Chapter 5.

Metabolism of Lipids

Lipids
Insoluble or immiscible

Triacylgerols
store and supply energy for metabolism.

Lipoids: phospholids, glycolipids, cholesterol and

cholesterol ester

membrane components

Metabolism of lipid

Fatty acids
esterified to some backbone molecules glycerol sphingosine cholesterol

Metabolism of Lipids

Fats
store in adipose tissue

Essential fatty acids: formation of membrane, regulation of chollesterol metabolism, precursors of eicosanoids (protaglandins, thromboxanes and leukotrienes. Necessary unsaturated fatty acids

Fat Facts
Dietary lipids are 90% triacylglycerols; also include cholesterol esters, phospholipids, essential unsaturated fatty acids; fat soluble vitamins (A,D,E,K)

Fat is energy rich and provides 9 kcal/gm

Normally essentially all (98%) of the fat consumed is absorbed, and most is transported to adipose for storage.

SIX STEPS OF LIPID DIGESTION AND ABSORPTION

Minor digestion of triacylglycerols in mouth and stomach by lingual (acidstable) lipase Major digestion of all lipids in the lumen of the duodenum/jejunum by pancreatic lipolytic enzymes Bile acid facilitated formation of mixed micelles that present the lipolytic products to the mucosal surface, followed later by enterohepatic bile acid recycling Passive absorption of the lipolytic products from the mixed micelle into the intestinal epithelial cell Reesterification of 2-monoacylglycerol, lysolecithin, and cholesterol with free fatty acids inside the intestinal enterocyte Assembly and export from intestinal cells to the lymphatics of chylomicrons coated with Apo B48 and containing triacylglycerols, cholesterol esters and phospholipids

Summary of the physiologically important lipases

Lipase lingual/acidstable lipase pancreatic lipase milk lipase Site of Action mouth, stomach small intestine small intestine Regulation ---colipase (+) Preferred Substrate TAGs with med. chain FAs TAGs with longchain FAs C cleaved 3 Product(s) FFA+DAG

1 and 3 FFA+2MG 1 and 2 FFA+ and 3 glycerol 2 1 and 2 and 3 3 Unsat FFA lysolecithin FFA+ glycerol FFA+DAG

bile acids (+) TAGs with med. chain FAs bile acids (+) PLs with unsat. Ca2+ (+) FA on position 2 apo CII (+) insulin (+) insulin (-) glucagon (+) Epineph. (+) TAGs in chylomicron or VLDL TAG stored in adipose cells

phospholipase small A2 (PLA2) intestine lipoprotein lipase capillary walls

Hormone-sens. adipose cell Lipase

Absorption of Lipids

Metabolism of Triacylglyerols

LIPOLYSIS

Mobilization of fats from triacylglycerols Hormone sensitive lipase

Rate-determining step
Specific for removing first fatty acid Phosphorylated form is active

cell membrane

Triacylglycerol
HORMONES Epinephrine Glucagon ATP HSL-a active +
protein kinase A

Fatty acid + Diacylglycerol OP

Adenylyl cyclase cyclic AMP

ADP

Insulin +
protein phosphatase

RECEPTORS

inactive
ATP

+ = activation - = inhibition

HSL-b phosphodiesterase OH - caffeine (inactive form) theophylline AMP HSL = hormone-sensitive lipase

Pi

Figure 1. Hormonal activation of triacylglycerol (hormone-sensitive) lipase. Phosphorylation brings about activation to HSL-a.

lipolysis

Glycerols and fatty acids

diffuse out of adipose cells and enter into circulation

Free fatty acids (FFA)

form fatty acid-albumin complexes

Glycerols
to form dihydroxyacetone phosphate (DHAP)

Figure. Page 176

Beta-Oxidation of Fatty Acids

Beta Oxidation Part I

The break down of a fatty acid to acetyl-CoA unitsthe glycolysis of fatty acids
STRICTLY AEROBIC

Occurs in the mitochondria

Acetyl-CoA is fed directly into the Krebs cycle

Overproduction causes KETOSIS Exemplifies Aerobic Metabolism at its most powerful phase

CH3CH2CH2COOH
ATP PPi [CH3CH2CH2CO-AMP] HS-CoA AMP Acyl-CoA synthetase

CH3CH2CH2CO~SCoA
Fatty acyl CoA

Prepares a Fatty Acid for transport and metabolism

Knoops Experiment
Diet (even chain) (odd chain)

CH2CH2CH2COO
Urine

CH2CH2COO

CH2COO
Phenylpyruvate Phenylacetate

COO
Benzoate Benzoate

Beta-Oxidation of Fatty Acids

THE ENERGY STORY

Glucose
C6H12O6 + 6O2 6CO2 + 6H2O Ho = -2,813 kJ/mol = - 672 Cal/mol = 3.74 Cal/gram Stearic Acid C18H36O2 + 26O2 18CO2 + 18 H2O Ho = -11,441 kJ/mol = - 2,737 Cal/mol = 9.64 Cal/gram

