Fatty Acid and Triacylglycerol Metabolism

Fatty Acid Physiological Roles

Fatty acids serve four major physiological roles:

Fuel molecules: Primary energy source during moderate exercise and rest.
Building blocks: Components of phospholipids and glycolipids.
Protein modification: Attachment targets proteins to membrane locations.
Hormones and messengers: Fatty acid derivatives act as hormones and intracellular messengers.

Triacylglycerols as Energy Stores

Triacylglycerols (triglycerides) are uncharged esters of fatty acids with glycerol, mainly stored in adipose tissue and muscle.

Concentrated energy: Triacylglycerols are highly concentrated stores of metabolic energy because they are reduced and contain no water.
Energy comparison: Complete oxidation of fatty acids yields $38 \text{ kJ g}^{-1}$ , compared to $17 \text{ kJ g}^{-1}$ for carbohydrates and proteins.
Anhydrous fat: A gram of nearly anhydrous fat stores $6.75$ times as much energy as a gram of hydrated glycogen.

Adipose Tissue and Adipocytes

Adipose tissue: Fuel-rich white tissue located throughout the body, under the skin (subcutaneous fat) and surrounding internal organs (visceral fat).
Adipocytes: Fat cells making up adipose tissue, specializing in triacylglycerol synthesis, storage, and mobilization into fuel.
Lipid droplets: Large globules formed by the coalescence of triacylglycerols within adipocytes.

Digestion of Dietary Lipids

Lipases: Intestinal enzymes secreted by the pancreas that degrade triacylglycerols into free fatty acids and monoacylglycerol.

Bile Acids and Colipase

Lipid emulsion: Lipids exit the stomach as an emulsion with a triacylglycerol core surrounded by cholesterol and cholesterol esters.
Bile acids: Amphipathic molecules synthesized from cholesterol in the liver and secreted from the gallbladder. They facilitate lipid digestion by orienting ester bonds for lipase accessibility.
Colipase: A protein secreted by the pancreas that binds lipase to the particle, permitting lipid degradation.

Transport of Dietary Lipids

Chylomicrons: Triacylglycerols are reformed in the intestine and packaged into lipoprotein particles called chylomicrons.
Absorption: Chylomicrons enter the blood for triacylglycerol absorption by tissues.
Micelles: Free fatty acids and monoacylglycerols are transported in micelles to the plasma membrane of intestinal epithelial cells.
FATPs: Fatty-acid transport proteins facilitate the transport of free fatty acids and monoacylglycerols inside the membrane.
FABPs: Fatty-acid binding proteins ferry free fatty acids and monoacylglycerols to the cytosolic face of the smooth ER for triacylglycerol resynthesis.

Chylomicron Formation and Degradation

Chylomicrons: Lipoprotein transport particles composed of newly synthesized triacylglycerols, with proteins, phospholipids, and cholesterol on the surface.
Degradation: Chylomicrons are released into the blood and degraded by membrane-bound lipases at adipose tissue and muscles.

Fatty Acid Utilization: Three Stages

Stage 1 (Mobilization): Triacylglycerol degradation to fatty acids and glycerol, release from adipose tissue, and transport to energy-requiring tissues.
Stage 2 (Activation and Transport): Activation of fatty acids and transport into the mitochondria.
Stage 3 (Breakdown into acetyl CoA): Degradation of fatty acids to acetyl CoA for processing in the citric acid cycle.

Mobilization of Triacylglycerols

Hormone-stimulated lipases: Catalyze the hydrolysis of triacylglycerols to fatty acids.
Glucagon and epinephrine: Act through 7TM receptors, leading to phosphorylation of perilipin and hormone-sensitive lipase.

Effects of Phosphorylated Perilipin

Restructuring: Phosphorylated perilipin restructures the fat droplet, making triacylglycerols more accessible to mobilization.
ATGL coactivator release: Phosphorylation of perilipin triggers the release of a coactivator for adipose triglyceride lipase (ATGL).
ATGL function: ATGL initiates mobilization by releasing a fatty acid from triacylglycerol, forming diacylglycerol, when bound to its coactivator.

