Fatty Acid Metabolism Notes

Fatty Acid as Major Fuel Storage

• A 70 kg man has the following fuel reserves:
  • Triacylglycerols: 420,000 kJ (100,000 kcal), 80%
  • Proteins: 100,000 kJ (24,000 kcal), 19%
  • Glycogen: 2,500 kJ (600 kcal), 0.5%
  • Glucose: 170 kJ (40 kcal), 0.03%

Fatty Acid Degradation Overview

• Utilization of fatty acids as energy stores requires three stages of processing.
• Unsaturated and odd-chain fatty acids require additional steps for degradation.
• Ketone bodies can be derived from fats.
• Fatty acid metabolism provides insight into various physiological states.
• Fatty acids are stored in adipose tissue as triacylglycerols (TAG), where they're linked to glycerol via ester linkages.
• Adipose tissue is the main site for synthesis, storage, and mobilization of fat droplets.

Three Stages of Processing for Fat Storage Utilization

1. Mobilization:
   • Triacylglycerols are hydrolyzed into their constituent molecules and transported to energy-requiring tissues.
2. Activation and Localization:
   • Fatty acids are activated and transported to the mitochondria for degradation.
3. Degradation:
   • Fatty acids are broken down, step-by-step, into acetyl-CoA.
   • This intermediate is oxidized by the TCA cycle.

Triacylglycerols Hydrolyzed by Hormone-Stimulated Lipases

• Hormones involved:
  • Glucagon
  • Epinephrine
• Key proteins:
  • Perilipin: A lipid droplet associated protein
  • HS lipase: Hormone-sensitive lipase
  • DAG: Diacylglycerol
  • MAG: Monoacylglycerol
  • ATGL: Adipocyte triacylglyceride lipase

Mechanism of Triacylglycerol Hydrolysis

• Binding of glucagon or epinephrine to receptors on adipose tissue results in a cAMP cascade.
• Protein kinase A phosphorylates perilipin and hormone-sensitive lipase.
• Phosphorylation of perilipin results in the activation of adipocyte triacylglyceride lipase (ATGL), initiating lipid breakdown.
• Fatty acids, being insoluble in aqueous solutions, bind to albumin in the blood, which delivers them to tissues in need of fuel.

Glycerol Release from Lipolysis

• Hydrolysis of triacylglycerols produces glycerol, which is absorbed by the liver and immediately phosphorylated.
• Following oxidation to dihydroxyacetone phosphate, it is isomerized to glyceraldehyde-3-P and processed by either the gluconeogenesis or glycolytic pathways.

Activation of Fatty Acids

• Upon entering the cell cytoplasm, fatty acids are activated by attachment to coenzyme A to form acyl-CoA.
• This reaction is catalyzed by acyl-CoA synthetase.
• Acyl-CoA is the carrier of activated acyl groups.
• This reaction is driven by the hydrolysis of PPi.

Carnitine's Role in Transporting Acyl-CoAs

• Fatty acids are activated on the outer mitochondrial membrane but must be transported to the matrix for oxidation.
• Fatty acyl-CoA transport requires a specialized mechanism due to the inner mitochondrial membrane's integrity.

Mechanism of Carnitine-Mediated Transport

1. Fatty acyl-CoAs are conjugated to carnitine, a zwitterionic alcohol, to form an acyl-carnitine intermediate.
   • This reaction is catalyzed by carnitine acyl-transferase I.
2. The acyl-carnitine intermediate is shuttled across the inner mitochondrial membrane through the action of acyl-carnitine translocase.
3. In the mitochondrial matrix, acyl-carnitine is re-esterified to CoA through the action of carnitine acyl-transferase II.
   • The regenerated carnitine molecule is translocated back to the cytoplasm to begin another round of transport.

Each Round of Fatty Acid Oxidation

• Generates an Acetyl-CoA, an NADH, and an FADH2
• The degradation of a saturated fatty acid chain with an even number of carbon atoms follows this sequence:
  1. Oxidation by FAD
  2. Hydration
  3. Oxidation by $NAD^+$
  4. Thiolysis by Co-A
• Additional enzymes are required to fully degrade odd-chain or unsaturated fatty acids.