On a per mole basis a typical fatty acid is 4 times more energy rich that a typical hexose

Sample calculation of energy produced for the cell via b-oxidation of palmitate (a C16 fatty acid):

Palmitoyl-CoA
Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O

8 Acetyl-CoA
7 FADH2 7 NADH + 7H+

80 ATP
10.5 ATP 17.5 ATP 108 ATP -2 ATP Total 106 ATP

Beta Oxidation Part II

3 Obstacles
Unsaturated fatty acid
Obstacle of cis double bonds

Polyunsaturated fatty acid

Obstacle of position of double bond

Odd number chain fatty acid

Obstacle of 3 carbons at the end

Oleic Acid
C18:cis9 4 3 2 1
H H C=C CH3CH2CH2CH2CH2C CH2CH2CH2CH2CH2CH2CH2CO~SCoA

Linoleic

Whoops! A cis D.B. will interfere 5 4 3

H H H H C=C C=C CH3CH2CH2CH2 CH2 CH2CH2CH2CH2CH2CH2CH2CO~SCoA

Unsaturated and Polyunsaturated Require Additional Enzymes

New b carbon
Cleavage here
CH3CH2CH2 H H 9 C=C 8 7 CH2CH2CH2-CO~SCoA

Enoyl CoA Isomerase

New COO group

9

H 8 7 CH2C CH3CH2CH2 C-CO~SCoA H

Trans double bond

4
H H C=C CH2-CH2

H H 9 C=C CH2 CH2C~SCoA 2-CH2-CH22C~SCoA 2C~SCoA -CH2-CH 2C~SCoA -CH2CH CH CH O O O O

Linoleic Acid C18 cis 9,12

6 5 4 3 2 1

CH3C~SCoA O

CH3C~SCoA O

CH3C~SCoA O

Poly Unsaturated (Continued)

9 C=C C=C -CH2 CH2 CH2CO~SCoA
H H H H

H H

H H

Round 4 starter

C=C C-C -CH2 CH2 C-CO~SCoA

H H H

Enoyl-CoA isomerase

C=C CH2CO~SCoA -CH2 CH2 Beta carbon to be

Round 5 starter

C=C CH2CO~SCoA -CH2 CH2

FAD

H H

Round 5 starter

beta 6
H H

FADH2

C=C

H C-CO~SCoA C H

Acyl-CoA dehydrogenase
C=C CO~SCoA CH2 Dead end
H H

New Strategy

Reduce near (bond), Shift far (bond)

H H

C=C

H C-CO~SCoA C H
NADPH + H+
NADP+

beta 6
H

2,4 dienoyl-CoA reductase

CH2 C
beta 6

C H

CH2CO~SCoA H C-CO~SCoA

3,2 enoyl-CoA isomerase

-CH2 CH2 C H beta 6

Continue Beta Oxidation

Ketone bodies formation and utilization

An excessive production of ketones in the blood 3 derivatives of acetyl-CoA Acetoacetate b-hydroxybutyrate

CH3CCH2COOO H CH3CCH2COOOH O b

What is Ketosis?

Acetone

CH3-C-CH3

What is the Significance of ketosis

Acidosis
Excessive acid in the blood

Overflow
Excessive oxidation of fatty acids

Metabolic Problem
Faulty Carbohydrate Metabolism

Metabolic fate of Acetyl CoA

Pyruvate Fatty Acids

Acetyl-CoA
major

minor

Ketone Bodies

Citrate

CH3C~SCoA O

CH3C~SCoA O HS-CoA

CH2C~SCoA CH3C + O rearrangement OH

b-Ketothiolase

CH3CCH2C~SCoA O O

Acetoacetyl-CoA

CH3CCH2C~SCoA O O HS-CoA CH2C-OO CH3CCH2C~SCoA HO O

CH3C~SCoA

HMG-CoA Synthase

OH OOC-CH2-C-CH2-C~SCoA CH3 O

b-hydroxy-b-methyl glutaryl-CoA
(HMG-CoA)

OH OOC-CH2-C-CH2-C~SCoA

HMG-CoA

CH3

Acetoacetate
CO2

CH3-C~SCoA O OH + OOC-CH2-C-CH2-C~SCoA HMG-CoA Lyase CH3 O

OOC-CH2-C-CH3 O NADH + H+

CH3-C-CH3 Acetone

NAD+
OOC-CH2-CH-CH3 OH b-hydroxybutyrate

Utilization of ketone bodies

1.

2.
3.

Acetoacetate/succinyl-CoA CoA transferase Acetoacetyl-CoA thiokinase Acetoacetyl-CoA thiolase Page 180

Pysiological Significance of ketogenesis

Ketone bodies produced by the liver are excellent fuels for a variety of extrahepatic tissues, especially during times of prolonged starvation. Reconversion of ketone bodies to acetylCoA inside the mitochondria provides metabolic energy.

