Lipid and Ketone Metabolism Week 12
Jan 11, 2016
Lipid and Ketone Metabolism
Week 12
2
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
3
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)
4
Functions of lipids• Fuel for energy metabolism• Membrane structure
• Phospholipid, cholesterol
• Hormone action• Steroids, prostaglandins
• Electron transfer• Ubiquinone
• Antioxidant• Vitamin E
5
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
6
• Linoleic, linolenic and arachidonic are essential fatty acids with >1 double bond
• Important for membrane function
• Cis double bond not trans
7
Lipid of di-acyl glycerol provides the hydrophobic nature of phospholipid membrane
8
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
9
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
10
Lipoproteins:
Central hydrophobic core of triglyceride, cholesterol ester
Monolayer of phospholipid in outer surface, hydrophilic head to outer-face
Apolipoprotein in phospholipid monolayer
11
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
12
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
13
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)
14
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
15
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
16
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
17
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
18
-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
19
-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
20
-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
21
Palmitic acid yields >2x ATP per gram compared to glucose
Fatty acids have no water of hydration
22
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
23
-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
24
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)
25
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
26
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
27
Ketosis of acetyl Co-A
28
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
29
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
30
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
31
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
32
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
33
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)
34
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
35
36
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
37
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
38
39
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
40
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
41
Example: palmitoyl CoA (C16) Palmitoleioyl CoA C16 C16, cis 9
In endoplasmic reticulum, NADH & cytochrome b5 required
42
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
*
43
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
44
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
45
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
46
Natural conformation of squalene encourages ring formation by cyclase
47
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)
48
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