Metabolism of Triacylglycerol John O. Thomas

Lipids are biological molecules that are insoluble or only sparingly soluble in water. These include triacylglycerols (also known as triglycerides), phospholipids, cholesterol (often in ester form), glycolipids, steroids, fat-soluble vitamins, and other compounds present in smaller amounts. These lectures will deal primarily with the metabolism of triacylglycerols. Cholesterol metabolism and steroid metabolism will be presented more extensively in the third part of this course, and phospholipids will be presented in the cell biology course.

O || O H2C - O - C - R1 || | R2 - C - O - CH | H2C - O - C - R3 || O Triacylglycerol

The body stores triacylglycerol for use as an energy source during times of starvation. A triacylglycerol is a glycerol esterified with three fatty acids, the fatty acids being the energy-rich part of the triacylglycerol. Triacylglycerols are highly insoluble in water, which is an advantage for storage since they can be stored without any added weight from associated water. Fatty acids consist of a hydrophobic hydrocarbon chain and a hydrophilic, terminal carboxyl group (-COOH;), which is ionized (-COO-) at physiological pH. Fatty acids are therefore amphipathic; that is, they have both hydrophobic and hydrophilic characteristics. Most naturally occurring fatty acids have a hydrocarbon chain of 14 - 20 carbons (these are commonly called long-chain fatty acids), and can be placed within one of three categories: saturated (no double bonds), monounsaturated (one double bond), and polyunsaturated (more than one double bond).

Palmitate Palmitic acid Cis-palmitoleic acid (18:1∆9) (an ω7 fatty acid) Trans-palmitoleic acid Figure 1 Fatty acid structures. The cis double bond of naturally occurring unsaturated fatty acids places a kink in the molecule that lowers the melting point of triacylglycerols and increases the fluidity of membranes. Trans-fatty acids have physical properties similar to saturated fatty acids.

∆9 bond

ω7 carbon

Carbon number 1 β-carbon (carbon number 3) ω-carbon

1

Fatty acids have both chemical names and common names. The common names are more frequently used in the medical literature and are listed in the following table. Linoleic acid and linolenic acid are required compounds that can not be synthesized by the body and must be taken in the diet. These are essential fatty acids. Arachidonic acid can be synthesized from linoleic acid so it is not an essential fatty acid.

Number of carbons: Number and positions of double bonds

Common name Physiological importance

Saturated Fatty Acids 1:0 Formic 2:0 Acetic 3:0 Propionic 4:0 Butyric 10:0 Capric

Important components of breast milk.

16:0 Palmitic 18:0 Stearic

Major components of triacy-lglycerols and structural lipids.

Monounsatruated Fatty acids

16:1 ∆9 (ω7) Palmitoleic 18:1 ∆9 (ω9) Oleic

Major components of triacyl-glycerols and structural lipids.

Polyunsaturated Fatty acids

18:2 ∆9,12 (ω6) Linoleic 18:3 ∆9,12,15 (ω3) Linolenic

Essential fatty acids: must be taken in diet.

20:4 ∆5,8,11,14 (ω6) Arachidonic Precursor of eicosinoids. The symbols ∆ and ω refer to two different systems for denoting the positions of double bonds in unsaturated fatty acids as illustrated in figure.1.

An omega-3 fatty acid

Nearly all naturally occurring unsaturated fatty acids have their double bonds in the cis configuration. Chemically processed fats such as margarine or fats heated to high temperatures contain a mixture of cis and trans double bonds. While the body is able to metabolize both cis and trans double bonds, cis and trans fatty acids have different physical properties that may affect their metabolism. Some epidemiological evidence correlates an increased intake of trans as opposed to cis fatty acids with an increased risk of coronary artery disease. The following table shows the fatty acid composition of some common foods.

Food: % Saturated % Trans % Monounsat. %polyunsat.Olive oil 16% 0.1% 74% 10% Sunflower oil 13% 0.5% 17% 70% Cheddar cheese 72% 2.6% 24% 4% Margarine - tub 19% 16.9% 43% 38% Margarine - stick 19% 31.8% 60% 20% French fries (typical values) 29% 32.3% 62% 9%

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Oxidative damage to polyunsaturated fatty acids (PUFA). Lipids are easily oxidized. Oxidized lipids are dangerous. They are taken up by macrophages, contributing to their transformation into foam cells. Foam cells are a major factor in the development of atherosclerosis. Vitamins E and C are important anti oxidants that, together with glutathione, protect against this damage.

I. A free radical (R·) can initiate a chain reaction that results in the formation of many molecules of PUFA peroxides. The acronym PUFA refers to polyunsaturated fatty acid. PUFA peroxides are highly reactive compounds that can cause further damage to lipids and cells.

R· RH O2 PUFA PUFA· PUFA PUFA radical PUFA peroxide radical PUFA preoxide

Chain reactionR· is any free radical, including a PUFA radical

II. Vitamin E, Vitamin C, Glutathione and NADPH function together to limit oxidative damage to PUFAs

2. Vitamin E, which is lipid soluble, reacts with the PUFA radical. The vitamin E radical that is formed is chemically stable

PUFA· PUFA

2 GSH G-S-S-G

Dehydro- vitamin C Vitamin C· Vitamin C

Vitamin E Vitamin E·

NADP+

NADPH

+

1. A dangerous PUFA free radical is formed.

4. Glutathione reduces the oxidized vitamin C.

3. Vitamin C picks up radicals from two molecules of vitamin E. Vitamin C is water soluble and forms a stable radical.

5. Oxidized glutathione is reduced by NADPH obtained from the pentose phosphate pathway.

III. Glutathione peroxidase detoxifies PUFA peroxides:

PUFA peroxide PUFA hydroxide

2 GSH G-S-S-G

NADP+

NADPH + H+

Glutathione peroxidase

Glutathione reductase

3

Overview of Tissues involved in Triacylglycerol Metabolism

FED STATE

Triacylglycerol metabolism in the liver

I. Fatty acid synthesis in the liver Overview: When a person is on a high carbohydrate diet, a major function of glycolysis in the liver is to provide acetyl-CoA for fatty acid synthesis. Acetyl-CoA gives rise to malonyl-CoA, which is the major source of carbons for fatty acid synthesis. Fatty acid synthesis also requires a large amount of NADPH, some of which is derived from the pentose pathway, and some of which is derived from the malic enzyme.

