids (TGs, cholesterol, fat soluble vitamins)

- VLDL: for lipids of the liver (biosynthesized TGs, biosynthesized or recycled cholesterol)

- IDL: intermediate (derives from VLDL, is converted to LDL)

- LDL: contains mostly cholesterol and cholesterol esters, should betaken up into the liver; desirable is < 120 mg/dL

- HDL: 'good cholesterol' (helps to clear the blood from cholesterol and from LDL; matures chylomicrons and VLDL); desirable is ~ 60 mg/dL

Which apoproteins does HDL provide, and for what purpose?

- ApoE: for chylomicron remnants, liver receptor ligand!

- ApoCII: for activation of LPL (digestion of fats to fatty acids for uptake by adipose or muscle tissue).

-Apo A1: for activation of LCAT (converts cholesterol to cholesterol esters, for recycling of cholesterol into the liver).

How does cholesterol (re)enter the liver? What regulatory effects has endocytosed cholesterol in the liver? What is it partially converted into?

- Mainly through LDL-receptor (backup = LRP receptor)

- Downregulates HMG-CoA reductase and LDL receptor synthesis (but not the macrophage scavenger receptor and not the LRP receptor! <-- insulin), upregulates ACAT synthesis

Bile acids (for the digestion of dietary fats)

What are PCSK9 inhibitors? How do they work as cholesterol lowering agents?

These are protein drugs, antibodies, that inhibit PCSK9 (= proprotein convertase subtilisin/kexin type 9). PCSK9 marks aging LDL receptors for degradation. The inhibitors keep these receptors alive thereby keeping the number of LDL receptors higher, which in turn keeps the re-uptake of cholesterol from the blood.

Which receptors are important in context with cholesterol re-uptake by the liver? What do these recognize, and how specific are these?

• LDL Receptor: Most important, many copies, specific for lipoproteins, recognizes apoE (chylomicrons, IDL, VLDL)and apo-100 (LDL, VLDL, IDL).

• LRP Receptor: Backup, less specific. Only recognizes apoE (for VLDL, IDL, HDL, chylomicrons), does not recognize LDL!

• Scavenger Receptors: broad specificity mostly found on macrophages, takes care of oxidized (damaged by ROS)LDL, loaded HDL. The process turns macrophages into foam cells atherosclerotic plaques!

What types of bile acids exists (names, pKa, structural characteristics in comparison to cholesterol)? Recycling (for what purpose)?

- The lipophilic side chain is changed into a (by three carbons) shorter acidic (COOH-containing) side chain; 5,6-double bond is saturated

- Cholic acids (-hydroxy groups at 3-, 7- and 12-position)

- Chenocholic acids (-hydroxy groups at 3- and 7-position)

- Conjugates (glycine, taurine): lower pKa from 6 to 4 (glycine) or from6 to 2 (taurine)

- Recycled into the liver to be reconjugated (but they cannot get rehydroxylated!)

What is the degree of deprotonation of a conjugated bile acid with a pKa of 4.5? What do you need to know to solve this? Why is that important?

- The bile pH is 6 (optimized for the pancreatic lipase).

- The Henderson Hasselbalch equation (pH= pKa - log [HA/A-])

- Thus: 6 = 4.5 - log[protonated/deprotonated]

1.5 = - log[protonated/protonated] = log [deprotonated/protonated]protonated/deprotonated = 10^1.5 = 31.6/1

- Thus, 97% of that bile acid is deprotonated. Only deprotonated bile acids can aid lipid digestion as detergents supporting micelle formation!

For a steroid, rings B/C and C/D are always ___.

trans-fused

What are the types of steroids?

• Cholesterol

- Provides membrane stability

- Precursor for steroid hormones and bile acids

• Steroid hormones

- Regulate growth, metabolism, and reproduction

• Bile Acids

- Are involved in lipid digestion and absorption

What are steroid hormones?

chemical messengers that provide communication from one part of the body to another. (ex: androgens, estrogens)

What are the complications associated with cholesterol?

