Biochemistry of Lipid Metabolism

Fat Metabolism Overview

• Fat is a significant energy store, containing a large amount of stored energy.
• Different hormones (Epinephrine and Insulin) regulate fat metabolism in Muscle and Liver cells.
• Muscle cells store glucose as glycogen.
• Liver cells share glucose from glycogen.
• Fats are broken down through a process known as beta-oxidation.
• Beta-oxidation produces Acetyl CoA, and the body derives ATP through the Citric Acid Cycle (CAC).
• During beta-oxidation, NADH and FADH2 are produced and carry electrons to the electron transport chain to generate ATP.
• Acyl CoA is produced in the cytoplasm, is important for the transportation of fats into the mitochondria for beta-oxidation.
• The process of transporting Acyl CoA into the Mitochondria requires a carnitine shuttle, which includes 2 enzymes (CAT 1& 2).
• The carnitine shuttle moves Acyl chain to carnitine in the intermembrane space to cross into the inner membrane, to then to be broken down using CAT II.
• The product of Beta-oxidation (Acetyl CoA) enters the CAC to produce energy.
• The overall process of beta-oxidation starts with 16:0 CoA and ends with 8 molecules of Acetyl CoA, as well as NADH, FADH2 and H+.

Learning Objectives

• Understand the processes and locations for fat creation/breakdown.
• Understand the ATP production through beta-oxidation via oxidative phosphorylation.
• Know fat synthesis enzymes ACC and FAS.
• Examine lipid synthesis and lipolysis (beta-oxidation) differences.
• Detail the reciprocal regulation of fat metabolism.

Triacylglycerol (Triglyceride, TG)

• Extra carbons are stored in molecules of triacylglycerol, mostly in fat cells and other tissues.
• Acyl chains are attached to a glycerol backbone via ester bonds..
• Fatty acyl chains are saturated to allow for close packing of the chains.
• When fatty chains are used, they must be hydrolyzed.

Fat Mobilization via GPCR signaling

• Epinephrine and glucagon bind to G-protein receptors.
• The signaling involves the activation of Adenylyl cyclase which produces cAMP.
• Protein kinase A (PKA) is activated.
• Triacylglycerol lipase (also known as hormone-sensitive lipase, HSL) is activated.
• TAGs are hydrolyzed to release fatty acids and glycerol.

Mitochondrial Structure

• Mitochondria has an outer membrane and an inner membrane that's folded to form cristae.
• The inner membrane contains porins (outer membrane proteins) permitting the passage of Acyl CoA molecules but, it doesn't cross the inner membrane.
• The intermembrane space lies between the outer and inner membrane.
• The matrix is inside the inner membrane.

Acyl CoA and β-oxidation

• Acyl CoA is produced in the cytoplasm, but β-oxidation happens inside the mitochondria matrix.
• Acyl CoA can move through the outer membrane via porins, but can't penetrate the inner membrane because it is too large and water-soluble.
• The function of making Acyl CoA outside the matrix lies in its necessity for the transportation through the inner membrane, to proceed to the matrix.

Carnitine Shuttle

• The carnitine shuttle assists in the transportation of acyl groups into the mitochondrial matrix to begin beta-oxidation.
• Carnitine Acyltransferase I (CAT I) moves the acyl group onto carnitine. The acyl chain moves from CoA to carnitine.
• Acyl carnitine is transported across the inner membrane in exchange for carnitine.
• Carnitine Acyltransferase II (CAT II) moves the acyl group from carnitine back onto CoA.
• Acyl CoA can be processed to generate energy through Beta-oxidation.

What's the difference between Acyl CoA and Acetyl CoA?

• Acyl CoA has a longer chain compared to Acetyl CoA.
• Acetyl CoA has two carbons, while Acyl CoA has more than two carbons.

Fatty Acid Activation in the Cytoplasm

• Acyl chains are trapped in the cell by the covalent addition of coenzyme A.
• Acyl CoA Synthetase is the enzyme catalyzing the reversible reaction using ATP (→ AMP) to form the fatty acyl CoA molecule.
• Pyrophosphate (PPi) hydrolysis to 2 Pi is favorable and drives this reaction in the forward direction.

ATP→AMP is Energetically Inefficient

• 95% of ATP production occurs through oxidative phosphorylation (using ADP as a substrate).
• AMP gets converted to ADP to form ATP, requiring additional ATP.
• Fatty acid activation requires 2 ATPs.

4 reactions to repeat throughout beta-oxidation

• Each round of beta-oxidation involves 4 reactions to shorten the fatty acid chain, producing NADH, FADH2, and Acetyl CoA.