Glucose Monoacylglycerol Fatty acid

Glucose Intestin Liver

Fatty acids

T.G. Synthesis

Muscle Adipose Use

Storage Glycogen T.G.(Small)

Ketone Bodies

Fatty acids Storage Energy

Brain Liver

Adipose Muscle

STARVED STATE

Energy

Energy Glucose Amino acids

(from muscle)

Glycogen

Triacylglycerol (CM/VLDL)

OVERVIEW OF SUBSTRATE FLOW

4

A. Production of cytosolic acetyl CoA Glycolysis, the pentose phosphate pathway and pyruvate dehydrogenase convert glucose to acetyl-CoA that is located inside of mitochondria. Cytosolic acetyl-CoA is required for fatty acid synthesis, but the mitochondrial acetyl-CoA can not pass through the mitochondrial inner membrane and the inner mitochondrial membrane has no transporter for acetyl-CoA. To obtain cytosolic acetyl-CoA, citrate leaves the mitochondria via the citrate-malate transporter (Fig. 2), citrate gives rise to acetyl-CoA and malate in the cytosol, and malate then re-enters the mitochondria via the citrate-malate transporter.

Fatty acid Glucose

Citrate lyase

CoA

Acetyl-CoA Citrate

Oxaloacetate Malate

NADH + H+ NAD+

Citrate

Malate Oxaloacetate

NAD+ NADH + H+

Acetyl-CoA

CoA ATP ADP + Pi

Citrate synthase

Malate dehydrogenase(mitochondrial)

Malate dehydrogenase(cytosolic)

Citrate – malate transporter

Outer membraneInner membrane

Mitochondrial matrix Cytosol

Pyruvate

+

CO2 ATP ADP + Pi

B. Formation of malonyl-CoA is the committed step of fatty acid synthesis. Fatty acids are synthesized by the sequential addition of two-carbon units. These carbons are

derived from malonyl-CoA, a three carbon compound. In the process, one carbon of malonyl-CoA is lost as CO2, providing some of the energy required for fatty acid synthesis. The concentration of malonyl-CoA in the cell limits the rate of fatty acid synthesis.

1. Malonyl-CoA is synthesized from cytosolic acetyl-CoA by acetyl-CoA carboxylase (ACC). Like other carboxylases, acetyl-CoA carboxylase contains biotin as a cofactor. 2. Acetyl-CoA carboxylase is regulated such that fatty acid production is stimulated when both blood glucose is high and the energy needs of the cell have been met.

a. Allosteric regulation: i. Activated by citrate (a precursor of fatty acid synthesis ii. Inhibited by palmitoyl-CoA (a product of fatty acid synthesis)

b. Phosphorylation inhibits the enzyme. Phosphorylation is by: i. Protein kinase A (PKA; stimulated by cAMP via glucagon) ii. AMP activated protein kinase (AMPK).

c. Transcriptional regulation: High carbohydrate diets increase the synthesis of acyetyl-CoA carboxylase, snd other enzymes involved in fatty acid synthesis are also increased (e.g. fatty acid synthase, ATP-citrate lyase, glucose-6-phosphate

5

dehydrogenase). The mechanism of this transcriptional regulation is described below

3. Malonyl CoA also functions as the primary regulator of fatty acid oxidation Thus, fatty acid synthesis and fatty acid oxidation do not occur at the same time. There are two isozymes of ACC.

a. ACC1 (also known as ACC-alpha) is located in cells that actively synthesize fatty acids in the liver and lactating mammary gland.

b. ACC2 (also known as ACC-beta) is located in most cells. It synthesizes small amounts of malonyl-CoA as a regulator of fatty acid oxidation.

C. Fatty acid synthesis requires reducing power in the form of NADPH + H+

1. Up to half of the required NADPH + H+ can be produced by the pentose phosphate pathway. Because of the large amount of NADPH that is required, the pentose phosphate pathway becomes a predominant pathway of glucose metabolism of the liver when blood glucose is high. The amount of glucose-6-phosphate dehydrogenase is increased in people who eat a diet rich in carbohydrate.

Malonyl-CoA

Acetyl-CoA

Acetyl CoA carboxylase

(Active)

Citrate

PP

Phosphorylated

(Inactive)

Acetyl CoA carboxylase

H2O Pi

ADP + Pi ATP

Xylulose-5-phosphate

Protein phosphatase

Transcription

PKA AMPK

+

Glucagon AMP

─+

+

+

+

Acetyl CoA carboxylase(Inactive)

Palmitoyl-CoA

Insulin

CO2 ATP ADP + Pi

Glucagon, AMP, Palmitoyl-CoA slow fatty acid synthesis Insulin, citrate, xylulose-5-P speed up fatty acid synthesis

6

2. The remainder of the needed NADPH is produced by the malic enzyme and a shuttle

system that transports malate out of the mitochondria and pyruvate back into the mitochondria:

H2PO4 H2PO4

hosphate carrier

NAD+

NADP+

Pyruvate carboxylase

Malate dehydrogenase

CO2 + ADP + Pi ATP NADH + H+

Malate Pyruvate Oxaloacetate

O

IMitochondrial matrix

Malate

CyMalic enzyme

Pyruv

HPO4 HPO4

Dicarboxylate carrier

Net half reaction: NADH + (mitochon

D. Fatty acids are synthesized by a single multifun Fatty acid synthesis is initiated using acetyl-CoA

of two carbon units derived from malonyl-CoA. reduced, dehydrated and reduced again. All of thenzyme, fatty acid synthase. The fatty acid is rellength of 16 or 18 carbons, the primary product bpalmitic acid. The overall reaction is

Acetyl-CoA + 7 Malonyl-CoA + 14NADPH + Fatty acid synthase Palmitic acid + 7CO2 + 14 NADP+ + 8CoA + 6

7

nner membrane

CO2 + uter membrane

Malic enzy

tosol

carr

ATP

H+ drion)

ctional e and procEach twoese reacteased froeing the

14H+

H2O

NADPH + H+

Pyruvateme

ADP + Pi

NADPH + H+

(cytosol)

nzyme eeds by the sequential ad-carbon unit that is addeions are catalyzed by a sm the enzyme once it rea16-carbon saturated fatty

H+

ate ier

H+

H+

H+

P

dition d is ingle ches a acid,

E. Fatty acid activation, elongation and desaturation. 1. Activation of fatty acids: formation of acyl-CoAs Metabolic reactions involving fatty acids require that the fatty acids be converted to

acyl-CoA. This is necessary for elongation and desaturation (described in this section), for β-oxidation (described in section III), for triacylyglycerol synthesis and for phospholipid and glycolipid synthesis. The thioester bond that joins the fatty acid and the CoA is a high energy bond.

