Lipid Metabolism: Beta-Oxidation, Ketogenesis & Fatty Acid Synthesis

What are the two major functional categories of lipids discussed in class, and which one is the focus of lipid catabolism?

Storage lipids (triacylglycerols) and membrane lipids; lipid catabolism focuses on the breakdown of triacylglycerols (TAGs).

In what specialized cells are storage triacylglycerols stored in the body?

Adipocytes (fat storage cells), within intracellular lipid droplets.

What are the two sources of triacylglycerols that converge on the beta-oxidation pathway?

1. Fats acquired from the diet; 2. Fats stored in adipocytes.

What are the initial breakdown products when a triacylglycerol molecule is completely hydrolyzed?

1 glycerol molecule (backbone) and 3 free fatty acids (FFAs).

What happens to the glycerol backbone released during triacylglycerol breakdown?

It travels through the blood to the liver and is converted into an intermediate of gluconeogenesis (or glycolysis).

Trace the 3 enzymatic steps that convert free glycerol into a gluconeogenic/glycolytic intermediate in the liver.

1. Glycerol -> Glycerol 3-phosphate (via glycerol kinase); 2. Glycerol 3-phosphate -> Dihydroxyacetone phosphate (DHAP); 3. DHAP -> Glyceraldehyde 3-phosphate (G3P).

Glycerol released by lipases during fat mobilization enters gluconeogenesis/glycolysis as which intermediate?

Glyceraldehyde 3-phosphate (G3P).

What is the ultimate product of free fatty acid breakdown via beta-oxidation, and what is its fate in extrahepatic tissues vs. the liver?

Acetyl-CoA; in extrahepatic tissues it enters the citric acid (TCA) cycle, whereas in the liver it cannot enter the TCA cycle and is used for ketogenesis.

Why must dietary triacylglycerols be broken down into free fatty acids in the digestive tract before being reassembled?

Ingested fats are foreign lipids that cannot be internalized intact; they must be broken down into free fatty acids to be absorbed by intestinal cells and repackaged into endogenous triacylglycerols the body can handle.

Where does the digestion of dietary fats occur, and what molecules emulsify large fat particles?

In the small intestine; bile acids (bile salts) secreted into the intestine emulsify large hydrophobic fat particles so they can be processed in a hydrophilic/aqueous environment.

What is the source and function of intestinal lipases during dietary fat digestion?

Secreted by the pancreas into the intestine; they hydrolyze the ester bonds of emulsified dietary triacylglycerols to release free fatty acids and glycerol.

What happens to free fatty acids immediately after they are absorbed by intestinal mucosal (epithelial) cells?

They are reassembled (reconverted) back into endogenous triacylglycerols (TAGs) and packaged into chylomicrons.

What are chylomicrons and what is their primary physiological job?

Large plasma lipoprotein carrier particles produced in intestinal cells that transport repackaged dietary triacylglycerols through the lymphatic system and bloodstream to target tissues.

Describe the membrane and surface structure of a chylomicron.

Covered by a single leaflet (monolayer, NOT a bilayer) of phospholipids embedded with recognition proteins called apolipoproteins (such as ApoCII).

What specific apolipoprotein on the surface of chylomicrons is required for recognition at target tissues?

ApoCII (ApoC2).

What enzyme at the target tissue recognizes ApoCII on chylomicrons, and what reaction does it catalyze?

Lipoprotein lipase (LPL); it recognizes ApoCII and hydrolyzes the chylomicron's triacylglycerols into free fatty acids and glycerol for absorption by the target tissue.

What are the two possible fates of free fatty acids once taken up by target cells from chylomicrons?

1. Broken down via beta-oxidation for energy (e.g., in muscle); 2. Re-esterified into triacylglycerols for storage (if the target cell is an adipocyte).

Under what physiological condition are stored fats in adipocytes mobilized, and what hormone initiates this signal?

When blood glucose (primary fuel) is low/unavailable; initiated by glucagon binding to a Gs-coupled GPCR on the adipocyte surface.

What intracellular signaling pathway is activated when glucagon binds its GPCR on an adipocyte?

Gs-coupled GPCR activates adenylyl cyclase -> converts ATP to cAMP -> cAMP activates Protein Kinase A (PKA).

What are perilipins, and what is their role in an unstimulated adipocyte?

Regulatory proteins arranged around the surface of intracellular lipid droplets that form a protective shield blocking lipases from accessing stored triacylglycerols.

Which two major protein targets in adipocytes are phosphorylated by PKA in response to glucagon signaling?