Regulation of Ketogenesis

Feeding status
In the hungry state, higher glucagon and other lipolytic hormones trigger the lipolytic process in adipose tissue with the result that free fatty acids pass into the plasma for uptake by liver and other tissues. This promotes fatty acid oxidation and ketogenesis in the liver.

Regulation of Ketogenesis

Metabolism of glycogen in the hepatic cells

once fats enter the liver, they have two distinct fates: activated to acyl-Co-A and oxidized, or esterified to glycerol in the production of triacylglycerols in cytoplasm. If the liver has sufficient supplies of glycerol3 phosphate by glucose metabolism, most of the fats will be turned to the production of triacylglycerols. In contrast, glucose deficiency will cause a lower triacylglycerols and ATP generation, with the majority of the FAs entering beta-oxidation leading to a increased production of ketone bodies.

Regulation of Ketogenesis

The fall in malonyl-CoA concentration can terminate the inhibition on carnitine acyltransferase I, such that long-chain fatty acids can be transported through the inner mitochondrial membrane to the enzymes of fatty acid oxidation and ketogenesis. This may happen during a hungry state. In contrast, administration of food after a fast, or of insulin to the diabetic subject, reduces plasma free fatty acid concentrations and increases liver concentration of malonyl-CoA, this will inhibit carnitine acyltransferase I and thus reverses the ketogenic process.

Fatty Acid Biosynthesis

Not exactly the reverse of degradation

by a different set of enzymes , in a different part of the cell

Primarily in the cytoplasm of the following tissues: liver, kidney, adipose, central nervous system and lactating mammary gland

Liver is the major organ for fatty acid synthesis

LIPID BIOSYNTHESIS

Fatty acid biosynthesis-basic fundamentals Fatty acid biosynthesis-elongation and desaturation Triacylglycerols Phospholipids Cholesterol Cholesterol metabolism

Fatty Acid Biosynthesis

Synthesis

Beta Oxidation

Cytosol Requires NADPH Acyl carrier protein D-isomer CO2 activation Keto saturated

Mitochondria NADH, FADH2 CoA L-isomer No CO2 Saturated keto

Rule:
Fatty acid biosynthesis is a stepwise assembly of acetyl-CoA units (mostly as malonyl-CoA) ending with palmitate (C16 saturated)

3 Phases
Activation

Elongation
Termination

ACTIVATION
O

Cofactor
Biotin
NH C 2C 2C 2C 2C H H H H O S N H C 2C 2C 2 E ZY E HC 2 H H H N M HN

CH3C~SCoA O

ATP
ADP + Pi
-OOC-CH

HCO3-

Biocytin
2C~SCoA

LY S

O
O

O C N

O NH

CO2

active carbon
S

C 2C 2C 2C 2C H H H H O

Acetyl-CoA carboxylase

Carboxybiocytin

Acetyl-CoA Carboxylase

The rate-controlling enzyme of FA synthesis

In Bacteria -3 proteins (1) Carrier protein with Biotin (2) Biotin carboxylase (3) Transcarboxylase In Eukaryotes - 1 protein (1) Single protein, 2 identical polypeptide chains (2) Each chain Mwt = 230,000 (230 kDa) (3) Dimer inactive (4) Activated by citrate which forms filamentous form of protein that can be seen in the electron microscope

Yeast Fatty Acid Synthase Complex 2,500 kDa Multienzyme Complex

6 molecules of 2 peptide chains called A and B

(6b6)

A: (185,000) Acyl Carrier protein b-ketoacyl-ACP synthase (condensing enzyme) b-ketoacyl-ACP reductase
B: (175,000) b-hydroxy-ACP dehydrase enoyl-ACP reductase palmitoyl thioesterase Fatty Acid Synthase Complex

Acyl Carrier Protein

Phosphopantetheine O HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-CH2-SerO OH H O Cysteamine
H

H HO CH3

ACP

Acyl carrier protein 10 kDa

O O HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-P-O-CH2 O O OH H O O O O-P-O OH
H

H HO CH3

Adenine
H

Coenzyme A

OH

Initiation

Overall Reaction
Malonyl-CoA + ACP
-OOC-CH 2C~S-

CH3C~SCoA O

ACP

+ HS-CoA

CO2

HS-CoA
ACP

Acyl Carrier Protein

CH3C-CH2C~SO O

NOTE: Malonyl-CoA carbons become new COOH end Nascent chain remains tethered to ACP CO2, HS-CoA are released at each condensation

b-Carbon
CH3C-CH2C~SACP

Elongation
O
O Reduction
b-Ketoacyl-ACP reductase

NADPH
D isomer

H CH3C-CH2C~SHO O

ACP Dehydration b -Hydroxyacyl-ACP dehydrase

-H2O NADPH

H CH3C- C- C~S- ACP = H O

Reduction

Enoyl-ACP reductase

CH3CH2CH2C~S- ACP O

TERMINATION
-KS

Ketoacyl ACP Synthase

Transfer to Malonyl-CoA

Transfer to KS -S-ACP

-CH2CH2CH2C~S- ACP O

Split out CO2

CO2

Free to bind Malonyl-CoA

When C16 stage is reached, instead of transferring to KS, the transfer is to H2O and the fatty acid is released