Completion of Fatty Acid Mobilization

Hormone-sensitive lipase: Converts diacylglycerol into a free fatty acid and monoacylglycerol.
Monoacylglycerol lipase: Converts monoacylglycerol into a free fatty acid and glycerol, completing fatty acid mobilization.
Ethanol inhibition: Ethanol can inhibit the signaling pathway, leading to fatty liver disease.

Release of Fatty Acids and Glycerol into the Blood

Albumin: Protein that transports fatty acids in the blood with seven binding sites of varying affinity.
Glycerol processing: Glycerol is absorbed by the liver, phosphorylated, oxidized to dihydroxyacetone phosphate, and isomerized to glyceraldehyde 3-phosphate.

Lipolysis and Glycerol Processing

Liver metabolism: Depending on metabolic needs, the liver processes glycerol via the glycolytic or gluconeogenic pathway.

Activation of Fatty Acids

FATPs and FABPs: Fatty acids enter intestinal cells through FATPs and are transported within the cell by FABPs.
Acyl CoA synthetase: Catalyzes the activation of fatty acids through thioester linkage to coenzyme A, requiring ATP and taking place on the outer mitochondrial membrane.

Transport of Activated Fatty Acids

Carnitine: An alcohol with both a positive and a negative charge (a zwitterion).
Carnitine conjugation: Fatty acids must be conjugated to carnitine to be transported across the inner mitochondrial membrane.

Acyl Carnitine Translocase

Translocase function: Mediates the entry of acyl carnitine into the mitochondrial matrix.

β-Oxidation Pathway

Four steps: Oxidation by FAD, hydration, oxidation by NAD+, and thiolysis by coenzyme A.
Chain shortening: Each repetition shortens the fatty acid chain by two carbons.
β-carbon oxidation: Named β-oxidation because oxidation occurs at the β-carbon atom.

Degradation of Fatty Acids: Reaction Sequence

Step 1: Acyl CoA is oxidized by FAD to trans-Δ2-Enoyl CoA, producing FADH2.
Step 2: trans-Δ2-Enoyl CoA is hydrated to L-3-Hydroxyacyl CoA.
Step 3: L-3-Hydroxyacyl CoA is oxidized by NAD+ to 3-Ketoacyl CoA, producing NADH.
Step 4: 3-Ketoacyl CoA undergoes thiolysis by HS-CoA to yield Acetyl CoA and a shortened Acyl CoA.

Unsaturated and Odd-Chain Fatty Acids

Unsaturated fatty acids: β-oxidation alone cannot degrade unsaturated fatty acids.
Isomerase: cis-Δ3-enoyl CoA isomerase converts the double bond into a trans-Δ2 double bond, yielding trans-Δ2-enoyl CoA, a normal substrate for β-oxidation.
Odd numbers of double bonds: Unsaturated fatty acids with odd numbers of double bonds require only the isomerase.

Odd-Chain Fatty Acid Degradation

Propionyl CoA: β-oxidation of fatty acids with odd numbers of carbons generates propionyl CoA.
Succinyl CoA conversion: Propionyl CoA is converted into succinyl CoA.
Succinyl CoA fates: Enter the citric acid cycle or convert into oxaloacetate for glucose biosynthesis.

Final Product Conversion

Odd-numbered fatty acid degradation product: Converted into a CAC (citric acid cycle) intermediate.

Fatty Acid Oxidation in Peroxisomes

Peroxisomes: Small membrane-bounded organelles present in most eukaryotic cells.
Function: Oxidation of long chain and branched fatty acids takes place in peroxisomes, shortening very long chains to make them better substrates of β-oxidation in mitochondria; oxidation halts with octanoyl CoA formation.

Peroxisomal Fatty Acid Degradation

Flavoprotein requirement: Initiation of peroxisomal fatty acid degradation requires a flavoprotein.