How many rounds of beta oxidation are needed?

• Determine the number of rounds required for breakdown of 16:0 CoA by calculating the difference in Carbon and divide it by two, because every round requires two carbons.

4 steps of β-oxidation

• Acyl CoA dehydrogenase produces FADH2 and creates a double bond in the fatty acid chain.
• Hydratase adds water across the double bond (β-OH).
• Dehydrogenase produces NADH and creates a keto group on the β-carbon in the chain.
• A thiolase uses CoA to release acetyl CoA, producing a fatty acyl CoA chain with 2 fewer carbons.

Overall β-oxidation of 16:0 CoA in the Mitochondria

• 16:0 CoA + 7 NAD+ + 7 FAD + 7 H2O + 7 CoA → 8 acetyl CoA + 7 NADH + 7 FADH2 + 7 H+

Calculate the number of ATPs and H2O made by the complete oxidation of 16:0 CoA.

• The calculation for ATP and H2O is based on Beta-oxidation, Citric Acid Cycle and Oxidative Phosphorylation.

P/O Ratios and H2O made

• Details the production of ATP and water through the electron transport chain.
• Complex I, III, and IV are crucial to the process.
• The Complexes are accompanied by the release of H2O.

Grand Total for the complete oxidation of 16:0 CoA in the Mitochondrial Matrix

• 16:0 CoA + 108 ADP + 108 Pi + 23O2 → 16 CO2 + 108 ATP + 123H2O +CoA.
• If starting from 16:0 (no CoA) activation of 16:0 requires 2 ATP and 1 H2O.

Summary - Fatty Acid Breakdown

• Epinephrine and/or glucagon trigger TAG breakdown.
• Fatty acids travel in the bloodstream bound to serum albumin, entering muscle cells.
• Fatty acids are activated using Acyl CoA synthetase.
• The enzymes CAT I, translocase, and CAT II control fatty acid import into the matrix, exchanging carnitine for free carnitine (inner membrane).
• Fatty acid chains are converted through β oxidation for energy producing ATP.
• β-oxidation does not produce a substantial amount of ATP directly (but using the Citric Acid Cycle and oxidative phosphorylation).

Using Fats as Fuel

• Acetyl CoA from fat degradation is processed through the Citric Acid Cycle (a crucial component for ATP production in mitochondria).
• CAC depends on the availability of Oxaloacetate.
• Oxaloacetate can be formed from amino acids or pyruvate via pyruvate carboxylase.

Hormonal Response and Fat Synthesis

• When glucose levels rise, insulin signals glucose uptake and breakdown.
• Insulin promotes glucose storage as glycogen in muscles or the liver.
• Once ATP/NADH levels are high in mitochondria, the CAC slows and fatty acid synthesis begins, using Acetyl CoA.
• Excess glucose, amino acids, and fats will be used as precursors to create more fatty acids.

Steps in Fatty Acid Synthesis

• Mitochondrial Acetyl CoA is transported to the cytoplasm for creating fatty acids.
• CoA Carboxylase converts Acetyl CoA to Malonyl CoA.
• Fatty acid Synthase uses Malonyl CoA to create fatty acid chains (16:0).

Step 1- Import & Export differ

• The transportation and movement of molecules like Acetyl CoA and citric acid between the mitochondrial matrix and cytosol are critical for cellular metabolism.
• The inner membrane of mitochondria plays a critical role in regulating transport.
• The Citrate shuttle transports Acetyl CoA (C2s) from mitochondria to the cytosol via the citrate in the form of C6.
• Then, it's converted back into Acetyl CoA again in the cytosol.

Step 1. Acetyl CoA is exported as Citrate

• CoA is unable to cross the mitochondrial membrane.
• It's transported outward through Citrate.
• Citrate is created in the CAC, moving to the cytoplasm and converted back into Acetyl CoA for fatty acid synthesis.
• Pyruvate returns to the mitochondria.

Fatty Acid Synthesis is an Anabolic Process

• Fatty acid synthesis happens in the cytoplasm.
• The committed and regulated enzyme for this synthesis is Acetyl CoA Carboxylase(ACC).
• This reaction uses ATP, while fatty acid synthase (FAS) uses NADPH for reducing power in the anabolic process.

Step 2: Acetyl CoA Carboxylase (ACC)

• Acetyl CoA carboxylase (ACC) is the enzyme responsible in synthesizing Malonyl-CoA from Acetyl-CoA, using ATP and bicarbonate. The addition of the COO- group to the malonyl-CoA promotes fatty acid synthesis by the Fatty Acid Synthase enzyme.