1. Perilipins; 2. Hormone-sensitive lipase (HSL).

What happens when PKA phosphorylates perilipins on the surface of a lipid droplet?

Phosphorylation changes the conformation/orientation of perilipins, opening physical access to the fat droplet and triggering downstream signaling (involving CGI-58) that activates ATGL.

Name the 3 lipases that sequentially hydrolyze triacylglycerols in adipocytes during fat mobilization, in order from Step 1 to Step 3.

1. ATGL (Adipocyte triacylglycerol lipase); 2. HSL (Hormone-sensitive lipase); 3. MGL (Monoacylglycerol lipase).

What specific reaction is catalyzed by ATGL (Adipocyte triacylglycerol lipase)?

Hydrolyzes Triacylglycerol (TAG) -> Diacylglycerol (DAG) + 1 Free Fatty Acid (FFA).

What specific reaction is catalyzed by HSL (Hormone-sensitive lipase)?

Hydrolyzes Diacylglycerol (DAG) -> Monoacylglycerol (MAG) + 1 Free Fatty Acid (FFA).

What specific reaction is catalyzed by MGL (Monoacylglycerol lipase)?

Hydrolyzes Monoacylglycerol (MAG) -> Glycerol backbone + 1 Free Fatty Acid (FFA).

Why can't the 3 free fatty acids released from adipocytes travel freely in the blood, and what protein transports them?

Free fatty acids are hydrophobic and blood is hydrophilic; they bind to the blood protein serum albumin (which carries about 10 free fatty acids per protein) for transport to target tissues.

Which three main target tissues take up free fatty acids from serum albumin to perform beta-oxidation?

Liver, skeletal/cardiac muscle, and kidneys.

Where in the cell are free fatty acids activated before catabolism, and what enzyme catalyzes this activation?

In the cytosol (at the outer mitochondrial membrane); catalyzed by Fatty acyl-CoA synthetase.

What is the reaction catalyzed by Fatty acyl-CoA synthetase, and what is its energetic requirement?

Free Fatty Acid + CoA-SH + ATP -> Fatty acyl-CoA (requires ATP).

What are the two biological purposes of converting a free fatty acid into fatty acyl-CoA in the cytosol?

1. Energizes the molecule by forming a high-energy thioester bond; 2. Traps the fatty acid inside the cytosol.

In what subcellular compartment does fatty acid beta-oxidation take place?

Inside the mitochondrial matrix.

Why is carnitine required for fatty acid catabolism?

Carnitine is required for the transport of fatty acyl-CoA into the mitochondrial matrix for beta-oxidation.

What cannot cross the inner mitochondrial membrane directly?

Long-chain fatty acyl-CoA

What is the function of carnitine?

It shuttles fatty acyl groups into the mitochondria.

What reaction is catalyzed by CPT1 / CAT1?

It catalyzes a transesterification reaction that replaces CoA with carnitine.

Where is CPT1 / CAT1 located?

On the outer mitochondrial membrane.

What is the rate-limiting step for the transport and beta-oxidation of fatty acids?

The conversion of fatty acyl-CoA to fatty acyl-carnitine by CPT1 / CAT1.

How does fatty acyl-carnitine enter the mitochondrial matrix?

Via a translocase transporter.

What converts fatty acyl-carnitine back into fatty acyl-CoA inside the matrix?

CPT2 / CAT2.

What 16-carbon saturated fatty acyl-CoA is used as the standard model for beta-oxidation?

Palmitoyl-CoA.

In a fatty acyl-CoA molecule, which carbon is designated C1?

C1 is the carbonyl (keto) carbon attached to CoA.

Why is the pathway named 'beta-oxidation'?

Because the beta-carbon (C3) is oxidized into a keto group before cleavage occurs.

What happens to the original C1, C2, and C3 carbons at the end of one round of beta-oxidation?

C1 and C2 are cleaved off as Acetyl-CoA, and C3 becomes the new C1 carbonyl carbon.

Name the 4 sequential reaction types that occur in every round of beta-oxidation.

1. Oxidation; 2. Hydration; 3. Oxidation; 4. Cleavage.

What occurs in Step 1 of beta-oxidation?

Acyl-CoA dehydrogenase oxidizes the bond between the alpha and beta carbons, forming trans-enoyl-CoA.

How do electrons from FADH2 produced by Acyl-CoA dehydrogenase enter the ETC?

Through Electron-Transferring Flavoprotein (ETF).

What occurs in Step 2 of beta-oxidation?

