Biochemistry: Structure and bioenergetics of lipids

Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**

Consortium leader

PETER PAZMANY CATHOLIC UNIVERSITY

Consortium members

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***

**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben

PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS

UNIVERSITY

Semmelweis University

ORGANIC AND BIOCHEMISTRY

Lipids: structure and bioenergetic role

http://semmelweis-egyetem.hu/

(Szerves és biokémia)

Biochemistry: Structure and bioenergetics of lipids

Lecture objectives

At the end of the presentation the participant will be able:

1) To define the term lipid

2) To discuss the classification of lipids in terms of their physicochemical properties 3) To interpret the relation between structure and biological role of lipids

4) To describe the absorption of lipids in the intestine

5) To discuss the storage and mobilization of fatty acids in adipose tissue 6) To interpret the general rules of lipid transport in blood

7) To define the term β-oxidation of fatty acids

8) To describe the reactions of fatty acid synthesis and degradation 9) To discuss the regulatory mechanisms in fatty acid metabolism

Biochemistry: Structure and bioenergetics of lipids

Lipids^DEF : compounds extractable from tissues

with non-polar solvents (ether, chloroform, carbon-tetrachloride)

Biochemistry: Structure and bioenergetics of lipids

Intermolecular interactions between lipids and water

Thermodynamic conditions for stable lipid/water systems

Δ ^G= Δ ^H-T Δ ^S<0

Small polar molecules (yellow balls) intermixed among water molecules (white-and-red balls) yield large negative ΔH term.

For lipids an alternative strategy

improves the thermodynamic stability of the lipid/water mixture: high increase in entropy.

Biochemistry: Structure and bioenergetics of lipids

Increase in entropy of lipid/water mixtures

Formation of larger lipid aggregates increases the disorder of the water molecules disrupting more H-bonds / unit mass.

Biochemistry: Structure and bioenergetics of lipids

Class 1. Lipids that are insoluble and do not form a stable association with water (e.g. cholesterol esters) or are just polar enough to form stable monolayers

at air-water interfaces (e.g. triacylglycerols TAG, diacylglycerols, protonated long- chain fatty acids, cholesterol).

Classification of lipids based on the nature of their interaction with water

Biochemistry: Structure and bioenergetics of lipids

Ultrastructure of triacylglycerol (TAG) aggregates in water environment Electron micrographs of adipocytes

Biochemistry: Structure and bioenergetics of lipids

Biological role of TAG related to their physicochemical properties

Energy content of the main fuels

Fuel ΔH (kJ.g ^-1 ) Protein

Fat

Carbohydrate Ethanol

17 37 16 29

N.B.! The fat is stored in 'dry' state

65 % of the weight of the stored glucose (glycogen) is H O, thus the

Biochemistry: Structure and bioenergetics of lipids

Evolutionary advantage of TAG as a storage form of energy

A top model in the “lipid world” and two top model candidates in the

“glycogen world”

Biochemistry: Structure and bioenergetics of lipids

Normal size of the TAG storage pool

TAG content of the human body (% body weight)

normal obesity

female 20-26 >30

male 12-15 >20

Biochemistry: Structure and bioenergetics of lipids

Classification of lipids based on the nature of their interaction with water Class 2. Lipids that are insoluble but sufficiently polar to allow association with water in a regular manner (to form liquid crystals) in the bulk phase (e.g. phospholipids, monoacylglycerols).

Biochemistry: Structure and bioenergetics of lipids

Spontaneous arrangement of phospholipids in water environment

Biochemistry: Structure and bioenergetics of lipids

Interactions of phospholipid bi-layers and water

water

phospholipid hydrophobic tail

phospholipid phospholipid polar head

Biochemistry: Structure and bioenergetics of lipids

Structural blocks of phospholipids 1.

Structural blocks of phospholipids 2.

Biochemistry: Structure and bioenergetics of lipids

R₃=

Biochemistry: Structure and bioenergetics of lipids

Biological role of phospholipids 1.

