Lipid and Ketone Metabolism Week 12. 2 Lipids Lipids: structure & function Transport of lipids: albumin-binding & lipoprotein Storage & release of lipids.

Lipid and Ketone Metabolism

Week 12

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Lipids

• Lipids: structure & function• Transport of lipids: albumin-binding & lipoprotein• Storage & release of lipids – adipose tissue• Catabolism: energy release from fatty acid• Activation of FA & carnitine shuttle• oxidation of palmitic acid• Ketone bodies

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What are lipids?

• Not soluble in water – man (or animal) is ~80% water

• Soluble in organic solvents – acetone, ether• Saponifiable (can be split by hyrolysis)

• Triglyceride (fats), waxes

• Phospholipids, sphingolipids, glycolipids, lipoproteins

• Non-saponifiable• Carotenoids, cholesterol, fat soluble vitamins (D, K, E, A)

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Functions of lipids• Fuel for energy metabolism• Membrane structure

• Phospholipid, cholesterol

• Hormone action• Steroids, prostaglandins

• Electron transfer• Ubiquinone

• Antioxidant• Vitamin E

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Importance of Lipid Metabolism

• In health– Provision of energy in fasting/starvation

– Provision of energy in pregnancy & for lactation

• In diseases– Endocrine disease

• Diabetes mellitus, Cushing’s syndrome

– Production/metabolic diseases• Ketosis, pregnancy toxaemia, peri-parturient syndrome, fatty

liver disease, hyperlipidaemia

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• Linoleic, linolenic and arachidonic are essential fatty acids with >1 double bond

• Important for membrane function

• Cis double bond not trans

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Lipid of di-acyl glycerol provides the hydrophobic nature of phospholipid membrane

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Transport of lipid

• Problem of solubility

• From intestine – lipoprotein (chylomicrons)

• From liver – very low density lipoprotein

• From tissues to liver – high density lipoprotein

• From adipose tissue – fatty acid binds to albumin

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Lipoproteins

• Chylomicrons: • from intestine to adipose & other tissues

• Very low density lipoprotein (VLDL)• TG, cholesterol ester & phospholipid to tissues

• Low density lipoprotein (LDL)• Remnant of VLDL after release of lipid

• High density lipoprotein (HDL)• Collects lipid, especially cholesterol for return to

liver

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Lipoproteins:

Central hydrophobic core of triglyceride, cholesterol ester

Monolayer of phospholipid in outer surface, hydrophilic head to outer-face

Apolipoprotein in phospholipid monolayer

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Lipoprotein composition (%)

Chylo VLDL LDL HDL

Protein 1 10 25 50

Triglyceride 90 60 10 3

Cholesterol ester & cholesterol

5 15 45 20

Phopholipid 4 15 20 27

Size (m) 1000 50 20 10

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Apolipoproteins

• Apolipoprotein:– Have many

hydrophobic amino acids

– Hydrophilic amino acid on surface

– Provide structure

– Some have activity in lipid metabolism

• Apo A: predominantly in HDL, Cofactor for lecithin cholestrol acyl transferase (LCAT)

• Apo B :Predominantly in HDL & VLDL

• Apo C:Predominantly in HDL VLDL; Activates lipoprotein lipase in transfer of TG to adipose tissue

• Apo D:Predominantly in HDL

• Apo E: Predominantly in HDL & VLDL

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Fats in diet carried by chylomicron to tissues eg adipose

Liver exports TG, CE PL in VLDL to tissues

VLDL release lipids and become LDL

LDL remnants to tissues and liver

HDL is collecting lipid from tissues & from LDL to return to liver (reverse transport)

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HDL released from liver

Collects excess cholesterol from tissues

Produces cholesterol ester and lyso-lecithin from cholesterol and lecithin (phosphotidyl choline)

Cholesterol ester to VLDL and TG from VLDL

HDL absorbed by liver

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Transport from Adipose tissue –When energy needed

• Hormones (adrenaline, glucagon, cortisol) activate hormone sensitive lipase in Adipose Tissue

• Triglyceride 3 Fatty acid + Glycerol • Released from adipose to blood• Fatty acid disrupts membranes• Fatty acid bound to albumin – protects membranes• Fatty acid goes to tissues (eg muscle & liver) for

energy• Glycerol goes to liver for gluconeogenesis

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In cell (eg liver, muscle), FA linked to coenzyme A by high energy bond

Required ATP, releases AMP and PPi

2 x high energy ~phos bonds used

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Carnitine Shuttle

CoA can not enter mitochondria

Outer membrane: After activation FA-CoA transfers FA to carnitine to produce fatty acyl-carnitine- enzyme inhibited by malonyl CoA