Step 1: Oxidation by Acyl-CoA Dehydrogenase

• The first reaction in every round of degradation is an oxidation reaction catalyzed by acyl-CoA dehydrogenase.
• This enzyme catalyzes the FAD-linked oxidation of a fatty acyl-CoA to a trans-D2-enoyl-CoA, with a double bond between C2 and C3.
• FAD is used as an oxidant instead of $NAD^+$ because the $\Delta G$ for this reaction is insufficient to drive the reduction of $NAD^+$ to NADH.
• The electrons from the resulting $FADH<em>2$ are transferred to $QH</em>2$ through Complex II, ultimately generating 1.5 ATP.

Step 2: Hydration by Enoyl-CoA Hydratase

• The second reaction is catalyzed by enoyl-CoA hydratase.
• This enzyme catalyzes the stereospecific hydration of the double bond between C2 and C3 to yield an L-3-hydroxyacyl-CoA.
• The enzyme is stereospecific, forming only one enantiomer of the product.

Step 3: Oxidation by L-3-hydroxyacyl-CoA Dehydrogenase

• Catalyzed by L-3-hydroxyacyl-CoA dehydrogenase.
• This enzyme catalyzes a second oxidation reaction, which converts the hydroxyl group at C3 to a keto group. The liberated electrons are transferred to $NAD^+$.
• The NADH generated enters the ETC through Complex I, forming 2.5 ATP per NADH utilized.
• By generating a keto group at position 3 of the fatty acyl-CoA chain, this reaction sets the stage for the final step.
• This enzyme is specific for the L-isomer of a 3-hydroxyacyl-CoA. FA oxidation is also called β-oxidation.

Step 4: Cleavage by β-ketothiolase

• The final reaction is catalyzed by β-ketothiolase.
• This enzyme catalyzes the cleavage of the 3-ketoacyl-CoA into two products: a fatty acyl-CoA chain shortened by two carbon atoms, and acetyl-CoA.
• The acetyl-CoA generated can proceed into the TCA cycle, generating 10 ATP equivalents.
• The shortened fatty acyl-CoA chain can proceed through another round of degradation.
• Once the chain is shortened sufficiently, a different acyl-CoA dehydrogenase with higher affinity for shorter fatty acyl-CoA chains is used for the first step; the other reactions are identical.

Summary of Fatty Acid Oxidation Cycles

• Cycle 1: remove C1-C2 as acetyl CoA, the shorter acyl chain has CoA attached to C3
• Cycle 2: remove C3-C4 as acetyl-CoA, the shorter acyl chain has CoA attached to C5
• Cycle 5: remove C9-C10, left with C11-C12 , two acetyl-CoA made
• For even number saturated fatty acids degradation: 5 $FADH_2$, 5 NADH, 6 acetyl-CoA

Complete Oxidation of Palmitate

• Yields 106 Molecules of ATP
• In each reaction cycle, a fatty acyl-CoA is shortened by two carbon atoms, and one molecule of $FADH_2$, NADH, and Acetyl-CoA are formed.
• The overall reaction:
  $C<em>n$-acyl-CoA + FAD + $NAD^+$ + $H</em>2O$ + CoA → $C<em>{n-2}$-acyl-CoA + $FADH</em>2$ + NADH + Acetyl-CoA + $H^+$
• Complete degradation of palmitoyl-CoA ($C<em>{16}$-acyl-CoA) requires 7 cycles: $C</em>{16}$-acyl-CoA + 7 FAD + 7 $NAD^+$ + 7 $H<em>2O$ + 7 CoA → 8 Acetyl-CoA + 7 $FADH</em>2$ + 7 NADH + 7 $H^+$
• Assuming 2.5 ATP/NADH, 1.5 ATP/$FADH_2$, and 10 ATP/acetyl-CoA, 108 molecules of ATP are generated.
• Because 2 ATP-equivalents were consumed in activating the palmitate to palmitoyl-CoA, the net yield of ATP from oxidation of palmitate is 106 ATP.