O O || ||

R-C-OH R-C-S-CoA Fatty acid acyl-CoA

Fatty acid + CoA + ATP Acyl-CoA + AMP + PPi

AcylCoA synthetase

A thioester

There are multiple isozymes of acyl-CoA synthase that differ in their subcellular location and in their specificity for the chain length of the fatty acid. a. The isozyme that is used for fatty acid biosynthetic reactions, including elongation

and desaturation. is located on the endoplasmic reticulum. b. Other isozymes of acyl-CoA synthetase are involved in fatty acid oxidation (see

below) and are located on the mitochondrial outer membrane, and on the inside of the mitochondrial inner membrane.

2. Elongation of fatty acids a. Palmitic acid (16 carbons) produced by fatty acid synthase can be elongated in two

carbon units. Most tissues can elongate palmitate to produce 18 and 20 carbon fatty acids.

b. Very long chain fatty acids (>20 carbons) are produced by neural tissues.for the synthesis of phosphlipids and glycolipids.

3. Desaturation of fatty acids Fatty acids can be desaturated by enzymes that insert double bonds between the carbons 9 and 10 from the COOH end. Desaturation can also occur closer to the COOH end, but not further away (hence linoleic acid and linolenic acid must be taken in the diet).

F. Regulation of fatty acid synthesis

1. Pyruvate carboxylase is activated by acetyl-CoA thus providing an adequate supply of oxaloacetate and hence citrate for transport to the cytosol.

2. The major point of regulation is acetyl-CoA carboxylase (see above). 3. The entire block of enzymes involved in fatty acid synthesis is regulated at the

transcriptional level, under control of a transcription factor termed carbohydrate response element binding protein (ChREBP). ChREBP is inactivated through phosphorylation by protein kinase A (via cAMP, which is elevated in response to glucagon). ChREBP is activated by removal of the phosphate by protein phosphatase2A (the same phosphatase that removes the phosphate from phosphofructokinase 2). As discussed in the carbohydrate lectures, this phosphatase

8

is stimulated by xyulose-5-phosphate, which is elevated under conditions of high glucose availability (much of the glucose passes through the pentose phosphate pathway in route to fatty acid synthesis).

II. Triacylglycerol production in the liver and export to the blood.

A. The synthesis of triacylglycerol is unregulated, and limited only by the availability of the substrates: glycerol-3-phosphate and acyl-CoAs. 1. Glycerol-3-phosphate for triacylglycerol synthesis is obtained from dihydroxyacetone

phosphate, an intermediate of glycolysis.

NADH + H+ NAD+

Dihydroxyacetone phosphate Glycerol-3-phosphate Glycerol-3-phosphate dehydrogenase

H2C-OH | O=C O - | | H2 C-O-P-O - || O -

Dihydroxyacetone phosphate

H2C-OH | HO-CH O - | | H2 C-O-P-O - || O - L-Glycerol 3-phosphate

2. In the liver, triacylglycerol is formed by the sequential addition of acyl-CoAs to glycerol 3-phosphate (a different pathway is used in the intestine).

2 Acyl-CoA 2 CoA H2O Pi Acyl-CoA CoA

Glycerol 3-phosphate Phosphatidate Diacylglycerol Triacylglycerol O

|| O H2C-O – C R1 || | R2 C –O -CH O | || H2 C-O – C R3

Triacylglycerol

O || O H2C-O – C R1 || | R2 C –O -CH

| H2 C-OH

Diacylglycerol

O || O H2C-O – C R1 || | R2 C –O -CH O -

| | H2 C-O- P-O - || O - Phosphatidate

B. Export of triacylglycerol. 1. The liver exports lipids into the circulation in the form of Very Low Density

Lipoprotein (VLDL) particles. These particles contain triacylglycerol, cholesterol

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cholesterol ester, phospholipids, and one molecule of apolipoprotein B-100. Several other proteins are also associated with the VLDL particle.

2. Apolipoprotein B-100 is a huge protein (4536 amino acids). As it is being synthesized, the N-terminus forms a nucleus for the generation of the VLDL particle.

3. As the apolipoprotein B-100 protein continues to be synthesized, microsomal triacylglycerol transfer protein (MTP) delivers triacylglycerol to be packaged by the still growing peptide chain. MTP also assists in the further maturation of the VLDL.

4. The rate of synthesis of apolipoprotein B-100 limits the rate at which triacylglycerol can be exported from the liver. If the rate of synthesis of triacylglycerol is faster than apolipoprotien B-100 synthesis, triacylglycerol will accumulate, giving rise to a fatty liver.

III. Fatty acid oxidation

A. The liver uses fatty acids as its primay energy source. Sources of the fatty acids that enter the cytosol of the liver include:

Fasting State: - free fatty acids released from adipose tissue

Fed State: - free fatty acids released from chylomicrons by lipoprotein lipase located on

extrahepatic tissues (lipoprotein lipase is discussed below). - fatty acids produced by the action of hepatic lipase on lipoproteins (primarily IDL

and LDL). Hepatic lipase is anchored to the outside of the cell. It is homologous in structure and function to lipoprotein lipase, which is discussed below.

- chylomicron remnants and low density lipoproteins (LDL) that have been taken up by the cell into lysosomes where triacylglycerol and phospholipids are hydrolyzed by lysosomal enzymes.

