Ring structure of cholesterol cannot be brown down into CO2 and H2O Cholesterol is either: 1. Converted to bile salts 2. Excreted as intact cholesterol in the bile

Watery heterogenous mix which includes: _phosphatidylcholine_ PC solubilizes cholesterol in bile _bile salts_ Functions as a surfactant to help emulsify dietary fats Reduction in PC production or increased cholesterol production can lead to formation of lithogenic bile

Cholesterol Flashcards by Quynh Tran

Cholesterol Structure

Steroid nucleus: four fused rings
Branched hydrocarbon tail attached to C-17 of ring D
Presence of -OH group at C-3 of ring A gives rise to term sterol
- Membrane cholesterols have free -OH at this position
- Most plasma cholesterols have a fatty acid esterified at the C-3 -OH producing a cholesteryl
Adding FA makes molecule more hydrophopic
- Lipoproteins required for transport throughout body

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Sources of Cholesterol

Dietary (~30%)
- Animal sources only
- Amount ingested has little effect on plasma levels
- Plant sterols (phytosterols) compete for absorption and can be used to reduced dietary cholesterol absorption
De Novo Synthesis (~70%)
- All tissues capable but primarily liver, adrenal cortex, intestine, and reproductive organs.
- Occurs in cytosol and SER
- All C’s from acetyl CoA and fatty acid β-oxidation
  - Requires a lot of NADPH & ATP
- Dietary fat is the main determinant of plasma cholesterol (source of Acetyl CoA)

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Cholesterol

De Novo Synthesis

Condensation of two acetyl-CoA to form acetoacetyl-CoA.
Addition of a third acetyl-CoA to form HMG-CoA by HMG CoA Synthase
- Cytosolic isozyme makes HMG-CoA that flows into cholesterol
- Mitochondrial isozyme makes HMG-CoA used in generation of ketone bodies
HMG-CoA reduced to Mevalonate by HMG-CoA Reductase
- Rate-limiting and regulated step of cholesterol synthesis
- Statin drugs act as competitive inhibitors
Mevalonic acid converted in multi-step reaction to produce Isopentenyl pyrophosphate (IPP)
- Requires 3 ATP per IPP made
IPP [5C] used to build Geranyl pyrophosphate (GPP) [10C] then Farnesyl pyrophosphate (FPP) [15C].
- FPP used to synthesize:
  - Dolichol: used in N-linked glycosylation
  - Ubiquinone (CoQ): ETC
  - Prenylated proteins: used in post-translational processing
Two FPP condensed to produce Squalene [30C]
- Requires a total of 18 ATPs
Squalene converted to Lanosterol through ring-closure by SER-associated Squalene monooxygenase
Several steps including removal of 3 methyl groups and movement of double bonds to produce Cholesterol.

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Cholesterol Biosynthesis

Regulation Overview

HMG-CoA Reductase is the major control point for regulation of cholesterol biosynthesis.

Transcriptional Control
Proteasomal Degradation
Phosphorylation/Dephosphorylation
Hormonal Regulation

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HMG-CoA Reductase

Transcriptional Regulation

Controlled by SREBP-2 (sterol regulatory element binding protein-2) binding to SRE (sterol response element)

SREBP-2 is an integral SER protein normally associated with another SER-membrane protein SCAP (SREBP cleavage activating protein)
SCAP contains a sterol-sensing domain
When [sterol] high:
- SCAP binds to a third ER membrane protein Insig (insulin-induced gene product), causing retention of SREBP-2/SCAP in the ER.
When [sterol] low:
- SCAP no longer interacts with Insig and SREBP-2/SCAP complex translocated to the Golgi apparatus.
In the Golgi, two proteases (S1P and S2P) cleave SREBP-2 to produce a soluble N-terminal domain that enters nucleus and acts as a transcription factor at the HMG-CoA Reductase gene.
Other genes upregulated by SREBP-2 include:
- HMG-CoA Synthase
- Low-density lipoprotein receptor (LDL-R)
- Proprotein convertase subtilisin kexin 9 (PCSK9)

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HMG-CoA Reductase

Proteasomal Degradation

When [sterol] high:

HMG-CoA Reductase interacts with Insig in the ER membrane leading to ubiquitination and subsequent proteasomal degradation of the reductase.

