Topics for Fatty Acid Biosynthesis

Regulation of Fatty Acid Biosynthesis

Dr. Susan C. Frost BCH 6206

! 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

Chapter 25 pgs 930-942

copyright: Susan C. Frost

Figure 1

Figure 2

Provision of Acetyl CoA via Citrate

Glucose Glucose 6-phosphate Fructose 6-phosphate Palmitate
NADP HEX - P NADPH + H+ Fatty Acid Synthase

Acetyl CoA Carboxylase

O CH3-C ~ S-CoA O HOOC-CH2-C ~ S-CoA ADP + Pi

biotin

ATP + CO2

Malonyl CoA Glyceraldehyde 3-P

NAD NADH + H+

Malate OAA

Acetyl CoA Carboxylase 1

Acetyl CoA

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

Pyruvate

Citrate

cytosol mitochondria

Pyruvate CO2

Acetyl CoA Oxaloacetate

Acetyl CoA Carboxylase 2

Malonyl CoA

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?

Citrate

Malate

" -Ketoglutarate

Figure 3

Figure 4

Effect of Insulin and Citrate on Polymerization of ACC

(1) 0.75 0.50 (2)
control

(1)

(2)
+ citrate

Effect of Fasting and Refeeding on ACC Phosphorylation and Activity

Acetyl CoA Carboxylase Activity (% of total) - Act.

60 40 20

A280

0.25 0 0.75 0.50 0.25 0 10 20 10 20 10 20

+ insulin

Pi

Phosphate Incorporation (mol Pi/mol subunit)

10
+ insulin + citrate

20 60 40 20

Carboxylase Activity (U/mg)

A280

5 0 fasting 0 12 24 48 Time (hours) 72

Adipose tissue was treated or not with insulin, extracts prepared and treated or not with citrate. Partially purified ACC (equivalent to 500mg of original tissue) was chromatographed on an FPLC. Fractions were then assayed for protein content or activity.

rf = refeeding

(1) Elution of PDH marker (10 x 106 ) (2) Elution of ferritin marker (450,000)
adapted from Borthwick etal. (1987)

ACC was prepared from fed (time 0) and fasted and refed animals. Activity (circles) and phosphate content (squares) was determined (in the absence of added citrate) as a function of time.
adapted from Thampy and Wakil (1988)

Figure 5

Figure 6

Functional Regions of ACC

Classification of Phosphorylation Sites on ACC

Class 1 sites Class 2 sites
cAMP-dependent PK (1200) 5'AMP-dependent PK (77) inactivation of ACC (in vitro) Calmodulin-dependent PK (25) Casein kinase (29) Protein kinase C (PKC) (95) no effect on activity

1200

1958 1990
acetyl CoA binding site

273

469

785

NH2
biotin interaction site of ATP and HCO3 Ser-P

2345
COOH

In vitro

1200

2345

200

ACC activity ( :U/ :g ACC)

NH2 1 (cAMP-dep PK)

COOH

150 100 50 0 10 Time (min)

no kinase cAMP-dep 5'-AMP-dep

23 25 29

77

95

100 3 (5'AMP-dep PK)

20

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 Kim et al. (1989)

adapted from Ha et al. (1994)

Figure 7

Figure 8

AICAR Inactivates ACC

(AICAR is an analog of AMP)

Inhibition of AMP-kinase and Activation of ACC by Insulin

1.6

Acetyl-CoA Carboxylase (nmol/min/g of cells)

40 30 20
10

AICAR ( :M) 0 50 100 500 200

AMPK (pmol/min/ g protein)

1.2 0.8
0.4

10

20

30

control + insulin

Acetyl CoA Carboxylase (relative activity)

20 10 Time after addition (min)

30

0.12 0.09 0.06

0.03

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 of ACC was measured in digitonin-permeabilized cells.