The transport processes that get these molecules to the liver are discussed below. Inside of the cell, fatty acids are associated with Fatty Acid Binding Protein (FABP), which facilitates their transport into and within the cell.

B. Fatty acids that enter the cell are converted to their CoA thioesters by an acyl-CoA synthetase located on the outer mitochondrial membrane.

O O || ||

R-C-OH R-C-S-CoA Fatty acid acyl-CoA

Fatty acid + CoA + ATP Acyl-CoA + AMP + PPi

AcylCoA synthetase

A thioester

C. Fatty acid oxidation takes place in the mitochondria. The rate of transport into the

mitochondria is the rate controlling step of fatty acid oxidation. 1. In order to be transported into the mitochondrion, acyl-CoA must first be converted to

acyl-carnitine. This occurs on the cytosolic side of the inner mitochondrial membrane, and is catalyzed by carnitine acyltransferase I (CAT-I). CAT-I is the rate limiting step in fatty acid oxidation. It is inhibited by malonyl CoA, the starting point of fatty acid

10

synthesis. This inhibition of CAT-I functions to prevent the oxidation of newly synthesized fatty acids.

2. Acyl-carnitine is transported into the mitochondria. 3. Inside of the mitochondria acyl-carnitine is converted back to acyl-CoA. This is

catalyzed by carnitine acyl transferase II (CAT-II) 4. Carnitine is transported back to the cytosol. The carnitine carrier protein catalyzes an

exchange of acyl-carnitine and carnitine

Outer mitochondrial membrane

CAT-I Acyl-CoA Acyl-carnitine Carnitine CoA

Mitochondrial membrane space

inner mitochondrial membrane

mitochondria Carnitine CoA

Acyl-CoA (mitochondria) Acyl-carnitine (mitochondria) CATII

Inhibited by malonyl-CoA

Carnitine carrier protein

D. The metabolic break down of acyl-CoA occurs in a series of steps that remove two carbon units at a time. The two carbon units appear as acetyl-CoA. Important aspects are summarized here. If you wish, you may refer to the text for the actual series of steps. 1. The first step in the removal of two carbons is catalyzed by acyl-CoA dehydrogenase.

This reaction is catalyzed by one of three enzymes, depending on the size of the acyl-CoA

- Long chain acyl-CoA dehydrogenase (LCAD) - Medium chain acyl-CoA dehydrogenase (MCAD) - Short chain acyl-CoA dehydrogenase (SCAD)

2. MCAD is of clinical importance since a deficiency of this enzyme can lead to sudden infant death if left untreated, and mutations affecting this gene are relatively common. The pattern of inheritance is autosomal recessive. Several states, including New York, routinely screen newborns for this deficiency. The Baby Ian tutorial available from the MGB web site focuses on this disorder. Like succinate dehydrogenase, this enzyme utilizes FAD as a hydrogen acceptor

3. The overall oxidation of palmitoyl-CoA can be summarized as: Palmitoyl-CoA + 7 FAD + 7 NAD+ + 7CoA + 7H2O → 8 Acetyl-CoA + 7FADH2 + 7NADH + 7 H+

Lots of energy in these hydrogens

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4. The FADH2 and NADH + H+ are oxidized by the electron transport chain to ultimately yield ATP and water. The acetyl-CoA can have a number of fates including oxidation via the TCA cycle. When all of these reactions proceed to completion, over 100 molecules of ATP are produced per palmitic acid oxidized.

E. Regulation

1. CAT-1 inhibition by malonyl-CoA is the major short term point of control for fatty acid oxidation in the liver as well as in other tissues. Malonyl-CoA concentration is determined by the availability of citrate and the activity of the highly regulated enzyme acetyl-CoA carboxylase.

2. In the liver, the genes involved in fatty acid oxidation are regulated at the level of transcription. A major regulator is PPARα (Peroxisome Proliferator-Activated Receptor alpha). This is a member of the nuclear receptor family of transcription factors, and works by the same mechanism as the estrogen receptor (described earlier in the course). PPARα is stimulated by a number of lipids, and when stimulated it increases the transcription of target genes including the fatty acid binding protein and enzymes of fatty acid oxidation. Refer to the estrogen receptor for a model of the mechanism of activation.

3. The fibrate class of lipid lowering drugs function by activating PPARα.

IV. Propionic acid metabolism A. Propionyl-CoA is a product of branched-chain amino acid metabolism and the

metabolism of fatty acids that contain an odd number of carbons. The amount of propionyl-CoA produced is significant and under some conditions medically important. While we have elected to present propionyl-CoA metabolism at this point in the Molecules to Cells module, it is important to note that the majority of the propionyl-CoA that is produced is derived from amino acid metabolism rather than odd-chain-length fatty acid metabolism.

B. Propionic acid is the end product of the metabolism of fatty acids that have an odd number

of carbons. When these compounds are metabolized by the beta-oxidation pathway described above, propionyl-CoA is the last product rather than acetyl-CoA.

C. Propionyl-CoA is converted into succinyl-CoA in a series of reactions. In the first

reaction, carbon dioxide is added. This is catalyzed by a biotin-dependent enzyme (propionyl-CoA carboxylase). The product, D-methylmalonyl-CoA is converted to the L isomer, then an intramolecular rearrangement (catalyzed by methylmalonyl-CoA mutase) produces succinyl-CoA.

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HCO3 - + ATP ADP + Pi

Propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA

Propionyl-CoA carboxylase Methylmalonyl-CoA mutase

Succinyl-CoA

Propionyl-CoA Methymalonyl-CoA Succinyl-CoA

Requirement for vitamin B12Requirement

for biotin

D. Methylmalonyl-CoA mutase requires vitamin B12. If not treated, a lack of vitamin B12

will cause irreversible neural damage due to the build up of toxic byproducts of propionyl-CoA metabolism. Propionyl-CoA metabolism is one of two places where vitamin B12 is required. The other, methionine synthase, will be presented in the amino acid lectures.