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HMG-CoA Reductase

Covalent Regulation

HMG-CoA Reductase is controlled by phosphorylation by AMP-activated protein kinase (AMPK).

Increased [AMP] leads to phosphorylation of HMG-CoA Reductase and inactivation.
AMPK also regulates Acetyl-CoA Carboxylase from fatty acid synthesis.
Thus, in conditions of low [ATP], fatty acid and cholesterol synthesis is decreased.

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HMG-CoA Reductase

Hormonal Regulation

Insulin and Glucagon indirectly mediate HMG-CoA reductase activity via cAMP levels.

Glucagon causes increased [cAMP] leading to inhibition of HMG-CoA reductase phosphatase via PKA
- Maintains the the reductase in its phosphorylated & inactive form.
Insulin causes decreased [cAMP] leading to increased HMG-CoA reductase phosphatase activity
- Results in increased reductase activity

Thyroxine can up-regulate HMG-CoA Reductase synthesis.

Glucocorticoids can down-regulate HMG-CoA Reductase synthesis.

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Cholesterol Removal

Ring structure of cholesterol cannot be brown down into CO₂ and H₂O
Cholesterol is either:
1. Converted to bile salts
2. Excreted as intact cholesterol in the bile

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Bile

Watery heterogenous mix which includes:
- phosphatidylcholine
  - PC solubilizes cholesterol in bile
- bile salts
Functions as a surfactant to help emulsify dietary fats
Reduction in PC production or increased cholesterol production can lead to formation of lithogenic bile

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Bile Salts

Synthesis & Metabolism

Two compounds represent the primary bile acids:

Cholic acid

Chenodeoxycholic acid

Bile acids produced in the liver from cholesterol through:
- Shortening
- Attachment of a carboxylate group to the hydrocarbon chain
- Attachment of -OH groups to the sterol ring
- Rate-limiting step:
  - addition of -OH group at C-7 of the B-ring by cholesterol-7-α-hydroxylase
    - Up-regulated by cholesterol
    - Down-regulated by cholic acid
- Modification makes the molecule amphipathic
Once bile acids formed, they are conjugated with glycine or taurine to produce bile salts.
- Conjugation further increases amphipathic nature
Bile salts secreted into the intestine where they are metabolized by bacteria.
Once in the intestines bile salts can either:
- Be excreted (~5%)
  - Increased with bile acid sequestrants sometimes used to treat hypercholesterolemia
- Be reabsorbed for reuse through process called enterohepatic recirculation

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Steroid Hormone Synthesis

Cholesterol is the precursor for all classes of steroid hormones including glucocorticoids, mineralocorticoids, and sex hormones:

Cholesterol converted to pregnenolone by sidechain cleavage enzyme (CYP11A1).
End product depends on which enzymes are present in a given tissue:
- Adrenal cortex produces cortisol, aldosterone, and androgens
- Ovaries and placenta produce estrogen and progesins
- Testes produce testosterone
Synthesis of all steroid hormones involve:
- Shortening of the hydrocarbon chain
- Hydroxylation of the steroid nucleus by a series of cytochrome P450 hydroxylases
Defects in specific enzymes of a given pathway can results in lack of a specific hormone or shunting to alternate pathways.
- Most common are the congenital adrenal hyperplasias (CAH)

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Congenital Adrenal Hyperplasias

(CAH)

Caused by a defect in 3-β-Hydroxysteroid Dehydrogenase
Results absence of all steroid hormones
Autosomal recessive

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17-α-Hydroxylase Deficiency

Virtually no sex hormones or cortisol
Increased production of mineralocorticoids (Aldosterone) causing:
- Sodium and fluid retention
- Hypertension
Female-like genitalia

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21-α-Hydroxylase Deficiency

Most common form of CAH
Partial and complete deficiencies known
Mineralocorticoids and glucocorticoids virtually absent or deficient
Overproduction of androgens leading to masculinization of external genitalia in females & early virilization in males