Compartmentation^DEF by bi-layer membranes

above melting temperature below melting temperature

lamellar α structure (fluid) lamellar β structure (rigid)

Biochemistry: Structure and bioenergetics of lipids

Typical composition of the two layers of cellular membranes

Biochemistry: Structure and bioenergetics of lipids Biological role of phospholipids 2.

Control of surface tension by phospholipid monolayers

Biochemistry: Structure and bioenergetics of lipids

Formation of the phospholipid monolayers in lung surfactant^DEF

TM: trabecular matrix (expanding LBs)

M: dipalmitoyl phosphatidyl choline monolayer (high melting temparature, stable Lβ structure)

Biochemistry: Structure and bioenergetics of lipids

Splitting the phospholipid bi-layers: structural rearrangements Requirement for HII (hexagonal structures at the air/water interface)

HII structures are formed by phospholipids with small polar heads (phosphatidyl

Biochemistry: Structure and bioenergetics of lipids

Background of the neonatal respiratory distress syndrome

The immature lung (before the 34^th gestational week) does not produce sufficient amounts of dipalmitoyl phosphatidylcholine and phosphatidyl ethanolamine.

Thus, the stable phospholipid monolayer cannot be formed at the air/water interface and the alveoli collapse at the end of expiration.

Transient treatment: aerosolic application of phospholipid mixtures with appropriate composition or biological surfactant.

Biochemistry: Structure and bioenergetics of lipids

Classification of lipids based on the nature of their interaction with water Class 3. Lipids (e.g. bile salts) that are soluble at low concentrations but at higher concentrations, that is, above the critical micellar concentration (CMC), form a micellar 'solution'. Micelles^DEF are aggregates of molecules which are similar to particles in an emulsion but are considerably smaller (typically 4-6 nm in diameter).

assembly of a bile acid micelle in water

Biochemistry: Structure and bioenergetics of lipids

Solubility of bile acids in water

Biochemistry: Structure and bioenergetics of lipids

Biological role of bile acids 1.

Formation of mixed micelles for absorption^DEF of free fatty acids and cholesterol in the lumen of the small intestine

bile acid

phospholipid

cholesterol

free fatty acids

Biochemistry: Structure and bioenergetics of lipids

Biological role of bile acids 2.

Emulsification of dietary fat

Biochemistry: Structure and bioenergetics of lipids

Regulation of the pancreatic lipase

activity 1.

active site (Ser¹⁵³, His²⁶⁴, Asp¹⁷⁷)

At the high pH of the pancreatic juice the lid peptide blocks the access of

substrates to the active site of the lipase.

Biochemistry: Structure and bioenergetics of lipids Regulation of the pancreatic lipase activity 2.

Pancreas produces a small cofactor

protein (procolipase), which binds to the C-terminal domain of lipase, but at pH>8 it does not interact with the lid domain.

Biochemistry: Structure and bioenergetics of lipids Regulation of the pancreatic lipase activity 3.

At neutral pH or if trypsin cleaves the N-terminal pentapeptide of procolipase, the cofactor protein interacts with the lid peptide and opens the active site of the lipase (pancreas lipase gains activity only after its release in the small intestine).

Biochemistry: Structure and bioenergetics of lipids

Regulation of the pancreatic lipase activity 4.

Interfacial activation: binding of pancreatic lipase (with the help of colipase) to the surface of TG droplets is a prerequisite for optimal activity (compare the hydrolysis by non-specific

esterase and pancreatic lipase at increasing TG concentrations;

saturation of 1 indicates the TG concentration at which

Biochemistry: Structure and bioenergetics of lipids

Overview of the fate of dietary fats in the human body

Biochemistry: Structure and bioenergetics of lipids

Absorption of TAG

1. Hydrolysis in the lumen

2. Resynthesis of TAG in the intestinal cells

Biochemistry: Structure and bioenergetics of lipids

Chylomicron

formation

in the intestinal cell

Biochemistry: Structure and bioenergetics of lipids

Intracellular localization of the TAG synthesis

The hydrophobic molecular domains of the substrates

are inserted in the ER membrane.

The highly hydrophobic product (TAG) accumulates between the two layers of the membrane.