Inner membrane: FA transferred back to CoA, carnitine recycles

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-Oxidation of saturated fatty acid

a) Dehydrogenase (oxidation) at -carbon with FAD reduced and trans == formed

b) Hydratase, hydrates double bond

c) Dehydrogenase with NAD reduced

d) Thiolase cleavage with release of acetyl CoA and CoA on shortened fatty acid

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-Oxidation of saturated fatty acid

a) Dehydrogenase (oxidation) at b-carbon with FAD reduced and trans == formed

b) Hydratase, hydrates double bond

c) Dehydrogenase with NAD reduced

d) Thiolase cleavage with release of acetyl CoA and CoA on shortened fatty acid

e) Note similarity to steps of TCA cycle

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-oxidation of palmitic acid

16 carbon fatty acid

7 cycles yields

8 acetyl CoA

7 FADH2

7 NADH + H+

Which gives 131 ATP

Equivalent of 2 ATP used in activation of FA:- net 129 ATP

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Palmitic acid yields >2x ATP per gram compared to glucose

Fatty acids have no water of hydration

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If FA has odd number of carbons, -oxidation proceeds till C3 fatty acyl CoA (propionyl CoA)

Carboxylase requiring biotin

Racemase (isomerase) converts between sterioisomers

Mutase – internal transfer of carboxyl group- required Vit B12

Succinyl CoA to TCA cycle and gluconeogenesis

THIS IS VERY IMPORTANT FOR RUMINANTS

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-Oxidation of poly unsaturated fatty acid

-oxidation operates for saturated FA (no double bonds)

When unsaturated other enzymes are involved for oxidation of double bonds

Reaction requires 2 x isomerases and NADPH dependent reductase as well as the usual enzymes

Altered reaction costs 5 ATP per double bond

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Catabolism of lipid

Stimulated by hormone, (glucagon, adrenalin)

Adipose tissue releases FA & glycerol

In liver & muscle FA is activated & enters mitochondria

Oxidised to acetyl CoA & enters TCA cycle

In liver, excess acetyl CoA forms ketone bodies, secreted to blood & used in other tissues (not brain)

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Control of lipid catabolism

• Whole animal level– Hormone sensitive lipase

• Triglyceride fatty acid + glycerol

– Activated by glucagon, adrenaline

• Cellular regulation– Carnitine shuttle inhibited by malonyl CoA– Malonyl CoA is a reactant in FA synthesis– Malonyl CoA elevated when cell is energy rich

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Formation of Ketone Bodies

In Liver (only organ with enzymes):

3 x acetyl CoA form 3-OH, 3-methyl glutaryl CoA

1 acetyl CoA released

Acetoacetate* formed

Reduction forms -hydroxybutyrate*

Decarboxylation forms acetone*

Exported to blood

Ketone bodies are more soluble that fatty acid & help to make energy available for tissues in fasting & starvation

* These are the ketone bodies

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Ketosis of acetyl Co-A

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Use of Ketone Bodies

In tissues (not brain in most species):

OH butyrate & acetoacetate to acetyl CoA

Enters TCA cycle for ATP production

Excess production of ketone bodies occurs in metabolic diseases – diabetes mellitus, bovine ketosis, ovine toxaemia

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Ketones in Ruminants

• Carbohydrate in diet of ruminants forms ‘volatile fatty acids’ in rumen

• These are acetate C2, propionate C3 and butyrate C4 fatty acids

• Butyrate converted to -OH butyrate (ie ketone body) in rumen wall before entry to blood

• Much energy for ruminant tissue metabolism come from this ketone body

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Lipid Anabolism – FA Synthesis• When body/cell is energy rich• Liver, adipose tissue & mammary gland• In cytoplasm, separate from -oxidation• Substrate is acetyl CoA (2 carbons)

• From glycolysis or amino acid catabolism

• Exported from mitochondria by citrate shuttle

• Primary product – palmitoyl CoA, (16 carbons)• Then modified - elongated, desaturated, conjugated

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Lipid Anabolism- FA Synthesis

• Co-factors: NADPH + H+, CoA

• Multi-enzyme complex• Fatty Acid Synthase complex

• Control point - Acetyl CoA carboxylase• Activated by citrate; inhibited by palmitoyl CoA

• Hormone activation (insulin); inhibition (glucagon)

• Nutrition: cabrohydrate activates; lipid inhibits

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FA Synthesis in Ruminants

• Fatty acid synthesis primarily in adipose tissue and mammary gland

• Little synthesis in liver• Substrate:

– Acetyl CoA from acetate in blood

– Acetate in blood is rumen product ‘volatile fatty acid’