E. Vitamin B12

1. The symptoms of vitamin B12 deficiency are related to the two reactions for which it is required.

a. Megaloblastic anemia (pernicious anemia) results from a block of folate metabolism and will be discussed in the amino acid metabolism lecture. These are the first symptoms that appear and can be reversed by administering B12. b. Methylmalonic acidosis and resulting irreversible neural damage are long term consequences of blocking methylmalonyl-CoA mutase

2. Vitamin B12 is present in bacteria and animal products, but not vegetables. Consequently, strict vegetarians are at risk for vitamin B12 deficiency, although the deficiency requires about a decade to develop due to the normally large stores maintained in the body.

3. Vitamin B12 deficiency is most often due to a problem with absorption of the vitamin rather than a lack of dietary intake. The absorption of B12 is dependent on the presence of “intrinsic factor”, a protein that is secreted by the parietal cells of the stomach. Loss of the parietal cells by autoimmune mechanisms is the most common cause of B12 deficiency. This deficiency can be treated by periodic injections of B12.

V. Glycerol utilization

A. Glycerol is released into the blood by the hydrolysis of triacylglycerol (see below under adipose tissue). The glycerol is taken up by the liver and converted to glycerol-3-

13

phosphate which is oxidized to give dihydroxyacetone phosphate, an intermediate of both gluconeogenesis and glycolysis.

B. Adipose tissue can not use glycerol since it lacks glycerol kinase.

VI. Ketone body synthesis: the liver is the major producer of ketone bodies, but it can not

use them. A. During the fasting state, the production of acetyl-CoA exceeds the capacity of the TCA

cycle to use it. This is because the liver derives most of its energy from the FADH2 and NADH produced by fatty acid oxidation. The excess acetyl-CoA is converted to acetoacetate and β-hydroxybuyrate which are exported to other tissues for use.

B. Synthesis of acetoacetate and β-hydroxybutyrate

O O O O OH O O O || || || || | || || || CH3-C-S-CoA CH3-C- CH2-C-S-CoA - O-C-CH2-C-CH2-C-S-CoA CH3-C- CH2-C-O – Acetyl-CoA Acetoacetyl-CoA | Acetoacetate

CH3 HMG-CoA

(3-hydroxy-3-methyl-glutaryl-CoA) OH O | ||

CH3-CH-CH2-C-O –

β-hydroxybutyrate

CoA H2O + Acetyl-CoA CoA Acetyl-CoA

2 Acetyl-CoA Acetoacetyl-CoA HMG-CoA Acetoacetate

Acetone is a by product: NADH + H+ NADH + H+ CO2

Acetoacetate Acetone NAD+ NAD+ (Not catalyzed) β-hydroxybutyrate

ATP ADP + Pi NAD+ NADH + H+ Glycerol Glycerol-3-phosphate Dihydroxyacetone phosphate Glycerol kinase Gluconeogenesis or Glycolysis

Not present in adipose

14

C. Synthesis of acetoacetate and β-hydroxybutyrate D. HMG-CoA that is produced in this pathway is in the mitochondria. There is a distinct pool of HMG-CoA that is present in the cytosol, which is used for the synthesis of cholesterol. E. The liver is the major source of ketone bodies Heart and renal cortex use large amounts of ketone bodies, and the brain will adapt to the utilization of ketone bodies during prolonged starvation. The liver is unable to utilize ketone bodies since it does not have the enzyme required to convert acetoacetate back to acetoacetyl-CoA (see below under Muscle), the first step in its utilization. F. Acetoacetate and β-hydroxybutyrate are moderately strong acids. The release of large quantities into the blood can cause a significant drop in blood pH. Under pathological conditions, such as uncontrolled diabetes, this acidosis can be life threatening. G. Acetoacetate is converted to acetone by a non-enzymatic chemical reaction. The small amounts of acetone produced as not physiologically important, but are noticeable on the breath of a diabetic in ketoacidosis.

Adipose Tissue

I. Triacylglycerol uptake and storage: Triacylglycerols are broken down outside of the cell, fatty acids are taken up by the cell and then resynthesized into triacylglycerol A. Lipoprotein lipase

1. Reaction: hydrolysis of the glycerol – fatty acid ester bonds of triacylglycerols found in VLDL and chylomicron particles.

2. Located on the surface of capillaries. Lipoprotein lipase is synthesized by adipose cells (as well as muscle cells and other fatty acid-utilizing tissues). It is exported from the cell and binds non-covalently to heparin sulfate located on the outside of endothelial cells.

3. The Km of the adipose isoform of lipoprotein lipase is higher than the lipoprotein lipase that is present in other tissues. Thus, when triacylglycerol is abundant it is stored in adipose tissue; when it is scarce, it is used by other tissues

4. Regulation: The synthesis of adipose lipoprotein lipase is increased by the action of insulin.

Capillary Capillary wall

Triacylglycerol Gycerol Fatty acids

LL

adipocyte

Lipoprotein lipase

15

B. Resynthesis of triacylglycerol for storage in the adipose cell 1. Triacylglycerol is formed from acyl-CoA and glycerol-3-phosphate by the same series

of reactions as in the liver (see above). 2. Adipose does not contain glycerol kinase. Hence, adipose can not reuse glycerol; it

must manufacture new glycerol-3-phosphate 3. Availability of glycerol-3-phosphate is limiting. Glycerol-3-phosphate is derived from

dihydroxyacetone, an intermediate of glycolysis. In adipose tissue, the rate of glycolysis, and hence availability of dihydroxyacetone phosphate, is limited by GLUT-4. As with the muscle, insulin causes intrecellualar membrane stores of GLUT-4, to move to the plasma membrane. Hence, insulin stimulates triacylglycerol synthesis and storage by the adipocyte.