Biochemistry: Structure and bioenergetics of lipids

Absorption of cholesterol

1. Hydrolysis of cholesteryl esters (CE) in the lumen and transport of free cholesterol (FC) through Nieman-Pick C 1 like protein 1 (NPC1L1)

2. Resynthesis of CE in the intestinal cells

3. Transport of CE to the lymph in the form of

Biochemistry: Structure and bioenergetics of lipids

Intracellular localization of the CE synthesis

The hydrophobic molecular domains of the substrates

are inserted in the ER membrane.

The highly hydrophobic product (CE) accumulates between the two layers of the membrane.

Biochemistry: Structure and bioenergetics of lipids

General structure of the lipoproteins

Biochemistry: Structure and bioenergetics of lipids Composition of the major classes of lipoproteins

VLDL: very low-density lipoprotein LDL: low-density lipoprotein

HDL: high-density lipoprotein

Biochemistry: Structure and bioenergetics of lipids

Apolipoproteins

in the major classes of lipoproteins

Biochemistry: Structure and bioenergetics of lipids

Relative size of the major classes of lipoproteins

Biochemistry: Structure and bioenergetics of lipids

Roles of the major classes of lipoproteins in the transport of lipids

Biochemistry: Structure and bioenergetics of lipids

Reactions catalyzed by lipoprotein lipase

(catalytic mechanism similar to that of pancreatic lipase)

Catalytic site:

Ser, His, Asp Cofactor:

ApoC-II Substrates:

chylomicron, VLDL

Biochemistry: Structure and bioenergetics of lipids

Roles of the lipoprotein lipase

(LPL) in the transformation of

lipoproteins

Biochemistry: Structure and bioenergetics of lipids

Storage of fatty acids in the form of triacylglycerol (overview)

Biochemistry: Structure and bioenergetics of lipids

Reactions of triacylglycerol synthesis in adipocytes 1.

Biochemistry: Structure and bioenergetics of lipids

Reactions of triacylglycerol synthesis in adipocytes 2.

LD: lipid droplet

PAT: Perilipin, ADRP (Adipose Differentiation Related Protein),

TIP47 (Tail Interacting Protein 47)

Biochemistry: Structure and bioenergetics of lipids

Formation of triacylglycerol droplets in adipocytes

Biochemistry: Structure and bioenergetics of lipids

Structure of triacylglycerol droplets in adipocytes

Biochemistry: Structure and bioenergetics of lipids

Mobilization of fatty acids from TAG in adipocytes

ATGL: adipose trigliceride lipase; HSL: hormone sensitive lipase; MGL: monogliceride

Biochemistry: Structure and bioenergetics of lipids

Transport of free fatty acids in blood

Following release from adipocytes free fatty acids enter the blood stream, where they bind to albumin, the most abundant serum protein.

Biochemistry: Structure and bioenergetics of lipids

Utilization of fatty acids for energy production 1.

Activation of fatty acids in the cytosol

Biochemistry: Structure and bioenergetics of lipids

Utilization of fatty acids for energy production 2.

Reversible formation of acyl-carnitine^DEF

R1=

Substrate ΔG⁰ of hydrolysis at 25 °C kJ/mol

Biochemistry: Structure and bioenergetics of lipids

Utilization of fatty acids for energy production 3.

Transport of fatty acids

through the two membranes of mitochondria

CPT: carnitine palmitoyl transferase

Biochemistry: Structure and bioenergetics of lipids

Utilization of fatty acids for energy production 4.

Overview of the mitochondrial stages Stage 1: Generation of acetyl-CoA (β-oxidation^DEF)

Stage 2: Oxidation of acetyl-CoA (citric acid cycle)

Stage 3: Synthesis of ATP (oxidative phosphorylation)

Biochemistry: Structure and bioenergetics of lipids

Reactions of β-oxidation

Biochemistry: Structure and bioenergetics of lipids

Yield of ATP in the course of complete oxidation of palmitoyl-CoA

β-oxidation

citric acid cycle

Regulation of β-oxidation 1.

The product of the ACC reaction (malonyl-CoA) inhibits the CPT-1 enzyme.

ACC is active in dephosphorylated state and inactive in phosphorylated state.

