Lecture 13 (Fatty acid Metabolism)

Proton pumping and ATP yield

10 NADH + 2 FADH₂

1 NADH → 10 H⁺ 2.5 ATP (Into intermembrane space)

1 FADH₂ → 6 H⁺ 1.5 ATP (into intermembrane space)

Total = 16 H⁺ 4 ATP (across intermembrane space)

Total NADH, FADH₂ and ATP produced

NADH: 10

FADH₂: 2

ATP made by ETC: 28 ATP

total ATP: 32 ATP

Fatty Acid Metabolism

Triacylglycerol (TAG) are a major dietary energy source and long-term energy storage.

Fatty acid oxidation supplies most of the energy required by heart and support liver metabolism.

Fatty acid oxidation produce Acetyl CoA, NADH, FADH₂, links fat metabolism to TCA and Oxidative phosphorylation.

Fats better for Long term energy storage

Fatty acids are highly reduced: more energy than carbohydrates

Fats are stored without associated water: stored in anhydrous lipid droplets

Energy density: 9 kcal/g (fats) vs 4 kcal/g (carbs)

Glycogen: short-term energy reserve

Triacylglycerols: long-term energy reserve, hydrophobic

Digestion and absorption of Dietary Fats in the small intestine

1. Emulsification: Bile salt, disperse large fat droplets into small fat droplets, increase surface area for digestion.

2. Enzymatic digestion: Pancreatic lipase, assisted by colipase, hydrolyzes TAGs, produce free fatty acid and 2-monoacylglycerols

3. intestinal absorption: Bile salt helps make fatty acid and monoacylglycerols. Deliver lipids to the intestinal epithelial cells (enterocytes)

4. Re-esterification: fatty acid and monoacylglycerols are reassembled to TAGs in the ER

Chylomicron formation: newly synthesized TAGs packaged with cholesterol, phospholipids, & apolipoproteins into chylomicrons

Transport of Dietary Fats to Tissues

5. Chylomicron secretion: chylomicrons released from enterocytes by exocytosis and enter intestinal lymphatic vessels

6. Lipoprotein lipase (LPL): activated by apolipoprotein C-II (apoC-II) on chylomicrons. Hydrolysis of chylomicrons; releasing free fatty acids

7. Fatty acid utilization: fatty acids go through β-oxidation to support ATP production

What are Lipoproteins and apolipoproteins

Lipoproteins are transport particles

Apolipoproteins are the ones that determine how those particles interact with enzymes and cells

What happens to Chylomicron remnants

taken by liver through receptor-mediated endocytosis

Structure and Functions of Lipoproteins

ApoC-II (apolipoprotein C-II): activates LPL

ApoB-100: binds LDL receptors, liver being a major site for LDL clearance.

ApoE: mediates uptake of chylomicron remnants by liver.

ApoB-48: Chylomicron assembly and secretion by enterocytes.

Lipoprotein and their function

Chylomicrons: Transports dietary TAGs from the intestine

VLDL: Transport liver-produced TAGs to tissues

LDL: Deliver cholesterol to cells cleared by liver

HDL: Participate in reverse cholesterol transport

Lipoprotein size and density

Size: Chylomicrons > VLDL > LDL > HDL

Density: HDL > LDL > VLDL > Chylomicrons

Mobilization of Stored Fats: Lipolysis in Adipose Tissue

During fasting or high energy demand, Lipolysis breaks down TGAs in adipocytes into 3 fatty acids and glycerol.

Lipolysis involve 3 sequential enzyme:

1. ATGL: TAG → DAG + fatty acid

2. HSL: DAG → MAG + fatty acid

3. MGL: MAG → glycerol + fatty acid

And That’s GoaL. He Shoot Left. Me GoaL. (numonic)

Released fatty acids undergo beta-oxidation

Glycerol is transported to liver to do gluconeogenesis

Catecholamine activates what

Catecholamine activates beta adrenergic receptors

What does Insulin do to lipolysis

Insulin inhibits Lipolysis

ATGL and HSL promote Lipid breakdown in the Liver

Question: can increase lipase activity reduce excess triglyceride accumulation in liver?

Experiment: done on obese mice that received adenoviruses expressing ATGL and HSL

Key finding: Hepatic triglyceride level dropped

Mechanism: increased lipase expression promoted fatty acid oxidation

yes, the lipase expression promotes fatty acid oxidation, breaking down the triglycerides.

Glycerol Metabolism: What happens to glycerol after TAG breakdown?

Glycerol released during lipolysis is transported mainly to liver and kidney

Glycerol kinase: Glycerol → glycerol-3-phosphate, consuming ATP

DHAP can enter glycolysis or gluconeogenesis

Glycerol contributes small fraction of the total energy from TAGs.

Fatty acid activation by conversion to fatty acyl-CoA

Fatty acid needs to be turned to acyl CoA before beta oxidation

Acyl CoA synthetase catalyzes fatty acid using ATP and CoA-SH

2 ATP is consumed. ATP is converted to AMP + PPi (pyrophosphate)

Long-chain fatty acyl groups are transferred to carnitine for transport.