Principal Reactions Required for Fatty Acid Degradation

*An AMP-forming ligase.

Unsaturated and Odd-Chain Fatty Acids

• Require Additional Steps for Degradation
• The oxidation of fatty acids containing double bonds, and those containing odd numbers of carbon atoms require additional enzymes.
• Many dietary fatty acids contain unsaturations (in particular those derived from plants), and their degradation proceeds according to a generally similar scheme to the one just discussed, with the exception of two enzymes.
• The two enzymes required to complete unsaturated fatty acid degradation are cis-D3-enoyl-CoA isomerase and 2,4-dienoyl- CoA reductase.
• Unsaturated fatty acids with double bonds at odd numbers require only the isomerase; at even numbers require both the isomerase and reductase.

Degradation of Palmitoleate

• An Unsaturated Fatty Acid with a Double Bond at an Odd Position
• Palmitoleyl-CoA ($C_{16}$) can undergo three cycles of degradation according to the standard mechanism. However, after the third round, the remaining carbon skeleton has an unsaturation between C3 and C4, and is not a substrate for acyl-CoA dehydrogenase.
• In this case, an enzyme called cis-D3-enoyl- CoA isomerase converts this double bond into a trans-D2 unsaturation.
• The resulting product, trans-D2-enoyl-CoA, is a regular substrate for fatty acid degradation. It can be processed as normal.

Degradation of Odd Chain Fatty Acids

• Yields Propionyl-CoA Following the Final Thiolysis Step
• In general, fatty acids with an odd number of carbon atoms are rare. This is due in part to how fatty acids are synthesized (as 2-C unit). Nevertheless, their breakdown occurs more-or-less similarly to their even chain counterparts until the final round. Here propionyl-CoA is formed along with acetyl-CoA.
• Propionyl-CoA enters the TCA cycle as succinyl-CoA following a series of peculiar reactions. There are three steps in this minipathway.
• First, propionyl-CoA is carboxylated to D- methylmalonyl-CoA in an ATP-requiring reaction catalyzed by propionyl-CoA carboxylase.

Conversion of Propionyl-CoA to Succinyl-CoA

• Next, D-methylmalonyl-CoA is racemized to the L- methylmalonyl-CoA form in a reaction catalyzed by methylmalonyl-CoA epimerase
• The final step is catalyzed methylmalonyl-CoA mutase. The product of this reaction is succinyl-CoA, which then enters the TCA cycle.

Ketone Bodies Formation

• Acetyl-CoA resulting from fatty acid oxidation enters the TCA cycle only when fat and carbohydrate breakdown are balanced.
• This is because the oxidation of Acetyl-CoA through the TCA cycle is dependent upon the availability of Oxaloacetate (OAA). Mitochondrial levels of OAA are dependent on an adequate supply of carbohydrates from glycolysis. In fasting or diabetic conditions, OAA is used to form glucose and not available for TCA cycle.
• When carbohydrates are unavailable (or utilized improperly, as in diabetes), mitochondrial levels of OAA drop and acetyl-CoA cannot proceed efficiently through the TCA cycle.
• Under these physiological or pathophysiological conditions that favor gluconeogenesis, the excess acetyl-CoA produced from fatty acid oxidation is diverted to the production of ketone bodies.

Ketone Bodies Production

• During the starved state or in uncontrolled diabetics, acetyl-CoA can be converted into either D-3-hydroxybutyrate and/or acetone.
• The 4-carbon ketoacid, acetoacetate, is the biochemical precursor to both D-3- hydroxybutyrate and acetone. It is formed from acetyl-CoA in three enzymatic steps:
  • Thiolase
  • HMG-CoA Synthase
  • HMG-CoA Lyase
• The sum of the reactions that yield acetoacetate is as follows:
  2 Acetyl-CoA + $H_2O$ → acetoacetate + 2 CoA + $H^+$
• The final product D-3-hydroxybutyrate forms by the NADH-dependent reduction of acetoacetate via D-3- hydroxbutyrate dehydrogenase. Its absolute levels depend on the amount of NADH in mitochondria.
• Acetone results from the slow, non- enzymatic decarboxylation of acetoacetate. Its levels mirror those of acetoacetate in the blood.

