fa synthesis
TRANSCRIPT
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Regulation of FattyAcid Biosynthesis
Dr. Susan C. Frost
BCH 6206
Chapter 25 pgs 930-942
copyright: Susan C. Frost
Topics for Fatty Acid
Biosynthesis
Substrate for Fatty Acid Biosynthesis
Acetyl CoA Carboxylase I and 2
Allosteric regulation
Covalent modification
Polymerization
Hormone action
Fatty Acid Synthase
Multifunctional catalysis
Transcriptional regulation
Overview of Pathways for FA and TG
synthesis
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Provision of Acetyl CoA via
Citrate
Figure 1
Glucose
Glucose 6-phosphate
Fructose 6-phosphate
Glyceraldehyde 3-P
Pyruvate
Pyruvate Acetyl CoA
CitrateOxaloacetate
Citrate
Acetyl CoA
OAA
Malonyl CoA
Palmitate
-Ketoglutarate"Malate
MalateNAD
NADH + H+
HEX - P
NADP
NADPH + H+
CO2
Acetyl CoA
Carboxylase 1
Fatty Acid Synthase
mitochondria
cytosol
Malonyl CoAAcetyl CoACarboxylase 2
Acetyl CoA Carboxylase
Figure 2
CH3-C ~ S-CoA
O
HOOC-CH2-C ~ S-CoA
O
ATP + CO2 ADP + Pi
biotin
Rate limiting step
Allosteric regulation (citrate and fatty acyl CoA's)
Polymerization (citrate, fatty acyl CoA, insulin)
Covalent Modification (phosphorylation)
Two different forms: ACC1 and ACC2
ACC1 is highly expressed in liver and adipose and is
localized to the cytosol
ACC2 is expressed in heart and skeletal muscle, and to
a lesser extent in liver and is localized to the
mitochondria
Malonyl CoA from either enzyme serves as a key metabolic
regulator
Question: Are there two different pools of malonyl CoA?
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Effect of Insulin and Citrate on
Polymerization of ACC
Figure 3
0
0.25
0.50
0.75
10 20
A280
10 20
20
40
60
AcetylCoAC
arboxylaseActivity(%
ofto
tal)
10 200
0.25
0.50
0.75
A280
10 20
20
40
60+ insulin
+ citrate
+ citrate
+ insulin
control
Adipose tissue was treated or not with insulin, extracts prepared and treated ornot with citrate. Partially purifi ed ACC (equivalent to 500mg of original tissue) was
chromatographed on an FPLC. Fractions were then assayed for protein content
or activity.
adapted from Borthwick etal. (1987)
(1) (2)
(1) Elution of PDH marker (10 x 106 )
(2) Elution of ferritin marker (450,000)
(1) (2)
Effect of Fasting and Refeeding
on ACC Phosphorylation and
Activity
Figure 4
24 48 72120
8
7
6
5
Time (hours)
PhosphateIncorporation
(molPi/molsubunit)
adapted from Thampy and Wakil (1988)
ACC was prepared from fed (time 0) and fasted and refedanimals. Activity (circles) and phosphate content (squares) wasdetermined (in the absence of added citrate) as a function of time.
rf = refeeding
CarboxylaseActivity
(U/mg)
0
Pi
-
Act.
fasting
Functional Regions of ACC
Figure 5
1
1200
2345
NH2 COOH
273
46
9
785
1958
1990
acetyl CoA
binding site
biotin
interaction site of ATP
and HCO3 -
Ser-P
adapted from Kim et al. (1989)
1 1200
2345
1
10025
29
77
95
3
NH2 COOH
(cAMP-dep PK)
(5'AMP-dep PK)
23
Classification of Phosphorylation
Sites on ACC
Figure 6
Class 1 sites Class 2 sites
Calmodulin-dependent PK (25)
Casein kinase (29)
Protein kinase C (PKC) (95)
cAMP-dependent PK (1200)
5'AMP-dependent PK (77)
no effect on activity inactivation of ACC (in vitro)
0 10 20
Time (min)
ACCa
ctivity
(U/g
ACC)
:
:
50
100
150
200
no kinase
cAMP-dep
5'-AMP-dep
ACC was purified from transfected HeLa cells. Phosphorylation by cAMP-dep PK
decreases Vmax and increases Km for citrate. Phosphorylation by 5'AMP decreases
Vmax (in fact to a greater extent than does cAMP-dep PK)
adapted from Ha et al. (1994)
In vitro
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0 10 30
Time after addition (min)
Ac
etyl-CoACarboxylase
(nmol/min/gofcells)
10
20
30
20
40
0
AICAR ( M):
500 200
100
50
0
AICAR Inactivates ACC
adapted from Henin et al. (1995)
Figure 7
Legend: Isolated rat hepatocytes were incubated in the
presence of 15mM glucose and specific concentrations of
AICAR, added at time 0. At the times indicated, activity ofACC was measured in digitonin-permeabilized cells.