Biochemistry: Structure and bioenergetics of lipids

Biochemistry: Structure and bioenergetics of lipids

Regulation of β-oxidation 2.

The activity of β-oxidation is coupled to the demand for ATP through the activity of AMPK, which phosphorylates the ACC.

Biochemistry: Structure and bioenergetics of lipids

β-oxidation of unsaturated fatty acids

Oleic acid: the cis double

bond at C⁹ cannot be handled by the enzymes of β-oxidation.

Solution: enoyl-CoA isomerase

Biochemistry: Structure and bioenergetics of lipids

β-oxidation of unsaturated fatty acids

Linoleic acid: the cis double bonds at C⁹ and C¹² cannot be handled by the enzymes of β-oxidation.

Solution: enoyl-CoA isomerase + 2,4-dienoyl-CoA reductase

Biochemistry: Structure and bioenergetics of lipids

β-oxidation of fatty acids with odd number of C-atoms

Each cycle of β-oxidation shortens the fatty acid chain by 2 C-atoms. Thus, the terminal C-atoms will remain in the form of propionyl- CoA. Following carboxylation and isomerization these C-atoms enter the citric acid cycle as succinyl-CoA.

Biochemistry: Structure and bioenergetics of lipids

Intracellular compartments and lipid metabolism

Biochemistry: Structure and bioenergetics of lipids

Substrates for the synthesis of fatty acids

1. Acetyl-CoA: primarily from oxidation of glycolysis and the action of pyruvate dehydrogenase complex (acetyl-CoA is generated in mitochondria and should be transported to the cytosol.

2. NADPH: primarily from the pentose phosphate pathway, malic enzyme and cytosolic isocitrate dehydrogenase

Biochemistry: Structure and bioenergetics of lipids

Export of acetyl-CoA from mitochondria to cytosol

for the synthesis of fatty acids

Sources of NADPH

for the synthesis of fatty acids

Biochemistry: Structure and bioenergetics of lipids

Activation of acetyl-CoA

for the synthesis of fatty acids

Biochemistry: Structure and bioenergetics of lipids

Biochemistry: Structure and bioenergetics of lipids

Functional domains in the single polypeptide chain of mammalian fatty acid synthase homodimer (FAS)

enzyme activity role

acyl carrier protein (ACP) carries acyl groups in thioester linkage malonyl/acetyl-CoA-ACP transferase

(MAT)

transfers malonyl and acyl groups from CoA to ACP

β-ketoacyl-ACP synthase (KS) condenses acyl and malonyl groups

β-ketoacyl-ACP reductase (KR) reduces β-keto group to β-hydroxy group β-hydroxyacyl-ACP dehydratase (DH) removes water from β-hydroxyacyl-ACP enoyl-ACP reductase (ER) reduces double bond

Biochemistry: Structure and bioenergetics of lipids

β-ketoacyl-ACP synthase reaction

β-ketoacyl-ACP reductase reaction

Biochemistry: Structure and bioenergetics of lipids

β-hydroxyacyl-ACP dehydratase reaction

Biochemistry: Structure and bioenergetics of lipids

Enoyl-ACP reductase reaction

Biochemistry: Structure and bioenergetics of lipids

Biochemistry: Structure and bioenergetics of lipids

Summary of the reactions of fatty acid synthesis

Biochemistry: Structure and bioenergetics of lipids

Regulation of fatty acid synthesis

Messages to take home

1) The term lipid covers all hydrophobic compounds of biological origin 2) Although all lipids are hydrophobic, their biological function is based on their weak interactions with water

3) Dietary fats are digested in the intestine prior to their absorption

4) Bile acids are essential in fat digestion as detergents, lipase activators and micelle forming agents

5) Lipid transport in blood requires lipoproteins as specialized vehicle particles with hydrophilic shells

6) Apolipoproteins determine the direction of lipid transport in blood

7) Fatty acids are degraded through oxidative removal of two C-atom units from the aliphatic chain

Biochemistry: Structure and bioenergetics of lipids

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 1

Which statement characterizes best the physicochemical properties of bile salts?