4. Fatty acid acids are activated to acyl-CoAs by acyl-CoA syntetase as described above.

II. TriacylglyA. The firs

Triacylgl

B. Hormon

phospho1. Inacti2. Activ

The pnorep

3. The 2fatty

4. The nof wh

5. In thetissuenerprod

C. Regulati

1. The pa. In

tb. In

Fatty acid + CoA + ATP Acyl-CoA + AMP + PPi

Acyl-CoA synthetase

cerol mobilization from adipose tissue. t, and most highly regulated enzyme is hormone sensitive lipase.

ycerol + H2O 2,3-Diacylglycerol + fatty acid Hormone sensitive lipase

Highly regulated

e sensitive lipase is inactivated by the action of insulin and activated by rylation by protein kinase A. vation by insulin is probably the most important level of control. ation by protein kinase A (PKA) catalyzed phosphorylation is also important. rimary activators of PKA in adipose are catacholamines (epinephrine and inepherine) acting through β-adrenergic receptors. ,3-diacylglycerol formed by hormone sensitive lipase is further hydrolyzed to acids and glycerol by other unregulated lipases. et products of triacylglycerol mobilization are free fatty acids and glycerol, both ich are released into the blood. blood, free fatty acids are transported as a complex with serum albumin. Other es (but not brain or red blood cells) use the free fatty acids for the production of gy. The liver uses these for both energy production as well as ketone body uction.

on rimary regulator of triacylglycerol metabolism in adipose is insulin. sulin increases storage of triacylglycerol by glucose entry into adipocytes and in he long term by increasing lipoprotein lipase levels. sulin inactivates triacylglycerol mobilization by hormone sensitive lipase.

16

2. The development of adipocytes and the regulation of their enzymes is controlled to a large extent by the activity of PPARγ, which is analogous to PPARα, (discussed above in the section on liver. Activation of PPARγ by fatty acids stimulates the synthesis of enzymes involved in glucose uptake, fatty acid uptake and storage. It also stimulates the production of several hormones that are released by adipocytes.

3. The thiazolidinedione class of insulin-sensitizing drugs used for treating type II diabetes function by stimulating PPARγ.

+

ATP cAMP AMP Adenylyl cyclase phosphodiesterase

Protein kinase A

Hormone sensitive lipase (active)

P

Hormone sensitive lipase (inactive)

ATP ADP

Lipase phosphatase

Pi H2O

Insulin

Epinepherine

Noepinepherine Insulin

H2O Pi PPi + -

Triacylglycerol

Diacylglycerol Other lipases 2 Fatty acid + Glycerol

H2O Fatty acid A

LBUMI N

+

+

Adipocyte

17

Muscle I. Muscle can utilize fatty acids as fuel.

A. Albumin-bound fatty acids mobilized from adipose tissue can be taken up from the circulation.

B. Fatty acids in chylomicrons and VLDL are released by hydrolysis of the triacylglycerol

This hydrolysis is catalyzed by lipoprotein lipase bound to the outside of capillary endothelial cells (see above section on adipose)

C. Once taken up, the fatty acids are imported into the mitochondria and metabolized by

beta-oxidation as described for the liver (see above section on liver III.A). D. Regulation

1. The amount of fatty acid that can be utilized is regulated by the rate of entry of fatty acids into the mitochondria. As in the liver, CAT-I is inhibited by malonyl-CoA (see above section on liver III.C). Muscle has a small amount of acetyl-CoA carboxylase that functions to synthesize regulatory amounts of malonyl-CoA. Muscle does not use the malonyl-CoA for fatty acid synthesis. As in liver, muscle acetyl-CoA carboxylase is stimulated by citrate. Therefore, when glucose is abundant, citrate increases, malonyl-CoA increases and oxidation of fatty acids is inhibited.

2. PPARα, (see Liver section V.E.3) stimulates the synthesis of the muscle enzymes required for lipid metabolism in response to increased concentration of a number of lipids.

E. Triacylglycerol. Muscle contains a small amount of triacylglycerol that can be used as an

immediate energy source. The regulation of muscle triacylglycerol storage and used is poorly understood, but of potential importance for the field of sports medicine.

II. Muscle imports and metabolizes ketone bodies

A. The reactions:

NAD+ NADH + H+ Succinyl-CoA Succinate β-hydroxybutyrate Acetoacetate Acetoacetyl-CoA

CoA transferase CoA 2 Acetyl-CoA

This enzyme is missing in liver. Hence liver produces, but does not use ketone bodies

18

Brain and Red Blood Cells I. Red blood cells lack mitochondria and therefore can not metabolize either fatty acids or

ketone bodies. They are dependent on glucose for their energy needs. II. The brain normally uses glucose, but during prolonged starvation it can use ketone bodies

for a considerable amount (but not all) of its energy needs. Neither triacylglycerols nor fatty acids are used by the brain for the production of energy.

Intestine

Triacylglycerol

Lymph

To tissues via lymph

2-Monoacylglycerol

Triacylglycerol

Mixed micelles

5

Jejunum

Enterocyte 6 Fatty acyl CoA Fatty acid AMP + PPi ATP

2-Monoacylglycerol

Bile Salts (to ileum)

Other lipids

CoA-SH

7

Chylomicron

Apolipoprotein B48 phospholipid, cholesterol Apolipoprotein A1

8

Apolipoproteins C, E

Enzymes: 1. acid-stable lipase (mouth) 2. pancreatic lipase (pancreas) 3. colipase 4. phospholipase A2/other lipases 5. intestinal fatty acid binding protein (required for transport to the endoplasmic reticulum) 6. acyl-CoA synthase 7. acyltransferase 8. microsomal transfer protein

1,2,3

Other lipids

Bile Salts (from liver)

4

+ Fatty acids

19

I. Digestion A. Digestion begins in the mouth with the action of lingual lipase. The free fatty acids,

together with phospholipids present in food emulsify the fat into small droplets. B. When the gastric contents are emptied into the duodenum, the presence of lipids and

proteins stimulates the release of the peptide hormone cholecystokinin from the lower duodenum and jejunum. This hormone stimulates the gall bladder to contract and release gall (micelles of bile salts, phospholipids and cholesterol) into the duodenum. The gall acts on the partially emulsified lipids from the stomach to form smaller mixed micelles. Cholecystokinin also stimulates the release of digestive enzymes from the exocrine cells of the pancreas. These enzymes include several that work at the surfaces of the mixed micells to remove fatty acids from various lipids.

C. Pancreatic lipase (not to be confused with lipoprotein lipase or hormone sensitive lipase)

catalyzes the hydrolysis of fatty acids from positions 1 and 3 of triacylglycerols. 1. Pancreatic lipase requires the protein colipase which is also released from the pancreas.

