LIPID MAPS Lipid Metabolomics Tutorial

Fatty Acid Oxidation

Professor Edward A. Dennis

Department of Chemistry and Biochemistry Department of Pharmacology, School of Medicine University of California, San Diego Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org

E.A. DENNIS 2010

©

Metabolism and Energy Overview

Proteins Carbo hydrates Amino Acids Simple Sugars Pyruvate Lipids Fatty Acids • Triglycerides (TG’s) are decomposed into Fatty Acids (FA’s).

• FA’s are b -oxidized to release energy and create acetyl CoA fragments.

• Acetyl CoA enters the power-producing Krebs cycle and electron transport chain.

Acetyl CoA Energy (CO 2 , H 2 O) E.A. DENNIS 2010

©

16 

Naming Conventions: Palmitic Acid

e 6 d 5 g 4 b 3 2 a 1

Carbonyl carbon

 e d g b a omega, always the last alkyl carbon epsilon, fifth carbon after the carbonyl delta, fourth carbon after the carbonyl gamma, third carbon after the carbonyl beta, second carbon after the carbonyl alpha, first carbon after the carbonyl E.A. DENNIS 2010

©

Beta Oxidation In a Nutshell...

Fatty Acid Acyl CoA (1) Enoyl CoA (2) L-hydroxyacyl CoA (3) Ketoacyl CoA (4) One iteration of

b

-Oxidation:

Make fatty acyl CoA.

Step 1: Oxidize the b -carbon (C3) Step 2: Hydrate the b -carbon Step 3: Oxidize the b -carbon, again!

Step 4: Thiolyze a-b bond, releasing acetyl CoA REPEAT from step 1, w/ 2 fewer carbons

Acyl CoA (shorter) Acetyl CoA TCA Cycle

E.A. DENNIS 2010

©

Make activated acyl CoA

Triglycerides

Hormone sensitive lipase

adipocyte

Albumin

Free FA’s Free FA’s

bloodstream

Free FA’s

Fatty acyl CoA synthase

Acyl CoA

target tissue cell

mitochondria Acyl CoA

Carnitine shuttle

• •

Hormone-sensitive lipase

– decomposes triglycerides – removes FA groups – free FA’s diffuse through membrane

Albumin

– carries FA’s to target tissue – FA’s diffuse into tissue cells • •

Fatty acyl CoA synthase

– adds CoA to the free FA

Carnitine shuttle

– moves fatty acyl CoA into the mitochondria E.A. DENNIS 2010

©

Regulation

• Hormone Sensitive Lipase is activated by cAMP • Cyclic AMP also turns off acetyl CoA carboxylase , stopping FA synthesis • Hormones like glucagon and epinephrine increase cAMP – FA synthesis slows – Triglycerides are broken down – FA’s enter b -oxidation faster E.A. DENNIS 2010

©

Location, Location, Location

• Fatty Acyl CoA is oxidized inside the mitochondrial matrix • The

carnitine shuttle

the matrix carnitine moves it into – Free carnitine is exchanged for acyl • •

Carnitine acyl transferases

catalyze reactions on both sides of membrane – driven by concentration gradient

CAT-1

is inhibited by high levels of malonyl CoA generated by acetyl CoA carboxylase in the pathway to Fatty Acid Synthesis.

Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Lif e at the Molecular Level, 2 nd with permission of John Wiley & Sons, Inc. ed. Reprinted E.A. DENNIS 2010

©

Step 1: Oxidize the

b

-carbon

Fatty acyl CoA FAD FADH 2 Acyl CoA dehydrogenase

• Acyl CoA dehydrogenase oxidizes the b -carbon – 3 versions of the enzyme exist – Specific to short, medium and long chain substrates • One FADH 2 is generated – makes 2 ATP’s • A

trans

double bond is created at the 2-carbon • Product is an Enoyl CoA D

2 -trans-enoyl CoA

E.A. DENNIS 2010

©

Medium-chain dehydrogenase deficiency

Fatty Acid Acyl CoA (1) Enoyl CoA (2) L-hydroxyacyl CoA (3) Ketoacyl CoA (4) Acyl CoA (shorter)

•

Incidence

: 1 in 10,000 live births – More common than phenylketonuria!

•

Symptoms

: – severe hypoglycemia >> lethargy, coma • little energy from FA’s • glucose reserves are immediately burned – contributes to sudden infant death syndrome (10% of cases) •

Mechanism

: – Normally, there are 3 separate fatty acyl CoA dehydrogenase enzymes for STEP 1 of b -oxidation • Specific for short, medium and long acyl chains, respectively – Autosomal recessive lack of medium chain enzyme •

Treatment

: Special diet and supportive care E.A. DENNIS 2010

©

Step 2: Hydrate the

b

-carbon

• Enoyl CoA hydratase adds a water molecule across the double bond • Product is b -hydroxyacyl CoA – S- stereoisomer E.A. DENNIS 2010

©

Step 3: Oxidize the

b

-carbon, Again!