A: completely insoluble in water, forming lipid droplets B: partially soluble in water, forming bilayer structures C: partially soluble in water, forming micelles

D: completely insoluble in water, forming bilayer structures

E: partially soluble in water, forming lipid droplets

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 2

What is the biological role of bile acids?

A: the end-product of cholesterol catabolism in mammalians B: the end-product of lecithin catabolism in mammalians

C: the end-product of purine catabolism in mammalians

D: the end-product of phospholipid catabolism in mammalians

E: the end-product of cardiolipin catabolism in mammalians

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 3

What is the biological role of bile acids?

A: contribute to protein digestion in mammalians

B: contribute to protein absorption in mammalians

C: contribute to fatty acid absorption in mammalians

D: contribute to fatty acid digestion in mammalians

E: contribute to cholesterol digestion in mammalians

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 4

Which factor is necessary for the activity of pancreatic lipase?

1: acidic pH 2: basic pH

3: protein cofactor 4: bile acids

5: triglyceride micelles

A: 1,3,4 B: 1,4,5 C: 2,3 D: 2,3,4 E: 2,3,4,5

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 5

The biological roles of lipids include 1: energy storage

2: maintenance of structural compartments 3: signal molecules

4: vitamins

5: transport of non-lipid substances in blood

A: 1 B: 1,2 C: 1,2,3 D: 1,2,3,4 E: all

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 6

The outer surface of micelles in the intestine contains 1: monoglycerides

2: diglycerides 3: triglycerides 4: phospholipids 5: bile acids

A: 1,2 B: 2,3 C: 3,4 D: 4,5 E: 3,5

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 7

Select the sequence that reflects best the route of fatty acid absorption in the intestine!

A: monoglyceride-diglyceride-triglyceride-VLDL B: diglyceride-monoglyceride-chylomicron

C: monoglyceride-diglyceride-triglyceride-chylomicron D: monoglyceride-cholesteryl ester-VLDL

E: diglyceride-cholesteryl ester-VLDL

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 8

VLDL contains 1: triglyceride

2: phospholipid bilayer

3: cholesteryl esters

4: apo B48 protein

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 9

Chylomicrons contain 1: triglyceride

2: phospholipid bilayer

3: cholesteryl esters

4: apo B48 protein

5: apo CII protein

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 10

Select the correct pairs of apolipoproteins and their function 1. apo AI – LCAT activator

2. apo AI – ABCA1 ligand 3. apo AI – CFTR ligand

4. apo B100 – LDL receptor ligand

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 11

Which statement is true regarding the product of the acetyl-CoA carboxylase catalyzed reaction?

1. regulates the β-oxidation of fatty acids 2. regulates the synthesis of fatty acids 3. it is a substrate of β-oxidation

4. it is a substrate of fatty acid synthesis

5. it is a substrate of bile acids

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 12

Which statement is true regarding the lipoprotein lipase?

1. it functions on the surface of intestinal cells 2. it functions on the surface of endothelial cells 3. triglycerides are its substrates

4. phospholipids are its substrates

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 13

Which metabolic pathway uses the reaction below?

A: glycolysis

B: citric acid cycle

C: fatty acid oxidation

D: fatty acid synthesis

E: bile acid synthesis

Biochemistry: Structure and bioenergetics of lipids

Comprehension problem 14

Which statement is true regarding the metabolism of fatty acids?

A: Their oxidation and synthesis occur in the cytosol

B: Their oxidation occurs in the cytosol, whereas the synthesis in mitochondria

C: Their synthesis occurs in the cytosol, whereas the oxidation in mitochondria

D: The intracellular localization of their oxidation and synthesis depends on hormonal effects (insulin/glucagon ratio)

E: The intracellular localization of their oxidation and synthesis

Recommended literature

Orvosi Biokémia (Ed. Ádám Veronika): pp. 143-162, 180-193

Biochemistry: Structure and bioenergetics of lipids

Biochemistry: Structure and bioenergetics of lipids

Answers to comprehension problems:

1. C; 2. A; 3. C; 4. E; 5. D; 6. D; 7. C; 8. C; 9. B; 10. C; 11. C; 12. D; 13. C; 14. C