Colpiase anchors the lipase to the surfaces of the miced micelles where digestion takes place the protein.

2. Pancreatic lipase is inhibited by the weight loss drug, orlistat At the recommended doses, Orlistat blocks about a third of dietary triacylglycerol from being digested

D. The resulting fatty acids and 2-monoacylglycerol are taken up by the enterocytes.

II. Resynthesis: Fatty acids and 2-monoacylglycerol enter the intestinal mucosal cells (entereocytes) where they are resynthesized into triacylglycerols.

A. Fatty acids are activated by conversion to acyl-CoA by acyl-CoA synthetase. This is the

same reaction described above for the liver.

B. Th R- 2-

Fatty acid + CoA + ATP Acyl-CoA + AMP + PPi

Acyl-CoA synthetase

e acyl-CoA combines with the 2-monoacylglyceride to reform triacylglycerol:

O CH2-O || | C-O-CH | CH2-Omonoaycl-

H 2 Acyl-CoA + 2-Monoacylglycerol Triacylglycerol + 2 CoA

H CoA

20

C. Triacylglycerol is exported from the intestine in combination with apolipoprotein B-48, phospholipids, cholesterol, cholesterol ester, and other apolipoproteins and lipids in the form of chylomicrons. 1. Apolipoprotien B-48 is a truncated form of apolipoprotein B-100 (B-100 is produced

in the liver and found in VLDL). 2. The modification that gives rise to B-48 as opposed to B-100 occurs at the level of

RNA editing. In intestinal cells, the mRNA sequence is modified so that an amino acid codon is converted to a stop codon.

3. The process of chylomicron formation is analogous to the formation of VLDL in the liver (see above; Liver I.E.3)

4. Chylomicrons enter the lymphatic system for distribution to the tissues After depletion of lipids, the chylomicron remnants are taken up by the liver (see below).

D. Chylomicrons that have been depleted of their triacylglycerol (chylomicron remnants) are

taken up by the liver.

Lipid transport

I. Overview. Lipids are transported between tissues as complexes with carrier proteins or as components of lipoprotein complexes.

A. Free fatty acids are transported in the blood bound to serum albumin, the major protein

present in the serum. B. There are three major lipid transport pathways

1. Chylomicrons are released from the intestine and carry one molecule of apolipoprotein B-48, dietary triacylglycerol, cholesterol ester, phospholipids and other water insoluble compounds. They mature in the blood through the addition of other apolipoproteins, notably apolipoproteins C and E. As a chylomicron passes through the tissues, the triacylglycerol is depleted through the action of lipoprotein lipase, resulting in a Chylomicron remnant. This is taken up by the liver.

2. Very Low Density Lipoproteins (VLDL) are released from the liver, and carry one molecule of apolipoprotein B-100, newly synthesized triacylglycerol, cholesrterol, cholesterol ester and phospholipid. They mature in the blood through the addition of other apolipoproteins, notably apolipoproteins C and E. In the blood the VLDL loses triacylglycerol to the tissues and gains cholesterol ester, progressing to Intermediate Density Lipoprotein (IDL) and then Low Density Lipoprotein (LDL). The LDL is taken up predominantly by the liver although all tissues are capable of taking up some of it.

3. High Density Lipoproteins (HDL) are released from the liver and intestine, and mature in the blood through the addition of other proteins and cholesterol which is picked up from the tissues. HDL functions primarily as a carrier of cholesterol, which it picks up from the tissues, and converts to cholesterol ester. The cholesterol ester is then donated to IDL and LDL.

21

C. Characteristics of the lipoproteins 1. Physical properties and composition (TG - triacylglycerol; PL - phospholipid; ChE -

cholesterol ester; the predominant compound(s) transported are in bold) Class Diameter (nm) % Protein % TG %PL %ChE Chylomicron 100-1000 1-2 86 8 3 VLDL 30-80 6-10 55 18 13 IDL 25-30 15-20 25 21 28 LDL 20-25 22 9 20 40 HDL 5-10 35-50 5 30 15

2. Functional properties (TG - triacylglycerol; ChE - cholesterol ester)

Class Source Apolipoproteins Transports: Fate Chylomicron intestine B-48, C, E, A Dietary TG TG to tissues, remnant to liver VLDL liver B-100, C, E New TG from liver TG to tissues/ becomes IDL IDL VLDL B-100, E New TG/

ChE from HDL TG to tissues/ ChE from HDL/ becomes LDL

LDL IDL B-100 ChE to liver uptake by liver HDL liver/

intestine A-I, A-II, C, E Cholesterol from

tissues/ ChE to IDL ChE to IDL/eventual uptake by liver

3. Functional properties of some of the apolipoproteins

Apolipoprotein Source Lipoproteins Function A-I, A-II liver, intestine HDL, chylomicrons Structural protein of HDL B-48 intestine Chylomicrons Structural protein of chylomicron B-100 liver VLDL, IDL, LDL Structural protein of VLDL, IDL, LDL C-I, C-II, C-III liver VLDL, IDL HDL,

CM Transferred between different classes C-II activates lipoprotein lipase

E liver HDL., VLDL, IDL, CM remnants

Mediates uptake of chylomicron remnants and IDL by liver

II. Transport by chylomicrons

A. Chylomicrons are formed in the intestine with apolipoprotein B-48 as the key structural protein. Apolipoproteins C and E are transferred to chylomicrons in the blood by HDL.

B. In addition to triacylglycerol, chylomicrons transport other dietary lipids and fat soluble vitamins.

C. Triacylglycerol is depleted from chylomicrons by the action of lipoprotein lipase lipase in adipose tissue, skeletal muscle, heart, and other tissues. Lipoprotiein CII stimulates this process. Apolipoproteins C and A are transferred to HDL leaving the chylomicron remnant, which contains aploliproteins B-48 and E.

D. Chylomicron remnants are taken up by the liver chylomicron remnant receptor (also known as LRP for LDL-receptor Related Protein). Processing by the liver is as for LDL described below.