Ketone Bodies as Fuel

Citric acid cycle entry: Acetyl CoA formed in fatty acid oxidation enters the citric acid cycle when fat and carbohydrate degradation are balanced.
Oxaloacetate combination: Acetyl CoA combines with oxaloacetate in the first step of the citric acid cycle, and oxaloacetate availability depends on carbohydrate availability.

Ketone Body Formation

Fasting or diabetes: Oxaloacetate is consumed to form glucose in the gluconeogenic pathway, diverting acetyl CoA to form acetoacetate and D-3-hydroxybutyrate.
Ketone bodies defined: Acetoacetate, D-3-hydroxybutyrate, and acetone are water-soluble, transportable forms of acetyl units.
Abnormal levels: Abnormally high levels of ketone bodies are present in the blood of untreated diabetics.

Ketone Body Formation in the Liver

Primary location: Ketone bodies are formed from acetyl CoA primarily in the liver.

Acetoacetate Reduction

D-3-hydroxybutyrate production: Reduction of acetoacetate in the mitochondrial matrix yields D-3-hydroxybutyrate, catalyzed by D-3-hydroxybutyrate dehydrogenase.
Hydroxybutyrate/acetoacetate ratio: The ratio depends on the NADH/NAD+ ratio inside mitochondria.
Acetone formation: Acetoacetate undergoes a slow, spontaneous decarboxylation to acetone, which may be detected in the breath.
Starvation conditions: Under starvation conditions, acetone may be captured to synthesize glucose.

Ketone Bodies as Fuel in Tissues

Liver production: The liver is the major site of production of acetoacetate and 3-hydroxybutyrate.
Transport: Acetoacetate and 3-hydroxybutyrate are carried from the liver mitochondria into the blood by transport proteins for delivery to other tissues (e.g., heart, kidney).
Brain adaptation: The brain adapts to the utilization of acetoacetate during starvation and diabetes.

Pathway Integration: Liver Supplies Ketone Bodies

During fasting or uncontrolled diabetes: The liver supplies ketone bodies to the peripheral tissues.

Acetoacetate as ATP Fuel

ATP Generation: Acetoacetate can be used as a fuel to drive ATP formation.

3-Hydroxybutyrate Conversion

3-step conversion: 3-Hydroxybutyrate must first be oxidized to produce acetoacetate and NADH for use in oxidative phosphorylation.

Fatty Acid Synthase

Enzyme complex: Fatty acid synthase is a complex of enzymes that synthesize fatty acids.
Tissue synthesis: Many tissues, such as liver and adipose tissue, are capable of synthesizing fatty acids.
Dietary sources: Most humans get enough fatty acids from the diet.
Physiological conditions: Fatty acid synthesis is required under certain physiological conditions such as during embryonic development and during lactation in mammary glands.

Fatty Acid Degradation vs Synthesis

Chemical similarity: The steps in fatty acid degradation and synthesis are chemically similar.

Fatty Acid Synthesis and Degradation Pathways

Location: Synthesis occurs in the cytoplasm, whereas degradation occurs in the mitochondrial matrix.
Acyl carrier: Intermediates in synthesis are covalently linked to the sulfhydryl groups of an acyl carrier protein (ACP), whereas intermediates in degradation are covalently attached to the sulfhydryl group of coenzyme A.
Two-carbon units: The activated donor of two-carbon units in synthesis is malonyl ACP, whereas degradation releases acetyl CoA.
Redox cofactors: The reductant in synthesis is NADPH, whereas the oxidants in degradation are NAD+ and FAD.
Isomers: The isomeric form of the hydroxyacyl intermediate in synthesis is D, whereas the form is L in degradation.

Malonyl CoA Formation

Committed step: Malonyl CoA acetyl CoA is carboxylated, yielding malonyl CoA which is catalyzed by acetyl CoA carboxylase 1 (ACC), a biotin-requiring cytosolic enzyme and proceeds through a carboxybiotin intermediate and requires ATP hydrolysis.
Two steps: The formation of malonyl CoA occurs in two steps.