Malonyl → Reciprocal Regulation for Fats

• When the molecule is active, fat synthesis occurs.
• Malonyl CoA stops the cycle of fat breakdown by inhibiting carnitine acyltransferase 1 (a key regulator for fat transportation through the mitochondrial inner membrane..
• Carboxylase regulation is important for fat metabolism (synthesis and breakdown).

ACC Regulation via Phosphorylation

• The regulation of Acetyl CoA Carboxylase (ACC) involves phosphorylation (adding a phosphate group).
• Phosphorylation generally inactivates the enzyme.
• Protein Kinase A (PKA) and AMP-activated protein kinase (AMPK) plays a role in such phosphorylations when the molecule is active.
• The enzyme phosphatase removes the phosphate group and reactivates the enzyme.
• This process is crucial to the reciprocal regulation of fat synthesis and breakdown.

What is the link between exercise and the inhibition of fat synthesis?

• Epinephrine through the G-protein coupled receptor (GPCR) pathway activates protein kinase A (PKA).
• High levels of AMP (indicating a lowered energy state) activate AMP-activated protein kinase (AMPK), which further inhibits Acetyl CoA carboxylase (ACC) to stop fat synthesis.

ACC Regulation via Phosphorylation

• Protein Kinase A participates in activating an inactive carboxylase.
• AMPK is activated when the energy state is low.
• Protein Kinase A and AMPK are activated in a catabolic (energy-requiring) state.
• Insulin activates the phosphatase to inactivate PKA and AMPK, activating the carboxylase.

Regulation of Fat Metabolism

• ACC is the regulated step in fat synthesis.
• Phosphorylation of ACC can decrease activity.
• Rising levels of citrate can increase ACC activity.
• The product of fatty acid synthesis, malonyl CoA, can shut down fat breakdown by inhibiting carnitine acyltransferase 1.

Step 3: Use of malonyl CoA to form fatty acid chains by Fatty Acid Synthase (FAS)

• The enzyme ACP will connect malonyl CoA and Acetyl CoA to form fatty acids.
• The Fatty Acid Synthase (FAS) catalyzes the synthesis of fatty acyl chains from malonyl-CoA.
• ACP is a carrier protein transporting the chain units.

Fatty Acid Synthase (FAS)

• FAS is a large multi-enzyme complex.
• The complex catalyzes the synthesis of fatty acids from acetyl-CoA and malonyl-CoA molecules.
• There are different steps involved in this reaction, using different redox reactions that include NADPH.
• An initial step involves condensation of acetyl-CoA and malonyl-CoA to form a four-carbon molecule.
• Following, there are some redox reactions, dehydration, and further condensation, repeating until a required length of the fatty chain is achieved.

Fat Synthesis

• The synthesis of fatty acids requires both Acetyl-CoA carboxylase (ACC) and Fatty Acid Synthase (FAS).
• Cycles of these, 7 FAS cycles and 1 acetyl-CoA to produce a 16-carbon fatty acid, followed by cycles of malonyl-CoA molecules each cycle.
• The reaction uses up 7 ATPs and 14 NADPHs at each cycle.
• Chain elongation to produce longer-chain fatty acids (greater than 16 carbons), using malonyl-CoA and NADPH.
• Desaturases, with NADPH and oxygen, introduce cis bonds to synthesize fatty acids.

Fatty Acid Synthase vs Beta Oxidation

• FAS (Fatty Acid Synthase), a major anabolic enzyme, works in the cytoplasm.
• β Oxidation, a major catabolic enzyme, works in the mitochondria.
• Different 2-carbon units, coenzymes, and number of reactions (not enzymes) distinguish the two processes.

Low Carb Diets

• Low carbohydrate intake results in lowered glycogen levels, pushing the body to rely on fat breakdown (for energy).
• The body converts pyruvate into Acetyl-CoA, allowing for fat breakdown and energy production though the citric acid cycle.
• Weight loss depends on lowered energy from glycogen stores.

Key Messages

• Excess carbons from fats, carbohydrates, and proteins become stored as triacylglycerol.
• GPCR signaling initiates TAG breakdown. Fatty acids are transported through the blood stream bound to serum albumin.
• Fatty acids are activated and transported into mitochondria where beta-oxidation occurs.
• Fatty acid synthesis, also known as lipogenesis, begins after acetyl-CoA moves out of the mitochondria. Acetyl-CoA carboxylase (ACC) is the main regulated enzyme.
• Fatty acid synthase (FAS) plays a part in this synthesis process.