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11-β1-Hydroxylase Deficiency

Decrease in serum cortisol, aldosterone, and corticosterone
Increased production of deoxy-corticosterone
- Causes fluid retention through RAAS suppression
Overproduction of androgens causing masculinization and virilization

Plasma Lipoproteins

Transports lipids through plasma in soluble form
Composed of:
- Neutral lipid core containing triacylglycerol and cholesteryl esters
- Shell of apolipoproteins, phospholipid, and free cholesterol.
Lipid:protein ratio determines size and density which is the basis for classification

Apolipoprotein Classification

Classified into several major classes with one or more members in each class.
Major classes as A, B, C, and E
Each type of apolipoprotein can be associated with one or more type of lipoprotein.

Chylomicrons (CM)

Characteristics

Assembled in intestinal mucosal cells
Carry dietary triglycerides (~90%), cholesterol and cholesteryl esters, fat-soluble vitamins, and other lipids to peripheral tissues
CMs only present in the plasma for severa hours after a meal
Apo B-48 produced through post-transcriptional mRNA editing and is unique to chylomicrons.

Chylomicron Lifecycle

Microsomal triacylglycerol transfer protein (MTP) loads Apo B-48 with lipids in the SER of intestinal cells.
Particle moved to the Golgi, secreted into the lymphatic system, then moved into the blood as a nascent chylomicron.
Apo E and Apo C-II from HDL added to the nascent CM forming complete CM.
Lipoprotein lipase (LPL) activated by Apo C-II and hydrolyzes up to 90% of triacylglycerol from lipoproteins releasing glycerol and free fatty acids
- LPL is a membrane-bound enzyme on endothelial cells of capillaries in most tissues other than liver
Apo C-II is returned to HDL.
Remaining particle is a CM remnant which are rapidly taken up by the liver via Apo-E receptor.
Endosome fuses with lysosomes where apolipoproteins and cholesteryl esters degraded releasing amino acids, free cholesterol, and fatty acids.

Abetalipoproteinemia

Defect in microsomal triacylglycerol transfer protein (MTP)
Results in an inability to load Apo B-48 with lipids
Causes marked reduction in CM production
- Also leads to decrease in VLDL formation
Inability to absorb fats from the diet
Leads to steatorrhea
Fat-soluble vitamin deficiency (especially Vit E)

Hyperlipoproteinemia Type I

Due to a defect in either lipoprotein lipase or apo C-II
Marked accumulation of CM particles in the plasma

VLDL

Characteristics

Produced in the liver
Consists predominantly of triacylglycerol
Functiosn to transport TAGs from the liver to the periphery

VLDL Lifecycle

Microsomal triglyceride transfer protein (MTP) loads lipids onto Apo B-100 in hepatocytes
- Defective in abetalipoproteinemia
Nascent VLDL released into blood where it picks up Apo E and Apo C-II from HDL.
As mature VLDL travels through the blood, lipoprotein lipase degrades the TAGs increasing the density of the particle to the intermediate density lipoprotein (IDL).
Some TAGs from VLDL exchanged for cholesteryl esters from HDL by cholesterol ester transfer protein (CETP).
Some IDLs can be taken up by the liver in an Apo E-mediated process.
Remainder is digested by LPL to produce low density lipoproteins (LDL).
Apo C-II and Apo E eventually returned to HDL.

LDL Characteristics

* LDLs have a higher concentration of cholesterol and cholesteryl esters than VDLDs * Main function to carry cholesterol as the ester to peripheral tissues

LDL Lifecycle

* **LDL receptors** (LDL-R) binds **Apo B-100** _on LDLs_ and **Apo E** _on IDLs_. * LDL-R found on the cell surface of various tissues including liver and muscle localized to clathrin-coated pits. * Binding triggers _endocytosis_ of LDL (or IDL). * Proton pump _reduces pH_ causing lipoprotein to _dissociate_ from the receptor. * Receptor-containing membrane recycled to the surface. * Lipoprotein _degraded_ in lysosomes to free cholesterol, fatty acids, phospholipids, and amino acids. * **Cholesterol** transferred to the SER where it can affect cellular cholesterol metabolism.