Fatty Acid Synthase

b-Ketoacyl -ACP synthase
KS

O S-C-CH2-CH2-CH3
KS

Acetyl-CoA HS CoA-SH

Acetyl-CoAACP transacylase

O CH3-CH2-CH2-C-S Enoyl-ACP reductase NADPH H+ O CH3-CH=CH-C-S b-Hydroxyacyl-ACP H2O

dehydrase

NADP+

O S -C-CH3
KS -SH
KS

Initiation or priming

ACP

O S-C-CH3

SH Malonyl-CoA
Malonyl-CoACoA-SH ACP transacylase

OH

CH3-CH -CH2-C-S NADP+ NADPH H+

S
C=O CH2 C=O CH3 CO2

O S -C-CH2-COOb-Keto-ACP synthase (condensing enzyme)

KS -SH

b-Ketoacyl -ACP reductase

Elongation

Overall Reactions
Acetyl-CoA + 7 malonyl-CoA + 14NADPH + 14H+ 7H + Palmitate + 7CO2 + 14NADP+ + 8 HSCoA + 6H2O

7 Acetyl-CoA + 7CO2 + 7ATP 7 malonyl-CoA +7ADP + 7Pi + 7H+

8 Acetyl-CoA + 14NADPH + 7H+ + 7ATP Palmitate + 14NADP+ + 8 HSCoA + 6H2O + 7ADP + 7Pi

PROBLEM: Fatty acid biosynthesis takes place in the cytosol. Acetyl-CoA is mainly in the Mitochondria
acetyl-CoA

How is acetyl-CoA made available to the cytosolic fatty acyl synthase?

SOLUTION: Acetyl-CoA is delivered to cytosol from the mitochondria as CITRATE

CH2COO HO-C-COO

HS-CoA

Acetyl-CoA

mitochondria
CH2COO HO-C-COO CH2COO Acetyl-CoA

CH2COO

Citrate lyase
COO C=O OAA Malate CH2 dehydrogenase COO
NADH

L-malate CO2

OAA
CO2 Pyr

COO HO-C-H L-malate CH2 COO Malic enzyme

NADP+

NADPH + H+ COO C=O Pyruvate CH3

Cytosol

Post-Synthesis Modifications

C16 satd fatty acid (Palmitate) is the product

Elongation Unsaturation Incorporation into triacylglycerols Incorporation into acylglycerol phosphates

Elongation of Chain (two systems)

R-CH2CH2CH2C~SCoA Malonyl-CoA* O (cytosol) HS-CoA OOC-CH2C~SCoA CH3C~SCoA O O CO2 Acetyl-CoA (mitochondria) R-CH2CH2CH2CCH2C~SCoA O O 1 NADPH Elongation systems are NADH found in smooth ER and 2 - H2O
3 NADPH

mitochondria

R-CH2CH2CH2CH2CH2C~SCoA O

Desaturation
Rules:
The fatty acid desaturation system is in the smooth membranes of the endoplasmic reticulum There are 4 fatty acyl desaturase enzymes in mammals designated 9 , 6, 5, and 4 fatty acyl-CoA desaturase Mammals cannot incorporate a double bond beyond 9; plants can. Mammals can synthesize long chain unsaturated fatty acids using desaturation and elongation

Triacylglycerol Synthesis

O O-C-R

O R-C-O

O O-C-R

Fatty acyl-CoA DHAP reduction to glycerol-PO4 or Glycerol kinase to glycerol-PO4 Two esterifications Diacylglycerol-PO4 intermediate Triacylglycerol

glycolysis CH2OH C=O CH2OP

DHAP O

Triacylglycerol Biosynthesis
CH2OH ADP ATP CH2OH HO-C-H HO-C-H glycerol kinase CH2OP CH2OH glycerol-PO4
dehydrogenase Glycerol-PO4
NADH + NAD+

2 R-C~CoA
Phosphatidic acid

O O CH2O-C-R R-C-O-C-H O CH2OP

H2O

Not in adipose tissue

R-C~CoA

O O CH2O-C-R Phospholipid R-C-O-C-H biosynthesis 1,2 Diacylglycerol CH2OH

(DAG)

PO4

O O CH2O-C-R R-C-O-C-H O CH2O-C-R

Question

Can a triacylglycerol (triglyceride) storage fat be synthesized entirely from glucose, i.e., every carbon in the fat comes from a sugar?
Answer: YES

Metabolism of Phospholipids

Phospholipid
phosphorous-containing lipids fatty acids, a phosphate group, and a simple organic molecule

Glycerolphospholipids (phosphoglycerides)
glycerol

Sphingolipid
sphingosine

Classification of structural features of glycerolphospholipids

Table 8-2

Phospholipids
hydrophilic head , hydrophobic tail

Membrane
phospholipid bilayer

Glycerolphospholipids

Phospholipid Biosynthesis (smooth ER)