10

20

30

Time (min)
adapted from Henin et al. (1995) adapted from Witters and Kemp. (1992)

Figure 9

Effect of ACC2 Knockout on Malonyl CoA Levels in Selected Tissue

Triglyceride Content in Liver Reduced in ACC2 Knockout Mice

Figure 10

oil-red stain indicates triglyceride droplets

Wild type

Knockout

Bar = 50 m White bars: ACC2 knockout; Black bars: wild type ACC1 compensates for loss of ACC2 in liver
Fram Abu-Elheiga, et al. (2001)

ACC1-generated malonyl CoA in the knockout did not block fatty acid oxidation, despite its abundance. This suggests that the malonyl CoA produced by ACC1 and ACC2 exists in two distinct compartments and that ACC2 is responsible for the pool which regulates fatty acid oxidation.
Fram Abu-Elheiga, et al. (2001)

Figure 11

Figure 12

Abdominal and Epididymal Adipose Tissue is Reduced in Knockout Mice

(one gene, one polypeptide, seven activities)

Function division
malonyl translacylase acetyl transacylase ketoacyl synthase enoyl hydratase hydratase

Dimeric Structure of FAS

ketoacyl reductase thioesterase ACP

Cys SH SH
Subun it divis ion

Pan SH SH

Bar = 1 cm Fatty acids are mobilized from adipose for oxidation in other tissue, particularly cardiac and skeletal muscle. Leptin, an adipocyte-specific cytokine, was reduced from 53 ! 9 ng/mL to 36 !3 ng/mL with an increase in appetite in knockout mice. Inhibition of ACC2 might allow humans to lose weight while maintaining normal caloric intake!?
Fram Abu-Elheiga, et al. (2001)

Pan
ACP thioesterase ketoacyl reductase

Cys
ketoacyl synthase acetyl transacylase malonyl translacylase

hydratase enoyl hydratase

Function division
see Smith (1994)

Figure 13

Figure 14

Fatty Acid Synthase

1 2 cys-SH pan-SH O 1 2 cys-S ~ C-CH3 O pan-S ~ C-CH2-COO Acetyl CoA
transferases

Malonyl CoA

Effect of Feeding and Starvation on FAS mRNA Abundance

2 days old and unfed to begin 11 days old

O 1 2 cys-S ~ C-CH2-CH2-CH3 pan-S ~ C-CH2-COO

FAS mRNA

ketoacyl synthase

*CO
1 2

cys-SH pan-S ~ C-CH2-C-CH3 O NADP O Malonyl CoA

1.5 1.0 0.5 0 24 12 Time (hours) 36

FAS mRNA (log scale)

2.0

0.5 0.2 0.1 0.05 0.02 0

STARVE

H + NADPH
ketoreductase

1 2

cys-SH H pan-S ~ C-CH2-C-CH3 O H OH OH 7 cycles

18 6 12 Time (hours)

dehydrase

FEED

cys-SH

mRNA was extracted from duck liver at the appropriate time during feeding or starvation, probed with FAS cDNA, and quantitated as relative abundance.

2 pan-S ~ C-CH=C-CH3 H + NADPH O

enoyl reductase

1 2

NADP cys-SH HH pan-S ~ C-C-C-CH3 HH O

thioesterase

Palmitate

adapted from Goodridge (1986)

Figure 15

O - C O
5

PUFA Synthesis
5 8 11 14 17 20 n6

Glucose

Pathways for FA and TG Biosynthesis

pentose phosphate shunt NADPH ME Malate

Figure 16

Glucose 6-phosphate

Pyruvate

linoleic acid

linolenic acid ER
Acetyl CoA Citrate

Oxaloacetate CL Citrate Acetyl CoA ACC Malonyl CoA FAS

elongation

elongation

Mitochondrial
arachidonic acid elongation elongation peroxisome 1-acyl glycerol 3-phosphate -oxidation docosahexaenoic acid
Wallis et al. (2002)

Palmitate Glycerol 3-phosphate GPAT

Endoplasmic Reticulum

GPAT 1-acyl glycerol 3-phosphate Phosphatidic acid

Cytoplasm

Triacylglycerol

Phospholipids

adapted from Sul and Wang (1998)

Regulation of FA Metabolism
Insulin Cytosol Citrate Acetyl CoA Acetyl CoA Mitochondrion Lactate Citrate

Figure 17

Pyruvate

Malonyl CoA Palmitate FACoA cAMP AMP

OAA Ketones

FACoA Liver Cell FFAs cAMP HSL

Free Fatty Acids

Triglyceride pool Adipocyte