Cholesterol deposition in arteries is associated with cardiovascular diseases and stroke. It is responsible for the two leading causes of death in Western culture.

Where can you obtain cholesterol?

-Obtained from diet: egg yolk, meat (red meat, liver)

-Biosynthesized by most of our cells (mostly by liver and intestinal cells).

-Not biosynthesized in plants (fruits and vegetables are cholesterol free!)

How can you avoid excess of cholesterol?

-Lose weight, exercise!

-Diets high in fruits and vegetables

-Examples: step 1 (< 300 mg/day) and step 2 (<200 mg/day) low cholesterol diets recommended by the NCEP.

-Cholesterol lower drugs (inhibitors of cholesterol biosynthesis, "cholesterol"-binding and removing resins, and cholesterol uptake inhibitors)

How can you avoid excess of dietary cholesterol?

-Study nutrition facts

-Be aware of what you eat

-Compare products

Where does cholesterol biosynthesis occur?

cytosol

What is the main precursor molecule for cholesterol biosynthesis?

acetyl CoA derived from the breakdown of glucose, fatty acids, or amino acids

In what form is acetyl-CoA transported from the mitochondria to the cytosol?

as citrate, which is then carried from the mitochondria to the cytosol using the tricarboxylic transport system

Once in the cytosol, what happens to citrate?

it is cleaved to oxaloacetate and acetyl-CoA using the enzyme ATP-citrate lyase

Why is the tricarboxylic transport system important?

Acetyl-CoA cannot be transported through the mitochondrial membranes

What happens during the 3 phases of cholesterol biosynthesis?

1. Three-step formation of the C6-metabolite mevalonate from three units of acetyl CoA.

2. Seven-step conversion of mevalonate via C5 phosphorylated isoprene units (isopentenyl-pyrophosphate, dimethylallylpyrophosphate) that can condensate into the C30 compound squalene.

3. Cyclization of squalene and further conversion to cholesterol (21 steps).

What reaction does HMG-CoA reductase catalyze?

reduction of HMG-CoA to mevalonate

What is the rate-limiting step in cholesterol biosynthesis?

The HMG-CoA reductase reaction (HMG-CoA --> mevalonate)

Why is liver HMG-CoA reductase especially effective?

because the liver is the major site of bile acid production, which increases demand for cholesterol synthesis

How many NADPH molecules are required for the HMG-CoA --> mevalonate reaction?

2 NADPH

What are the 2 reduction steps in the HMG-CoA reductase reaction?

a) Thioester --> thioacetal

b) Thioacetal --> primary alcohol

Which drugs are inhibitors of HMG-CoA reductase?

statins

What happens first to mevalonate in Phase II of cholesterol biosynthesis?

It is phosphorylated twice by ATP (requires 2 ATP) to form mevalonate diphosphate (pyrophosphate).

After phosphorylation, what reaction converts mevalonate into an activated isoprene unit?

decarboxylation to isopentenyl pyrophosphate (IPP)

What is the first isoprene unit formed in cholesterol synthesis?

isopentenyl pyrophosphate (IPP)

Isopentenyl pyrophosphate (IPP) is in equilibrium with which other isoprene unit? Both are C5 isoprene units.

dimethylallyl pyrophosphate (DMAPP)

Geranyl pyrophosphate (C10) + IPP (C5) combine to form what C15 intermediate?

farnesyl pyrophosphate (FPP)

What molecules combine to form squalene?

two farnesyl pyrophosphate molecules (C15 + C15=C30)

What is the first major step in Phase III for converting squalene to cholesterol?

formation of squalene epoxide (uses squalene epoxidase and NADPH cofactor)

How does oxygenase act on squalene during epoxide formation?

It inserts one O-atom of O2 into the 2,3-double bond; the second O-atom becomes water.

What structure is formed from squalene epoxide during cyclization?

The tetracyclic steroid skeleton, through the prosterol cation intermediate.