22

Chylomicron

Intestine

TGPL, Ch, ChE

B-48

TGPL, Ch, CE

B-48

CE

TGPL, Ch, ChE

B-48

CE

Endo Ct eh le llialMG + FA

TG PL, Ch, ChE

B-48

E

C

HDL

CHDL

Liver cell

E

Lipoprotein Lipase

A

A

A

AChylomicron remnant

LRP

A

III. Transport by VLDL, IDL, LDL A. VLDL is formed in the liver with apolipoprotein B-100 as the key structural protein.

Apolipoproteins C and E are transferred to VLDL from HDL in the blood. B. Triacylglycerol is depleted from VLDL by the action of lipoprotein lipase in adipose

tissue, skeletal muscle, heart, and other tissues. Lipoprotein CII stimulates this process.. C. Considerable remodeling of the VLDL takes place to produce IDL and LDL. This

includes the transfer of cholesterol ester from HDL (see below), the transfer of apolipoproteins C to HDL, and hydrolysis of excess triacylglycerol and phospholipid by hepatic lipase. Hepatic lipase is analogous to the lipoprotein lipase found in other tissues, but it does not hydrolyze triacylglycerol present in chylomicrons or VLDL.

D. LDL is taken up by liver via the LDL receptor.. After binding to the receptor, the LDL is taken into the cell by endocytosis where it is digested by lysosomal enzymes (this process will be covered in detail in the cell biology course). In the lysosome, cholesterol esterase hydrolyses cholesterol ester to cholesterol which then enters the cholesterol pool of the liver cell. Phospholipids are also hydrolyzed and the B-100 protein is digested to amino acids.

23

Liver

VLDL

TG PL, Ch, ChE

B-100

E n d o Ct eh le ll i a l MG + FA

TGPL, Ch, ChE

B-100

E

CLDL

C

HDL

E

Liver

ChE

Ch

Bile S Salts

TG PL, Ch, ChE

B-100

CE

E

TG PL, Ch, ChE

B-100

C E

Fatty Acids

ChE ChTissues

LDL receptor

V

HDL

TG PL, Ch, ChE

B-100

V

Liver

ChE

LDL receptor

E

cell

V IDL

FA + glycerol

FA + glcerol

E. Most of the cholesterol of the liver is exported to other cells in the form of VLDL (largely after being converted to cholesterol ester) or is used for the synthesis of bile, which consists of cholesterol, phospholipids and bile salts. Bile salts are derived from cholesterol. The synthesis and entero-hepatic circulation of bile will be discussed later in the course.

IV. Overview of cholesterol metabolism

A. Cells obtain cholesterol from a combination of endocytosis mediated by the LDL receptor and de novo synthesis. Although nearly all cells can synthesize cholesrterol, most of the body’s cholesterol is produced by the liver; on a typical western diet about a third comes from food. The synthesis of cholesterol will be discussed later in the course. Cholesterol is stored within liver cells and some other cells that are active in cholesterol metabolism (such as the adrenal cortex) as cholesterol ester. This is formed by the action of ACAT (acyl-CoA-cholesterol acyl transferase). Free cholesterol induces the enzyme.

24

Acyl-CoA CoA ACAT Cholesterol Cholesterol ester

B. Cholesterol is lost from the body through the death of intestinal cells and through the inadvertent loss of bile.

C. Regulation of intracellular cholesterol levels: 1. Free cholesterol represses the de novo synthesis pathway by inhibiting the rate limiting

step catalyzed by HMG-CoA reductase (this enzyme will be discussed later in the course).

2. Free cholesterol represses cholesterol uptake from the blood by repressing the synthesis of the LDL receptor and promoting the degradation of the LDL receptor.

D. Familial hypercholesterolemia is due to a severe mutation in the LDL receptor gene. Heterozygotes have half the normal ability to synthesize LDL receptors. The decreased amount of receptors leads to an increase in the amount of cholesterol in the blood by two mechanisms. 1. Low number of LDL receptors leads to low uptake of cholesterol into cells.. 2. This leads to low intracellualar cholesterol. 3. This stimulates cholesterol synthesis. 4. Additional cholesterol is exported from the liver 5. Blood cholesterol is increased.

E. Macrophages remove LDLs that contain oxidized lipids via lipoprotein receptors that are only are only marginally affected by cholesterol concentrations. Since these LDLs also contain cholesterol ester, macrophages can accumulate large amounts of cholesterol when the blood cholesterol is high. This accumulation leads to the progression of macrophages to lipid rich foam cells; an important event in atheroschlerosis.

F. Excess cholesterol is eliminated from cells by an ATP dependent transport pathway catalyzed by the ATP Binding Cassette A1 (ABC-A1) transporter. Homozygotes who lack this protein have Tangier Disease.

V. Transport by HDL

A. HDL functions primarily in the transport of cholesterol between tissues, the intestine and the liver.

B. Apolipoprotein A, which forms the nascent HDL particle, is released by the liver and apolipoprotiens C and E quickly join. Aplolipoprotein A is also released by the intestine. In the blood the nascent HDL acquires two key enzymes: Cholesterol Ester Transfer Protein (CETP) and lecithin-cholesterol acyl transferase (LCAT).

C. The HDL particles complete their maturation by picking up cholesterol exported from cells by the ABC-A1 transporter. The cholesterol is rapidly converted to cholesterol ester by LCAT.

25

D. The HDL particle then donates the cholesterol ester to LDL particles in exchange for lecithin. The process is mediated by CETP.

E. The LDL particles that are now enriched with cholesterol ester, are taken up by liver cells. F. Triacylglycerol and phospholipid acquired by HDL is removed by hepatic lipoprotein

lipase. G. HDL particles are also metabolized by the liver through the Scavenger Receptor B1

(SRB-1). This receptor binds the HDL, removes most of the cholesterol ester then releases a greatly depleted HDL. The cholesterol ester is taken up by the liver.

E C

E C

A

A

Intestine

Liver cell

Liver

ChE

Ch

Bile S Salts

PL, Ch

Tissues

ABC-A-1transporter

Ch

ChE ChE

LDLreceptor

LDL

PL, ChE

CETPCh

LCAT

IDL

TG ChE

TG

HDL

LDL receptor

SR-B-I receptor

LDL

hepatic lipase

A PL, Ch

26