– Activated by acetyl CoA synthase• Acetate + CoASH + ATP Acetyl CoA + AMP + PPi

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Acetyl CoA +

OxaloacetateADP, Pi

Citrate + CoA

Citrate + CoA+ ATP

Oxaloacetate+

Acetyl CoA +

ADP + Pi

MalatePyruvate

Pyruvate

Mitochondria

Cytoplasm

COO- , ATP

carboxylaseCitrate synthase (TCA cycle)

FATTY ACID SYNTHESIS

Citrate lyase

NAD+ NADH+ + H+

Malate DH

Malate enzyme

NADPH+ + H+ NADP+

CITRATE SHUTTLE

(Malate Shuttle also operates)

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Acetyl CoA carboxylase, activated by citrate, inhibited by palmitoyl CoA (product)

Activated by insulin, inhibited by glucagon

Upregulated by hi carbon/low fat diet

Downregulated by hi fat/low carbon diet

Control site for FA synthesis

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Fatty acid synthase complex: 7 enzymes and acyl carrier protein (ACP) in dimer formation

ACP has active –SH and binds growing FA chain

Cys with –SH also present

Each enzyme required for FA synthesis

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Transacylase: acetyl CoA to cys-SH, malonyl CoA to ACP-SH

Condensation by ketoacyl synthase: acetyl replaces carboxyl grp of malonyl, becomes C4

Reductase: C3 keto group becomes hydroxyl (NADPH)

Dehydratase: hydroxyl grp lost, double bond in place

Reductase: double bond is hydrogenated (NADPH)

Acetyl Transacylase: C4 FA transferred to cys-SH, new malonyl to ACP-SH

Reactions catalysed by fatty acid synthase

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Fatty Acid Synthesis

• Continues for 6 cycles

• C16 palmitoyl Co A is released by thioesterase

• Released palmitoyl CoA further modified– Elongated by addition of acetyl groups– Desaturated to give unsaturated FA– Conjugated to triglyceride and phospholipid

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In endoplasmic reticulum

C2 groups from malonyl CoA with loss of CO2

Multi-enzyme complex needed – FA elongase

Very long chain FA (22-24 carbons) needed for central nervous system

In Mitochnodria

Elongation by reversal of -oxidation

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Example: palmitoyl CoA (C16) Palmitoleioyl CoA C16 C16, cis 9

In endoplasmic reticulum, NADH & cytochrome b5 required

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In endoplasmic reticulum

In Liver: glycerol-3-phos from glycerol, product (TG) goes to VLDL synthesis

In adipose:glycerol-3 phos from glucose via glycolysis (no glycerol kinase), therefore only when animal has high glucose

* Note error in fig: Pi released from phosphatidic acid not taken up

*

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Control of FA synthesis

• In fed state with excess of energy– Increased insulin from pancreas

• Activates acetyl CoA carboxylase

• Activates lipoprotein lipase in adipose tissue, increasing TG breakdown in VLDL & transfer of Fatty acid to adipocyte

– Decreased glucagon – (insulin antagonist)

– High glucose • Enters adipose tissue, provides glycerol, essential for TG re-

synthesis

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Synthesis of Phospholipid

• In endoplasmic reticulum• A) diacyl glycerol (as in TG formation) reacts

with CDP-choline giving phosphatidyl choline and CMP

• Same with CDP-serine & CDP-ethanolamine

• B) phosphatidic acid (TG formation), react with CTP to form CDP-diacyl glycerol then with inositol to form phosphatidyl inositol

• From endoplasmic reticulum to membranes

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Synthesis of cholesterol

Liver predominant site

Acetyl CoA to mevalonate

3 x acetyl CoA forms HMG CoA (as in ketone formation)

Reduction to mevalonate (C6)

Converted to isopentenyl pyro phosphate (C5; diphosphate)

5 x isopent pyrophos condensed to give squalene

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Natural conformation of squalene encourages ring formation by cyclase

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Cholesterol

• Forms cholesterol ester for transport in VLDL (enzyme is LCAT)

• Forms bile acids with glycine, taurine for digestion

• Modifications lead to hormones

• No degradation in body (ie no enzyme to break ring structure)

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Regulation of cholesterol synthesis

• Committing enzyme:- 3-hydroxy 3-methyl glutaryl reductase (formation of mevalonic acid)

• Control by cholesterol concentration in cell– Hi chol inhibits: low chol activates

• Control by hormones– Insulin & thyroid hormone activates: glucagon & cortisol inhibit

• Control by diet – Enzyme synthesis increases in fasting– Enzyme synthesis decreases when dietary cholesterol is high

• Target for ‘statin’ drugs