(AICAR is an analog of AMP)
0 10 30
AMPK
(pmol/
min/
gprotein)
0.4
0.8
1.2
20
1.6
0
0 10 30
Time (min)
AcetylC
oA
Carboxylase
(rela
tivea
ctivity)
0.03
0.06
0.09
20
0.12
0
+ insulin
control
Inhibition of AMP-kinase and
Activation of ACC by Insulin
Figure 8
adapted from Witters and Kemp. (1992)
Effect of ACC2 Knockout onMalonyl CoA Levels in
Selected Tissue
Fram Abu-Elheiga, et al. (2001)
White bars: ACC2 knockout; Black bars: wild type
ACC1 compensates for loss of ACC2 in liver
Figure 9
Bar = 50 m
Wild type
Knockout
Fram Abu-Elheiga, et al. (2001)
Triglyceride Content in LiverReduced in ACC2 Knockout
Mice
oil-red stainindicates
triglyceridedroplets
ACC1-generated malonyl CoA in the knockout did not block
fatty acid oxidation, despite its abundance. This suggeststhat the malonyl CoA produced by ACC1 and ACC2 exists intwo distinct compartments and that ACC2 is responsible forthe pool which regulates fatty acid oxidation.
Figure 10
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Bar = 1 cm
Abdominal and Epididymal
Adipose Tissue is Reduced in
Knockout Mice
Fram Abu-Elheiga, et al. (2001)
Leptin, an adipocyte-specific cytokine, was reduced from
53 9 ng/mL to 36 3 ng/mL with an increase in appetitein knockout mice.
! !
Inhibition of ACC2 might allow humans to lose weight whilemaintaining normal caloric intake!?
Fatty acids are mobilized from adipose for oxidation in othertissue, particularly cardiac and skeletal muscle.
Figure 11
Dimeric Structure of FAS
Figure 12
Cys
SH
SH
Pan
Cys
SH
SH
Pan
Function division
Subunitdivision
Function division
ketoacylsynthase
acetyltransacylase
malonyltranslacylase
enoyl
hydratase
hydratase
ketoacyl
reductase
ACP
thio-
esterase
thio-esterase
ACP
ketoacylreductase
hydrataseenoyl
hydratase
malonyltranslacylase
acetyl
transacylase
ketoacylsynthase
(one gene, one polypeptide, seven activities)
see Smith (1994)
Fatty Acid Synthase
Figure 13
1
2
cys-SH
pan-SHAcetyl CoA
Malonyl CoA
1
2
cys-S ~ C-CH3
pan-S ~ C-CH2-COO-
O
O
*
1
2
cys-SH
pan-S ~ C-CH2-C-CH3
O
*CO2
O
1
2
cys-SH
pan-S ~ C-CH2-C-CH3
O O
1
2
cys-SH
pan-S ~ C-CH=C-CH3
O
1
2
cys-SH
C-C-C-CH3
O
1
2
C-CH2-CH2-CH3
O
pan-S ~ C-CH2-COO-
O
*
Malonyl CoA
Palmitate
ketoacylsynthase
ketoreductase
dehydrase
enoyl reductase
thioesterasepan-S ~
cys-S ~
NADP
NADP
NADP
7 cycles
transferases
H
H
H
H +
NADPHH +
H
OH
H
H
H
H
Effect of Feeding and Starvation
on FAS mRNA Abundance
Figure 14
0Time (hours)
FASmRN
A
0.5
1.0
1.5
2.0
12 24 360.02
0.05
0.1
0.2
0.5
FASmRNA(logsc
ale)
0 6 12 18Time (hours)
mRNA was extracted from duck liver at the appropriate time during feeding or
starvation, probed with FAS cDNA, and quantitated as relative abundance.
FEED STARVE
2 days old and unfed to begin 11 days old
adapted from Goodridge (1986)
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linoleic acid linolenic acid
PUFA Synthesis
Wallis et al. (2002)
Figure 15
elongationelongation
elongation
elongation
arachidonic acid
docosahexaenoic
acid
-oxidation
peroxisome
ER
C
O
-O
5 8 11 14 17
20
5 n6
Acetyl CoA Citrate Citrate Acetyl CoA
Malonyl CoA
Oxaloacetate
Malate
Pyruvate
NADPH
FASACC
CL
ME
GPAT GPAT
Palmitate
Glycerol 3-phosphate
1-acyl glycerol 3-phosphate 1-acyl glycero l 3-phosphate
Phosphatidic acid
PhospholipidsTriacylglycerol
Glucose
Glucose 6-phosphatepentose phosphate shunt
Mitochondrial
Cytoplasm
EndoplasmicReticulum
Pathways for FA and TG
Biosynthesis
adapted from Sul and Wang (1998)
Figure 16
MitochondrionCytosol
Liver Cell
Adipocyte
Citrate
Acetyl CoA
Triglyceride pool
Free Fatty Acids
HSL
FFAs
FACoA FACoA
Pyruvate
OAA
cAMP
Citrate
Acetyl CoA
Malonyl CoA
KetonesPalmitate
cAMP
AMP
InsulinRegulation of FA Metabolism
Figure 17
Lactate