What is the first steroid intermediate formed in cholesterol biosynthesis?

lanosterol

How many steps convert lanosterol into cholesterol?

19 steps

What are the 3 key modifications occurring during the lanosterol --> cholesterol conversion.

1. Removal of 3 methyl groups

2. Repositioning of the double bond in ring B

3. Saturation of the side-chain double bond

What final product is produced after the 19-step modification of lanosterol?

cholesterol

What enzyme catalyzes the reaction from 2,3-oxidosqualene to lanosterol?

squalene oxidocyclase

Cholesterol synthesized in the liver is either converted into ___ or into ___ (3-OH group can be esterified).

bile acids; esters

-Esterification increases the lipophilicity, advantageous for packaging into lipo-proteins and lipid droplets.

Which enzymes perform esterification?

LCAT and ACAT

Cholesterol and its esters are highly insoluble in water and are transported in ___.

lipoprotein complexes

How is biosynthetic cholesterol transported from the liver?

cholesteryl ester --> VLDL--> IDL--> LDL

Dietary cholesterol is transported in ___ from the intestines.

chylomicrons

What is the primary function of lipoproteins such as chylomicrons and VLDL?

to transport lipids and prevent accumulation of insoluble fat droplets in the blood

Which apoprotein is found on chylomicrons?

ApoB-48

Which apoprotein is found on VLDL?

ApoB-100

Why are amphipathic molecules necessary on the surface of lipoproteins?

they make the lipoprotein water-soluble, allowing it to travel in the bloodstream

What are chylomicrons and how do they enter the blood?

A lipoprotein complex that contains dietary lipids including dietary cholesterol. They enter the blood via the lymphatic system and mature by requiring proteins from HDL.

What are VLDL (very low-density lipoprotein)?

Lipoprotein complex that channels lipids from the liver. It contains liver lipids including cholesterol, either from biosynthesis in liver or taken up by the liver through endocytosis of chylomicron remnants.

What is the major purpose of lipoproteins?

to transport triacylglycerols and to deliver fatty acids (for storage and energy); cholesterol delivery is secondary

What is LDL (low-density lipoprotein)?

"Bad Cholesterol"

- Produced via VLDL and IDL (intermediate density lipoprotein).

- Docks to LDL receptors of target cells and enters these cells via endocytosis.

- Can also reenter the liver.

- LDL can also be damaged by oxidation and then needs to be taken up by scavenger receptors on macrophages.

What is HDL (high-density lipoprotein)?

"Good Cholesterol"

- Helps retard atherosclerotic process.

- Interacts with chylomicrons and VLDL to exchange lipids and proteins.

- Takes up cholesterol from surface of cells and from other lipoproteins and converts it to cholesteryl esters.

- Cholesteryl esters are returned to liver by 'reverse cholesterol transport'.

___ transfers apoE and apoCii to chylomicrons and VLDL, thereby maturing them.

HDL

___ on HDL stimulates LCAT to form cholesteryl esters.

apoAi

What does CETP stand for and what is its function in lipid metabolism?

- Cholesteryl Ester Transfer Protein

- CETP transfers cholesteryl esters from HDL to VLDL/LDL in exchange for triglycerides.

What was the intended purpose of CETP inhibitors?

to raise HDL levels by blocking the CETP-mediated transfer of cholesteryl esters

True or False: CETP inhibitors are natural products.

False

As VLDL is degraded to IDL and then on to LDL, it ___.

transfers apoCii and then apoE back to HDL

___ has low content of triacylglycerols and ___.

LDL; high content of cholesteryl esters

What is the most important and best-characterized lipoprotein receptor?

The LDL receptor (usually 20,000-50,000 receptors per cell)

Where are LDL receptors synthesized?

In the endoplasmic reticulum (ER) and golgi complex; located on the cell surface in clathrin-coated pits

Which apoprotein does the LDL receptor recognize?

ApoE and ApoB-100

The LDL receptor is quite ___ in contrast to the LRP (=LDL receptor related protein) and the scavenger receptor.

specific for blood lipoproteins