Ketone Bodies as Fuel

• The major site of ketogenesis is the liver. Once produced, ketone bodies can diffuse into the bloodstream to be taken up by peripheral tissues.
• Both acetoacetate and 3- hydroxybutyrate are normal substrates for respiration and are important energy sources.
• During starvation and diabetic states, the brain can meet up to 75% of its energy requirement through ketone bodies.
• Heart muscle and the renal cortex use acetoacetate in preference to glucose.

Utilization of Ketone Bodies

• Utilization of ketone bodies as metabolic fuels essentially involves the reverse chemistry as in their formation. Acetoacetate is converted into acetyl-CoA in two enzymatic steps.
• First, acetoacetate is activated to acetoacetyl- CoA by transferring the CoA group from succinyl-CoA via a specific CoA transferase enzyme. This enzyme is notably missing in liver!
• Acetoacetyl-CoA is then cleaved by thiolase to form two molecules of acetyl-CoA.
• 3-hydroxybutyrate requires one additional step, where it is oxidized by $NAD^+$ to form acetoacetate.

Ketone Bodies as Transportable Acetyl-CoA

• Because they can be reversibly formed from and reconverted into acetyl-CoA, ketone bodies represent a water-soluble, transportable form of acetyl-CoA units.
• The central molecule in this entire process is acetoacetate. It has a regulatory function on the enzymes which control the metabolic flux from triacylglycerols to ketone bodies.
• High levels of acetoacetate in the blood are a biochemical signal that acetyl units are abundant. This results in a decrease in the rate of formation of free fatty acids from triacylglycerols in adipose tissue, since free fatty acids are the ultimate precursor for ketone bodies.
• 2 Acetyl-CoA + $H_2O$ ® acetoacetate + 2 CoA + $H^+$
• D-3-hydroxybutyrate and acetone

Ketone Bodies During Starvation

• Glucose is the predominant fuel for the brain.
• During starvation, protein degradation is initially the source of carbons for gluconeogenesis in the liver. The glucose is then released into the blood for the brain to use.
• After several days of fasting, the brain begins to use ketone bodies as a fuel. Ketone body use curtails protein degradation and thus prevents tissue failure. Moreover, ketone bodies are synthesized from fats, the largest energy store in the body.
• After depletion of triacylglycerols, protein degradation accelerates, and death inevitably results from a loss of heart, liver, or kidney function. A person’s survival time is mainly determined by the size of the triacylglycerol depot.

Fuel Metabolism in Starvation

Glucose Synthesis Limitations

• Animals Cannot Convert Fatty Acids into Glucose
• An important biochemical limitation of mammals is that they are unable to synthesize glucose from fatty acids.
• This is because acetyl-CoA cannot be converted into either pyruvate or oxaloacetate, since the pyruvate dehydrogenase reaction (which converts pyruvate into acetyl-CoA) is irreversible.
• While fatty acid oxidation is used to provide ATP for gluconeogenesis, the actual carbon atoms used to form glucose are derived from non-fatty acid substances such as lactate, amino acids, and glycerol.

High Blood Ketone Levels

• High blood concentrations of ketone bodies are seen in certain pathological conditions, such as diabetic ketosis. This is because the acidic nature of ketone bodies can drop blood pH to dangerously low levels.
• Type-I diabetics do not produce insulin, which signals to take up additional blood glucose AND to stop lipolysis by adipose tissue. Lack of glucose uptake means the liver can’t make OAA from glucose, the TCA cycle slows, and ketones accumulate. Continued TAG break- down by adipose tissue results in further ketogenesis by the liver.
• As blood ketone levels rise, blood pH drops. High Blood Levels of Ketone Bodies Can be Life Threatening.