Acyl Carrier Protein

Intermediates: Intermediates in fatty acid synthesis are linked to an acyl carrier protein.
ACP characteristics: Acyl carrier protein (ACP) is a 77 residue polypeptide with a phosphopantetheine group attached to Ser.
Function: The phosphopantetheine group serves as the fatty acid attachment point within ACP, just as it serves as the fatty acid attachment point in the much smaller CoA molecule, therefore, the much larger ACP molecule can be envisioned as a “macro CoA” molecule.

Fatty Acid Synthesis Steps

Elongation: Elongation begins with the formation of acetyl ACP and malonyl ACP (catalyzed by acetyl transacylase and malonyl transacylase), intermediates are attached to ACP.
Odd-numbered fatty acids: Synthesis of odd-numbered fatty acids begins with propionyl ACP, formed from propionyl CoA by acetyl transacylase.

Condensation of Acetyl ACP and Malonyl ACP

Product formation: Acetyl ACP and malonyl ACP are condensed to form acetoacetyl ACP, catalyzed by β-Ketoacyl synthase (condensing enzyme): Acetyl ACP + malonyl ACP $\rightarrow$ acetoacetyl ACP + ACP + CO2

Reduction and Dehydration Reactions

Acetoacetyl ACP Reduction: Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP by β-ketoacyl reductase, NADPH is the reducing agent.
D-3-hydroxybutyryl ACP Dehydration: D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP by 3-hydroxyacyl dehydratase.
Crotonyl ACP Reduction: Crotonyl ACP is reduced to butyryl ACP by enoyl reductase, NADPH is the reducing agent.

Second Round of Fatty Acid Synthesis

Condensation: Butyryl ACP condenses with malonyl ACP to form a C6-β-ketoacyl ACP.
Conversion: Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl ACP into a C6-acyl ACP.
Elongation cycles: Elongation cycles continue until C16-acyl ACP is formed.

Saturated Fatty Acid Synthesis

Acetyl ACP and Malonyl ACP are condensed, reduced with NADPH, dehydrated, and reduced again with NADPH to form Butyryl ACP

Citrate Transport

Location: Acetyl CoA must be transferred from mitochondria to the cytoplasm for fatty acid synthesis because mitochondria are not readily permeable to acetyl CoA.
Citrate formation: Citrate is formed from acetyl CoA and oxaloacetate in the mitochondrial matrix and can be transported to the cytoplasm.
ATP-citrate lyase: ATP-citrate lyase cleaves citrate into acetyl CoA and oxaloacetate: Citrate + ATP + CoA + H2O $\rightarrow$ acetyl CoA + ADP + Pi + oxaloacetate

Acetyl CoA Transfer

Transfer mechanism: Acetyl CoA is transferred from the mitochondrial matrix to the cytoplasm.

NADPH Sources

Impermeability: The inner mitochondrial membrane is impermeable to oxaloacetate.
Bypass reactions: A series of bypass reactions generate much of the NADPH needed for fatty acid synthesis.
Malate dehydrogenase: Catalyzes the reduction of oxaloacetate to malate by NADH.
Malic enzyme: Catalyzes the oxidative decarboxylation of malate to pyruvate, yielding NADPH: Malate + NADP+ $\rightarrow$ pyruvate + CO2 + NADPH

NADPH Sources Continued

Pyruvate carboxylase: Pyruvate enters the mitochondria, and pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate: Pyruvate + CO2 + ATP + H2O $\rightarrow$ oxaloacetate + ADP + Pi + 2 H+
Net reaction: NADP+ + NADH + ATP + H2O $\rightarrow$ NADPH + NAD+ + ADP + Pi + H+
NADPH production: One molecule of NADPH is generated for each molecule of acetyl CoA transferred from mitochondria to cytoplasm, and additional NADPH is synthesized by the pentose phosphate pathway.

Pathway Integration: Fatty Acid Synthesis

Cooperation: Fatty acid synthesis requires the cooperation of various pathways in different cellular compartments.