• Beta-hydroxyacyl CoA dehydrogenase oxidizes the b -carbon again • One NADH is created – makes 3 ATP’s • A ketoacyl CoA is produced E.A. DENNIS 2010

©

Step 4: Thiolyze off acetyl CoA

CoA-SH Acyl CoA: Acyltransferase (“thiolase”)

• Thiolase (aka: Acyl CoA acyltranferase ) splits the ketoacyl • A new CoASH is consumed • Acetyl CoA is released • A new, shorter Acyl CoA remains and re enters the cycle E.A. DENNIS 2010

©

REPEAT with a Shorter Acyl CoA

• Palmitoyl CoA (16 carbons) becomes myristoyl CoA (14 carbons).

• Each iteration releases another acetyl CoA.

• 7 iterations will release 8 acetyl CoA fragments from the original palmitoyl CoA.

• Net equation:

Palmitoyl CoA + 7CoASH + 7FAD + + 7NAD + + 7H 2 0 yields: 8 Acetyl CoA + 7 FADH 2 + 7NADH + 7H +

E.A. DENNIS 2010

©

b

-Oxidation Cycle

Removal of 2 carbons from acyl chain

[4]

Acyl CoA: acyltransferase Fatty acyl-CoA Acyl CoA dehydrogenase

[1]

Acetyl-CoA

b

-ketoacyl CoA

[3]

b-hydroxyacyl CoA dehydrogenase

D

2 -trans-enoyl CoA enoyl CoA hydratase

[2] TCA cycle b

-hydroxyacyl CoA

E.A. DENNIS 2010

©

How Much Energy?

Each palmitoyl CoA group released from a triglyceride: – directly produces 7 NADH & 7 FADH 2 • which generate 21 and 14 ATP’s, respectively – releases 8 acetyl CoA molecules for TCA Cycle • which each generate 12 ATP Grand total is 131 ATP per fully oxidized palmitoyl CoA Efficiency of Energy Recovery = 40% ** Recently, some have calculated energetically less ATP equivalents per NADH/FADH2 [2.5 and 1.5 instead of 3 and 2] which lowers the numbers somewhat (Berg).

E.A. DENNIS 2010

©

Efficiency of Fat Storage

Fat has 9 kcal/gram = 9 kcal/cc Both Carbohydrate and Protein have 4 kcal/gram = 4 kcal/3cc or 1.33 kcal/cc

Fat/(Carbohydrate or Protein) = 6x/cc!!!

E.A. DENNIS 2010

©

Problem:

Oxidizing Unsaturated FA’s

Linoleic Acid C12 (Even # Unsat) C9 (Odd # Unsat) 3 NAD + + 3FAD + CoA-SH 3 NADH + 3FADH2 + 3Acetyl-CoA 3 rounds of

b

- oxidation Problem:

b

-

g

cis double bond

E.A. DENNIS 2010

©

Special Case 1: Odd Unsaturations

The GOOD News: • Enzyme enoyl-CoA isomerase moves the double bond over 1 position.

• Oxidation process resumes The BAD News: • The first oxidation step is skipped • One less FADH 2 is made – 2 fewer ATPs E.A. DENNIS 2010

©

Special Case 2: Even Unsaturations

4 2 H + + NADPH NADP + 3

The GOOD News: • Enzymes 2,4 dienoyl-CoA reductase isomerase a-b a-b and 3,2 dienoyl-CoA convert the unsaturation.

g-d and double bonds into a single • Oxidation process resumes

3 4 2 3

The BAD News: • The conversion costs one NADPH directly.

• Net effect is the loss of one NADH – 3 fewer ATPs

4 2

E.A. DENNIS 2010

©

FA Synthesis vs. FA Oxidation

Cell Location Acyl carrier 2-Carbon Piece b -hydroxyl acyl step Electron carriers Primary tissue site

Synthesis

Cytosol ACP Malonyl CoA R-config NADPH Liver

Oxidation

Mitochondria CoA Acetyl CoA S-config NADH, FADH Muscle, liver E.A. DENNIS 2010

©

FA Synthesis vs. FA Oxidation (cont)

1. They are

not

the reverse of one another.

– Different subcellular locations – R and S isomers of b -hydroxyacyl intermediates cannot easily jump to the other pathway – Electrons donated from oxidation (NADH, FADH 2 ) cannot directly enter synthesis, which uses NADPH.

2. Separate, semi-independent pathways allow more sophisticated regulation.

– Accelerating one pathway does not mean slowing the other.

E.A. DENNIS 2010

©

Acknowledgement

This tutorial is based on an evolving subset of lectures and accompanying slides presented to medical students in the Cell Biology and Biochemistry course at the School of Medicine of the University of California, San Diego.

I wish to thank Dr. Bridget Quinn and Dr. Keith Cross for aid in developing many of the original slides, Dr. Eoin Fahy for advice in applying the LIPID MAPS nomenclature and structural drawing conventions [Fahy et al (2005) J Lipid Res, 46, 839-61; Fahy et al (2009) J Lipid Res, 50, S9-14] and Masada Disenhouse for help in adopting to the tutorial format.

Edward A. Dennis September, 2010 La Jolla, California E.A. DENNIS 2010

©