O O CH2O-C-R R-C-O-C-H O CH2OPO O

Phosphatidic Acid

Ester linkage - or +

- or + Polar component

= choline, serine, ethanolamine, etc

Glycerophospholipids O O CH2O-C-R R-C-O-C-H O + CH2OPO-CH2CH2-N(CH3)3 O

Phosphatidylcholine or lecithin

Strategy of Glycerophospholipid Biosynthesis

Activate diacylglycerol N2 H Activate appending moiety (salvage)

N O N

PPP- Ribose CTP

Eukaryotes
DHAP 2
FA-CoA

1 Glycerol-3-PO4

ATP

Glycerol

1-Acyl-DHAP
NADPH

1-Acyl-glycerol-3-PO4

O O CH2O-C-R Phosphatidic acid 3 R-C-O-C-H DAG CH2OP ATP

CTP

O O CH2O-C-R R-C-O-C-H CH2OH 1,2 DAG

Pi

CDP-diacylglycerol ethanolamine (CDP-ethanolamine) choline (CDP-choline) Serine (phosphatidylethanolamine) Glycerol (CDP-diacylglycerol) Inositol (CDP-diacylglycerol) Cardiolipin (phosphatidylglycerol)

N2 H N O
+

(C 3) N C 2C 2 O P O P O C 2 H H H H 3 O O O O C ytidine diphosphate (C P) choline H D O H

N2 H N O
+

O O

O O C2 H O H O

H N C 2C 2 O P O P H H 3

C tidine diphosphate (C P)ethanolamine y D

O H

Regulation of Triacylglecerol Metabolism

Pancreas
primary organ involved in sensing the organisms dietary and energetic states.

monitoring glucose concentrations in the blood.

Low blood glucose stimulates the secretion of glucagon Elevated blood glucose calls for the secretion of insulin

Acetaly-CoA carboxylase (ACC)

Committed enzyme in fatty acid synthesis

activated by citrate inhibited by palmitoyl-CoA, long-chain fatty acyl-CoAs

Affected by phosphorylation
glucagon or epinephrine
decreased activity of ACC by phosphorylation

insulin
increases the synthesis of triacylglycerols

Important Derivatives of Unsaturated Fatty Acids- Arachidonic Acid

EICOSANOID FACTS
20-carbon compounds Include prostaglandins, prostacyclins, thromboxanes, leukotrienes

Physiological effects at very low concentrations

Many of their effects mediated by cyclic AMP or calcium second messengers Unlike hormones, not transported in the blood Local mediators that act where synthesized or in adjacent cells

The Actions of Prostaglandins and Leukotrienes a. the inflammatory response involving primarily the joints (rheumatoid arthritis) and skin (psoriasis); the production of pain and fever; the regulation of blood pressure (vaso-constrictors/dilators) and blood clotting (platelet function); decreased gastric acid secretion (prostacyclins may be an ideal way to control the symptoms of peptic ulcer, but prostanoid synthesis inhibitors, like aspirin, increase acid secretion causing peptic ulcer); the control of several reproductive functions such as the induction of labor and delivery - this has led to the use of PGF2 as a mid-trimester abortifacient drug or as a labor-inducing agent; the regulation of the sleep/wake cycle; hypersensitivity allergic reactions (a primary action of leukotrienes).

b. c.

d.

e.

f. g.

Dietary linoleic acid

metabolism
Arachidonic acid esterification

Cell Activation Events: mechanical trauma, cytokines growth factors

Anti-inflammatory glucocorticoids GC induce lipocortin that inhibits PLA2 Aspirin, Indomethacin, Ibuprofen NSAIDs

Membrane phospholipids Phospholipase A2 (PLA2) Arachidonic acid

Zyflo

Cyclooxygenase (COX)

Lipooxygenase (LOX) Leukotrienes (Linear product) Zyflo competes with AA for binding

Prostaglandins and thromboxanes (Cyclic/ring product)

Aspirin inhibits irreversibly Indomethacin forms a salt bridge in the binding site Ibuprofen competes for substrate binding

Figure 1. Liberation of arachidonic acid and its metabolism to prostaglandins/ thromboxanes or to leukotrienes

LEUKOTRIENE FACTS
leukotriene synthesis inhibited by Zyflo, a lipooxygenase inhibitor leukotriene action blocked by accolate, a receptor antagonist peptidoleukotrienes: leukotrienes with short peptides added components of slow reacting substances of anaphylaxis (SRS-A) anaphylaxis violent (potentially fatal) allergic reaction 10,000 times more potent than histamine SRS-A released from lung following immunological stress SRS-A contract smooth muscle causing constriction of bronchi implicated in hypersensitivity reaction such as insect sting

Arachidonic Acid (6) derived from membrane phospholipids

aspirin indomethacin ibuprofen

X
PGG2 2GSH

O2 Cyclooxygenase Prostaglandin endoperoxide synthase Hydroperoxidase

GSSG
PGH2 central intermediate (Head of pathway)

