fa synthesis

7/29/2019 Fa Synthesis

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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


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


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

fa synthesis

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