Acyl-CoA:Cholesterol Acyltransferase | (ACAT)

ACAT esterifies cholesterol with a fatty acid so it can be stored.

Intracellular Cholesterol Effects

Increased intracellular cholesterol can lead to a reduction in cholesterol content via several mechanisms: 1. HMG-CoA Reductase activity is inhibited by end-product inhibition, reducing de novo synthesis. 2. A rise in cholesterol increases proteosomal degradation of the reductase. 3. Transcription of both HMG-CoA Reductase and LDL-R genes is reduced via a SRE & SREBP mechanism. * Dec. reductase decreased de novo synthesis * Dec. LDL-R reduces cholesterol entry via inhibition of LDL internalization 4. Increases in cholesterol increase ACAT activity.

PCSK-9 (Proprotein Convertase Subtilisin Kexin 9)

* PCSK-9 is a serine protease which blocks LDL-R recycling to the cell surface. * Increased [cholesterol] → decreased PCSK-9 expression via SREBP-2 → increased LDL-R recycling → greater internalization of LDL → reduction in plasma LDL levels.

LDL & Atherosclerosis

* Macrophages have a non-specific scavenger receptor that is capable of internalizing LDL that has been oxidized by exposure to superoxide or other oxidants. * In response to endothelial cell injury, macrophages accumulate at the site of injury and phagocytize oxidized LDL becoming foam cells. * Foam cells form plaques in vessels leading to atherosclerosis * Narrowing of coronary vessels can lead to hypoxic damage and MI.

Familial Hypercholesterolemia | (FH)

* Genetic defects in the LDL pathway lead to accumulation of LDL in the plasma leading to increased atherosclerosis, angima, and MI. * LDL-R - most common * Apo B * PCSK-9 * Autosomal dominant * Heterozygous FH treated with statin inhibitors of HMG-CoA Reductase. * Homozygous FH usually requires more drastic treatments such as LDL apheresis or liver transpolant.

HDL Lifecycle

HDL plays an important role in reverse cholesterol transport & removal from periphery. * Lipid to added to **Apo A-1** in the blood to form **nascent HDL** * Apo A-1 produced by liver and intestine. * N-HDL particles consist of phospholipid and Apo A-1, Apo A-II, Apo C-II, and Apo-E. * Discoid shape * Cholesterol released from non-hepatic tissues by the **ABCA1 transporter** (aka cholesterol efflux regulatory protein - **CERP**) * Cholesterol _taken up_ by nascent HDL. * Cholesterol then _esterified_ to plasma enzyme **lecithin:cholesterol acyltransferase (LCAT)**. * _LCAT activated by Apo A-1_ on HDL surface and transfer the fatty acid from C-2 of **Phosphatidylcholine** to **cholesterol** to produce **lyso-PC** and **cholesteryl ester**. * As cholesteryl ester accumulate in HDL it becomes spherical. * Mature spherical HDL carries the cholesteryl esters to the liver and steroidogenic cells where they are taken up via the **scavenger receptor cell B type 1 (SR-B1)** * Cholesterol used for bile acid synthesis, disposal via bile, or hormone synthesis.

Lipoprotein(a)

* Lp(a) consists of LDL and a glycoprotein Apoprotein (a) covalently attached to Apo B-100 * Function of Lp(a) unknown * Elevated circulating Lp(a) levels associated with increased risk of CAD

Cholesterol & Heart Disease

High levels of LDL and low levels of HDL are independent risk factors for cardiovascular disease. * Total cholesterol and lipoprotein levels measured after 12 hour fast (no CM present) * Then again after precipitation of Apo B containing lipoproteins (VLDL and LDL) giving HDL-cholesterol * Seperate assay to determine triglyceride (TG) levels * _Friedewald equation_ used to obtain an estimate of HDL levels

Hypercholesterolemia Treatments

* Diet changes * Bile sequestrants (Cholestyramine) * Bind bile acids and prevents absorption * HMG-CoA Reductase inhibitors (statins) * Reduce cholesterol synthesis * Both approaches increase synthesis of LDL-Rs resulting in reduction in LDL-chol levels