Figure 3. Conversion of arachidonic acid to PGH2

Figure 4. Structure and mechanism of action of aspirin

CH2 OH Ser

COOH O O C CH3 O C CH3 Cyclooxygenase O (active) C O 3 CH O C CH3 O O C CH3 CH2 C C 3 3 O CH CH COOH OH Ser

Acetylated Cyclooxygenase (inactive)

COX-1 VS COX-2 DRUG ACTION

Aspirin: works on both isoforms COX-1 effect reduces platelet aggregation (TXA2) COX-2 effect reduces inflammation Side effects due to COX-1 inhibition stomach irritation Specific COX-2 inhibitors Celebrex/Vioxx Target inflammatory response No COX-1 inhibition to produce aspirin-induced side effects

Metabolism of Cholesterols

Biosynthesis of Cholesterol Introduction

Functions of cholesterol. Important cell membrane component. Precursor for 3 biologically active compounds. Bile. Steroid hormones. Vitamin D. Disease implications. Cardiovascular disease. Diet control and synthesis manipulation = < heart disorders.

Biosynthesis of Cholesterol Introduction

Disease implications.

Gall stones. Steroidogenic enzyme deficiency. Meat. Eggs. Dairy products. De novo liver synthesis.

Source of cholesterol.

Cholesterol Synthesis

OH CH2 C CH3 CH2

O C SCoA

hydroxymethylglutaryl-CoA

Hydroxymethylglutaryl-coenzyme A (HMG-CoA) is the precursor for cholesterol synthesis. HMG-CoA is also an intermediate on the pathway for synthesis of ketone bodies from acetyl-CoA.

The enzymes for ketone body production are located in the mitochondrial matrix.
HMG-CoA destined for cholesterol synthesis is made by equivalent, but different, enzymes in the cytosol.

O H2O O H3C SCoA HSCoA O

O CH2 C SCoA

acetoacetyl-CoA HMG-CoA Synthase

OH CH2 C CH3 CH2 O C SCoA

acetyl-CoA

H3 C

hydroxymethylglutaryl-CoA

HMG-CoA is formed by condensation of acetyl-CoA & acetoacetyl-CoA, catalyzed by HMG-CoA Synthase. HMG-CoA Reductase catalyzes production of mevalonate from HMG-CoA.

The carboxyl of HMG that is in ester linkage to the CoA thiol is reduced to an aldehyde, and then to an alcohol. NADPH serves as reductant in the 2-step reaction. Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA.

HO C H2C

CH3 CH2 C O SCoA

C O

HMG-CoA HMG-CoA Reductase

CH3 CH2 H2 C OH

2NADPH 2NADP+ + HSCoA HO C H2C

C O

mevalonate

HMG-CoA Reductase is an integral protein of endoplasmic reticulum membranes. The catalytic domain of this enzyme remains active following cleavage from the transmembrane portion of the enzyme. The HMG-CoA Reductase reaction, in which mevalonate is formed from HMG-CoA, is ratelimiting for cholesterol synthesis. This enzyme is highly regulated and the target of pharmaceutical intervention.

Mevalonate is phosphorylated by 2 sequential Pi transfers from ATP, yielding the pyrophosphate derivative.

HO C H2C

CH3 CH2 CH2 OH

C O

mevalonate
2 ATP (2 steps) 2 ADP O CH2 O P O ATP ADP + Pi O CH2 CH2 O P O O O P O O O O P O O

HO C H2C

CH3 CH2

ATP-dependent decarboxylation, with dehydration, yields isopentenyl pyrophosphate.

C O

5-pyrophosphomevalonate
CO2

CH3 C H2C

isopentenyl pyrophosphate

CH3

Isopentenyl pyrophosphate is the first of several compounds in the pathway that are referred to as isoprenoids, by reference to the compound isoprene.

C H2C C H2

H2 C O

O P O O

O P O O

isopentenyl pyrophosphate

CH3 C H2C C H CH2

isoprene

CH3 C H2C CH2 CH2 O O P O O O P O O

isopentenyl pyrophosphate

CH3 C H3C CH CH2 O O P O O O P O O

dimethylallyl pyrophosphate

Isopentenyl Pyrophosphate Isomerase inter-converts isopentenyl pyrophosphate & dimethylallyl pyrophosphate. Mechanism: protonation followed by deprotonation.

Condensation Reactions
Prenyl Transferase catalyzes head-to-tail condensations:

Dimethylallyl pyrophosphate & isopentenyl pyrophosphate react to form geranyl pyrophosphate. Condensation with another isopentenyl pyrophosphate yields farnesyl pyrophosphate. Each condensation reaction is thought to involve a reactive carbocation formed as PPi is eliminated.