Elongation and Unsaturation of Fatty Acids

Palmitate: The major product of the fatty acid synthase is palmitate.
Eukaryotes: Longer fatty acids are synthesized through elongation reactions by enzymes attached to the cytoplasmic side of the ER.
Elongation reactions: Sequentially add two-carbon units to the carboxyl ends of saturated and unsaturated fatty acyl CoA substrates (Malonyl CoA is the two-carbon donor, and Condensation is driven by decarboxylation of malonyl CoA.)

Unsaturated Fatty Acid Generation

Enzyme complex: The introduction of double bonds is catalyzed by a complex of three ER membrane-bound proteins: NADH-cytochrome b5 reductase, cytochrome b5, and stearoyl CoA desaturase.
Stearoyl CoA conversion: Stearoyl CoA + NADH + H+ + O2 $\rightarrow$ oleoyl CoA + NAD+ + 2 H2O

Fatty Acid Desaturation

Electron Transport Chain: Fatty acids are desaturated by an electron-transport chain in the ER Membrane.

Essential Fatty Acids

Unsaturated fatty acids in mammals are derived from: palmitoleate (16:1 cis-∆9), oleate (18:1 cis-∆9), linoleate (18:2 cis-∆9, ∆12), linolenate (18:3 cis- ∆9, ∆12, ∆15).
Mammalian limitation: Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C-9 in the fatty acid chain.
Essential fatty acids: Linoleate and linolenate are essential fatty acids because mammals cannot synthesize them, so they must be supplied in the diet.

Acetyl CoA Carboxylase in Fatty Acid Metabolism

Metabolic control: Fatty acid metabolism is controlled so synthesis and degradation are responsive to physiological needs.
Malonyl CoA production: ACC1 and ACC2 catalyze the committed step in fatty acid synthesis (the production of malonyl CoA).
Regulation: ACC1 and ACC2 play essential roles in regulating fatty acid synthesis and degradation.

Regulation of Acetyl CoA Carboxylase

ACC1 Regulation: Inhibited when phosphorylated by AMP-dependent kinase (AMPK). Activated when dephosphorylated by protein phosphatase 2A. Allosterically stimulated by citrate. Inhibited by palmitoyl CoA.
AMPK Regulation: AMPK is activated by AMP and inhibited by ATP.

Regulation of ACC2

AMPK Inhibition: ACC2, a mitochondrial enzyme, is phosphorylated and inhibited by AMPK.
Carnitine acyltransferase I Inhibition: The product of ACC2, malonyl CoA, inhibits carnitine acyltransferase I, preventing the entry of fatty acyl CoAs into the mitochondrial matrix.

Acetyl CoA Carboxylase Inhibition

Phosphorylation: Acetyl CoA Carboxylase Is Inhibited by Phosphorylation by AMPK.

Acetyl CoA Carboxylase Activity

Polymerization: Citrate facilitates the polymerization of the inactive dimers of ACC1 into active filaments (facilitated by the protein MIG12).
Disassembly: Palmitoyl CoA causes the filaments to disassemble.

Citrate concentration

Inhibition reversal: Polymerization can partly reverse the inhibition produced by phosphorylation.

Hormonal Control of Acetyl CoA Carboxylase

Glucagon and epinephrine: Inhibit ACC by augmenting AMPK activity, ultimately inhibiting fatty acid synthesis.
Insulin: Activates ACC by enhancing phosphorylation and inactivation of AMPK by protein kinase B, ultimately stimulating fatty acid synthesis. Insulin also promotes the activity of a protein phosphatase that dephosphorylates and activates ACC.

AMP-Activated Protein Kinase

Metabolic regulator: AMPK inhibits fatty acid synthesis and stimulates fatty acid oxidation.
Trimeric protein: Exists as a trimeric protein (αβγ ) in several isozymic forms.
Functions: Activates ATP-generating pathways, inhibits ATP-requiring pathways, moderates the inflammatory response, helps initiate nonshivering thermogenesis, and is required in early embryo development.