Chemistry of fatty acid activation

carboxylate ion is adenylated by ATP; forming acyl-adenylate and PPi, PPi immediately hydrolyzes to 2 molecules of Pi

Thiol group of coenzyme A attack acyl-adenylate

Why is ΔG° of all 3 step in converting fatty acid to acyl CoA negative?

fatty acid activation is coupled with ATP consumption and pyrophosphate hydrolysis, making the overall process energetically favorable

Carnitine shuttle (not required for short/medium chain)

Long chain fatty acyl-CoAs (14 or more carbons; majority) cannot cross inner mitochondrial membrane

CPT1: transfers fatty acyl group from CoA to carnitine; forms acylcarnitine

Carnitine-acylcarnitine translocase (CACT): Transports acylcarnitine across inner membrane

CPT2: Regenerates fatty acyl-CoA in the matrix; releases carnitine

β-Oxidation Pathway

β-Oxidation occurs primarily in mitochondrial matrix

each cycle has 4 reactions: oxidation, hydration, oxidation and thiolysis

each cycle generates 1 FADH₂, 1 NADH, and 1 acetyl-CoA, while shortening the fatty acyl-CoA by 2 carbons.

β-Oxidation: Step 1--- FAD-Dependent Dehydrogenation

1st reaction: Fatty acyl-CoA goes through dehydrogenation; lose 2 hydrogen atoms from the alpha (C2) and beta (C3) carbons.

Trans double bond forms between C2 and C3. produces trans-Δ²-enoyl-CoA.

Enzymes: chain-length-specific acyl-CoA dehydrogenases

• VLCAD: inner mitochondrial membrane-associated

• MCAD and SCAD: soluble mitochondrial matrix enzymes

Electron transfer: enzyme bound FAD turns to FADH₂ (accept 2 hydrogen)

Connection to ETC:

electrons from FADH₂ → electron transfer flavoprotein (ETF) → ETF: ubiquinone oxidoreductase → CoQ, entering the ETC without passing through Complex I or II

β-Oxidation: Step 2--- Hydration of the Double Bond

2nd reaction: trans-Δ²-enoyl-CoA undergoes hydration, adding H₂O across the double bond

mechanism:

• H is added to α-carbon (C2)

• OH is added to β-carbon (C3)

Enzyme: enoyl-CoA hydratases

• Short and medium chain substrates: Soluble mitochondrial matrix enzyme

• Long-chain substrates: Enoyl CoA hydratase

Stereospecificity: enzyme produces L-isomer of 3-hydroxyacyl CoA

Similarity to TCA cycle: going from fumarate to L-malate

Energy production: No NADH, FADH₂, ATP produced.

β-Oxidation: Step 3---NAD -Dependent Dehydrogenation

3rd reaction: L-3-hydroxyacyl-CoA oxidizes to a ketone

mechanism: -OH group in C3 is converted to a carbonyl (C=O)

• NAD+ turns to NADH

• product is 3-ketoacyl-CoA

Enzyme: L-3-hydroxyacyl-CoA dehydrogenase

• short and medium chain substrates: Soluble mitochondrial matrix enzyme

• Long chain: Dehydrogenase mitochondrial trifunctional protein

Stereospecificity: enzyme specifically recognizes L-isomer of 3-hydroxyacyl-CoA

Similarity to TCA cycle: malate to oxaloacetate, malate dehydrogenase oxidizes alcohol group to carbonyl group

Energy production: 1 NADH

β-Oxidation: Step 4 ---Thiolysis (Release of Acetyl-CoA)

4th reaction: β-Ketoacyl-CoA goes through thiolytic cleavage break C-C bond

Enzyme: β-ketothiolase (3-ketoacyl-CoA thiolase)

Reaction mechanism: cysteine attacks β-ketoacyl-CoA, form covalent acyl-enzyme intermediate and releases Acetyl CoA

Products:

• 1 acetyl-CoA

• 1 fatty acyl-CoA shortened by 2 carbons, undergoes another thiolysis

Final cycle: when fatty acyl CoA contain 4 carbon, thiolysis generates 2 acetyl-CoA molecules

Energy production: does not directly generate NADH, FADH₂ or ATP

Clinical relevance of lack of LCHAD (L-chain-3-hydroxylacyl-CoA dehydrogenase)

deficiency of LCHAD impairs fatty acid oxidation

Total ATP production from fatty acid oxidation

108 ATP from palmitoyl-CoA

Net 106 ATP from free palmitate after activation

Fatty acid Catabolism

• Produces acetyl-CoA

• produces NADH and FADH₂

• takes place in mitochondria.

Fatty acid Anabolism

requires Acetyl CoA and malonyl CoA

requires NADPH

takes place in cytosol for animals and plastids for plants

Malonyl-CoA (3C) supplies 2 C unit after decarboxylation

2 reduction consumes 2 NADPH per elongation cycle

principal product for mammalian fatty acid is palmitate

Malonyl-CoA formation is catalyzed by Acetyl-CoA carboxylase

Acetyl-CoA carboxylase catalyzes acetyl-CoA (2C) to malonyl-CoA (3C)

ACC requires Biotin (vitamin B7) as coenzyme

2 step mechanism:

• Step 1: ATP-dependent carboxylation of biotin using bicarbonate

• Step 2: transfer carboxyl group to acetyl-CoA, forming malonyl-CoA

Malonyl-CoA provides the 2 carbons for fatty acid chain elongation.

Added carboxyl group is released as CO₂, helping drive C-C bond formation.

ACC catalyzes the committed, major regulatory step of fatty acid synthesis

Fatty acid synthesis is catalyzed by fatty acid synthase

FAS catalyzes stepwise elongation of fatty acid chain

Acetyl group starts the synthesis, malonyl addes 2 carbon through each elongation cycle

2 NADPH is provide reducing power

repeated until palmitate is formed.

FA synthesis is regulated by acetyl-CoA carboxylase
(The reaction catalyzed by ACC is the rate-limiting step)

Citrate activates ACC, promoting fatty acid synthesis

Palmitoyl-CoA inhibits ACC, providing feedback inhibition

AMPK phosphorylates and inhibits ACC when cellular energy is low

Insulin favors ACC activation and fatty acid synthesis.