CH3 H3C C CH CH2 O

O P O O

O P O H2C PPi O CH3 C CH2 CH2 O O P O O O P O O

dimethylallyl pyrophosphate

isopentenyl pyrophosphate
O O O P O O CH2 CH2 O P O O O P O O O P O CH3

CH3 H3C C CH CH2 CH2

CH3 C CH CH2 O

geranyl pyrophosphate
H2C PPi CH3 H3C C CH CH2 CH2 CH3 C CH CH2 CH2

isopentenyl pyrophosphate
CH3 C CH CH2 O O P O O O P O O

farnesyl pyrophosphate

Each condensation involves a carbocation formed as PPi is eliminated.

CH3

CH3 CH CH2 CH2 C CH CH2

CH3 CH2 C CH CH2 O

O P O O

O P O O

2 H3C

NADPH NADP+ + 2 PPi

2 farnesyl pyrophosphate

NADP+ NADPH

H+

O2

H2O

HO

squalene

2,3-oxidosqualene

lanosterol

Squalene Synthase: Head-to-head condensation of 2 farnesyl pyrophosphate, with reduction by NADPH, yields squalene.

NADP+ NADPH

H+

O2

H2O

HO

squalene

2,3-oxidosqualene

lanosterol

Squaline epoxidase catalyzes conversion of squalene to 2,3-oxidosqualene. This mixed function oxidation requires NADPH as reductant & O2 as oxidant. One O atom is incorporated into substrate (as the epoxide) & the other O is reduced to water.

Squalene Oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization.

H+

HO

2,3-oxidosqualene

lanosterol

Structural studies of a related bacterial enzyme have confirmed that the substrate binds at the active site in a conformation that permits cyclization with only modest changes in position as the reaction proceeds. The product is the sterol lanosterol.

19 steps
HO HO

lanosterol

cholesterol

Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes in ER membranes. Additional modifications yield the various steroid hormones or vitamin D. Many of the reactions involved in converting lanosterol to cholesterol and other steroids are catalyzed by members of the cytochrome P450 enzyme superfamily.

Regulation of cholesterol synthesis

HMG-CoA Reductase, the rate-limiting step on the pathway for synthesis of cholesterol, is a major control point. Short-term regulation: HMG-CoA Reductase is inhibited by phosphorylation, catalyzed by AMP-Dependent Protein Kinase (which also regulates fatty acid synthesis and catabolism). This kinase is active when cellular AMP is high, corresponding to when ATP is low. Thus, when cellular ATP is low, energy is not expended in synthesizing cholesterol.

Long-term regulation is by varied formation and degradation of HMG-CoA Reductase and other enzymes of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:

Degradation of HMG-CoA Reductase is stimulated by cholesterol, oxidized derivatives of cholesterol, mevalonate, & farnesol (dephosphorylated farnesyl pyrophosphate).
HMG-CoA Reductase includes a transmembrane sterol-sensing domain that has a role in activating degradation of the enzyme via the proteasome (proteasome to be discussed later).

Long-term regulation is by varied formation and degradation of HMG-CoA Reductase and other enzymes of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:

Degradation of HMG-CoA Reductase is stimulated by cholesterol, oxidized derivatives of cholesterol, mevalonate, & farnesol (dephosphorylated farnesyl pyrophosphate).
HMG-CoA Reductase includes a transmembrane sterol-sensing domain that has a role in activating degradation of the enzyme via the proteasome (proteasome to be discussed later).

Lipid transport
triacylglycerides, cholesterol, phospholipids

dietary lipid transport chylomicron endogenous lipid transport (VLDL, IDL, LDL, HDL)

Dietary uptake and distribution of fatty acids

intestinal lumen triacylglycerols epithelial cells triacylglycerols FFA + monoacylglycedrols bile acids absorbed by intestinal cholesterol epithelial cells and micelles reconverted to triacylglycerols pancreatic lipases

Packaged into chylomicron Released into lymphatic system and then via capillaries to blood stream chylomicron acted upon by lipases on cell walls of capillaries in tissues

energy production
FFA taken up by tissues reconversion to TAGs in adipocytes for storage hormone sensitive lipases

FFA released to circulatory system and combine with albumin for delivery to tissues

Why do we need lipoproteins?

Triacylglycerides (TAGs) + cholesterol (Chol) are nonpolar molecules insoluble in H2O TAG + Chol must be packaged within a polar shell in order to be transported through the blood to the various tissues This is accomplished by combining nonpolar lipids w/ amphipathic lipids (a polar water-soluble terminal group attached to an H2O -insoluble hydrocarbon chain)

Lipoproteins & Apolipoproteins

Lipoproteins (LP) function: transport of cholesterol + esterified lipids in blood structure: 1) polar shell ---single phospholipid (PL) layer: head groups directed outward -Chol -apolipoproteins 2) nonpolar lipid core -hydrophobic TAG(triacylglycerol) -cholesteryl ester (CE)

apolipoproteins
Provide structural stability to Lp
Act as cofactors for enzymes involved in plasma lipid and Lp metabolism

Serve as ligands for interaction w/Lp receptors that help determine disposition of individual particles

There are many types of apolipoproteinsa

Apoprotein Lipoproteins Function(s) Secretion of VLDL from liver 2) Structural protein of VLDL, IDL, and HDL 3) Ligand for LDL receptor (LDLR)
1)

Apo B-100 VLDL, IDL, LDL

Apo B-48 Apo E Apo A-I Apo A-II

Chylomicrons, remnants Chylomicrons, VLDL, IDL, HDL HDL, chylomicrons HDL, chylomicrons

Secretion of chylomicrons from intestine; lacks LDLR binding domain of Apo B-100 Ligand for binding of IDL & remnants to LDLR and LRP Major structural protein of HDL 2) Activator of LCAT
1)

Unknown

Apo C-I
Apo C-II
Apo C-III

Chylomicrons, VLDL, IDL, HDL

Chylomicrons, VLDL, IDL, HDL Chylomicrons, VLDL, IDL, HDL

Modulator of hepatic uptake of VLDL and IDL (also involved in activation of LCAT)
Activator of LPL Inhibitor of LPL activity

Lipoprotein Structure

Lipoproteins
hydrophobic core (TAGS, cholesterol esters) hydrophilic surface (P-lipids, cholesterol, and apolipoproteins)

Function
transport of lipids in blood

Types of lipoproteins
(classified according to density) very low density (VLDL) intermediate density (IDL) low density (LDL) high density (HDL) Protein content increase, lipid decreases as density increases.

85% Chylomicron 2%

% TAGS VLDL IDL % Protein

8%
LDL HDL 33%

nm

Lipoproteins
Chylomicron:
85% TAG, 4% chol., 8% protein

formed in intestinal epithelial cells deliver exogenous TAGS to tissue 80 -500nm ApoCII activates lipases in capillary cell walls releasing FFA to tissue chylomicron remnants return to liver where they bind to ApoE receptor and are taken up 1/2 life in blood - 4-5 minutes

 VLDL:
50% TAGs, 22% choles., 10% protein

30 -100 nm formed in liver deliver endogenous lipids to other tissues (mainly muscle and fat cells) ApoCII activates lipases in capillary cell walls releasing FFA to tissue converted to IDLs and LDL as lipids are released

Lipoproteins
IDL: (31% TAGs, 29% choles., 18% protein) formed from VLDLs as lipids removed some IDLs return to liver rest converted to LDLs by further removal of lipids

 LDL: bad cholesterol 10% TAGs, 45% choles., 25% protein 25 - 30 nm formed as lipids removed from VLDLs and IDLs. all apolipoproteins lost except ApoB100 bind to LDL receptor via ApoB100 and taken up by endocytosis by hepatic and other tissues (50-75% taken up by liver). Primary mode of cholesterol delivery to tissues. Synthesis of LDL receptor is inhibited by high levels of intracellular cholesterol and stimulated by low levels of cholesterol. Therefore, cholesterol uptake is closely matched to intracellular cholesterol levels.

Lipoproteins
HDL: good cholesterol

8% TAGs, 30% choles., 33% protein 7.5 - 10 nm formed in liver scavenge cholesterol from cell surfaces and other lipoproteins and deliver it to liver. Convert cholesterol to cholesterol ester bind to scavenger receptor on liver cell surface - cholesterol esters taken up and HDLs released and reenter circulation.

Intestine Dietary lipids

Liver
Triacylglycerols cholesterol Cholesterol esters

HDL chylomicron

VLDLs

LDLs
Triacylglycerols FFA monoacylglycerols Cholesterol Cholesterol esters

HDLs

Peripheral tissues

Distribution of endogenous lipids The Exogenous Pathway Liver Intestine Dietary lipids

ApoE/LDLR mediated uptake

chylomicron

acquire ApoE, CII and others

Chylomicron remnants

LPLs activated by ApoCII

Triacylglycerols FFA monoacylglycerols Cholesterol Cholesterol esters

Peripheral tissues

Distribution of endogenous lipids The Endogenous Pathway Liver

Triacylglycerols cholesterol Cholesterol esters acquire ApoE, CII and others

VLDLs IDLs
LDLR/ApoE LDLR/ApoB100 LPLs activated by ApoCII

LDLs
Triacylglycerols FFA monoacylglycerols Cholesterol Ester Cholesterol

Peripheral tissues

Distribution of endogenous lipids The HDL Pathways Transport of excess cholesterol from peripheral tissues back to liver for excretion in bile HDLs act as acceptors for excess chol, Apo, PL derived from CM, VLDL and LDL HDLs synthesized by both liver and intestine

Distribution of endogenous lipids The HDL Pathways Liver scavenger receptor

Triacylglycerols cholesterol Cholesterol esters

uptake of cholesterol

HDL
VLDLs IDLs
CEs

LDLs
Triacylglycerols FFA monoacylglycerols

TAGs

HDLs VLDL Choles.

Peripheral tissues

Cholesterol Ester Cholesterol

Abnormal Metabolism of Lipoprotein

Hyperlipoproteinemia Genetic diseases