Enoyl-CoA hydratase adds water across the trans double bond to form beta-hydroxyacyl-CoA.

What occurs in Step 3 of beta-oxidation?

Beta-hydroxyacyl-CoA dehydrogenase oxidizes the beta-hydroxyl group to a beta-keto group.

What occurs in Step 4 of beta-oxidation?

Acyl-CoA acetyltransferase cleaves the alpha-beta carbon bond, releasing Acetyl-CoA.

How many rounds of beta-oxidation are required to completely oxidize Palmitoyl-CoA?

7 rounds.

What are the total products generated from the complete beta-oxidation of 1 molecule of Palmitoyl-CoA?

8 Acetyl-CoA, 7 FADH2, and 7 NADH.

Write the complete balanced overall equation for the beta-oxidation of Palmitoyl-CoA.

Palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H2O -> 8 Acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+.

How many ATP are generated directly from the 7 FADH2 and 7 NADH produced during beta-oxidation?

28 ATP from reducing equivalents alone.

For any even-numbered saturated fatty acid with N carbons, what are the formulas for total Acetyl-CoA produced?

Total Acetyl-CoA = N / 2.

How many rounds of beta-oxidation and how many Acetyl-CoA are produced from an 18-carbon vs. a 20-carbon saturated fatty acid?

18-carbon: 8 rounds yielding 9 Acetyl-CoA; 20-carbon: 9 rounds yielding 10 Acetyl-CoA.

What happens in the final round of beta-oxidation of an odd-numbered fatty acid?

The final cleavage yields 1 Acetyl-CoA and 1 Propionyl-CoA.

What is the metabolic fate of the 3-carbon Propionyl-CoA produced from odd-chain fatty acid oxidation?

It is carboxylated to Methylmalonyl-CoA and then to Succinyl-CoA.

What two additional enzymes are required for the beta-oxidation of unsaturated fatty acids?

Enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase.

Which statement is true regarding the beta-oxidation of palmitoyl-CoA?

It produces both NADH and FADH2 and requires 7 rounds, not 8.

What are the three ketone bodies synthesized from acetyl-CoA?

1. Acetoacetate; 2. Beta-hydroxybutyrate; 3. Acetone.

In what organ and specific subcellular compartment does ketogenesis occur?

In the mitochondria of the liver.

Why can't the liver oxidize its own acetyl-CoA from beta-oxidation during fasting?

The liver is performing gluconeogenesis, depleting mitochondrial oxaloacetate.

What is the physiological purpose of ketogenesis in the liver?

To package excess hepatic acetyl-CoA into ketone bodies for use in extrahepatic tissues.

Trace Step 1 of ketogenesis.

Thiolase condenses 2 Acetyl-CoA to Acetoacetyl-CoA + CoA-SH.

Trace Step 2 of ketogenesis.

HMG-CoA synthase condenses Acetoacetyl-CoA + Acetyl-CoA to HMG-CoA + CoA-SH.

How does the synthesis of HMG-CoA for ketogenesis differ from cholesterol synthesis?

Ketogenesis occurs in the mitochondria, while cholesterol synthesis occurs in the cytosol.

What pathways convert acetyl-CoA to HMG-CoA?

Both ketogenesis and cholesterol synthesis use similar reactions.

Where does ketogenesis occur?

In the mitochondria of the liver.

Where does cholesterol synthesis occur?

In the cytosol.

What enzyme cleaves HMG-CoA in ketogenesis?

HMG-CoA lyase.

What are the products of HMG-CoA cleavage?

Acetoacetate and Acetyl-CoA.

How is Acetoacetate converted into Acetone?

Acetoacetate decarboxylase removes CO2 from Acetoacetate.

What happens to Acetone produced during ketogenesis?

It is exhaled in the breath and not used for fuel.

How is Acetoacetate converted into Beta-hydroxybutyrate?

Beta-hydroxybutyrate dehydrogenase reduces Acetoacetate.

What two ketone bodies are exported from the liver?

Acetoacetate and Beta-hydroxybutyrate.

Why do patients with uncontrolled diabetes overproduce ketone bodies?

Lack of insulin or insulin resistance shifts hormone balance toward glucagon.

Where does ketone body oxidation occur?

In the mitochondria of extrahepatic tissues.

What are the steps of ketone body oxidation?

1. Beta-hydroxybutyrate -> Acetoacetate; 2. Acetoacetate + Succinyl-CoA -> Acetoacetyl-CoA + Succinate; 3. Acetoacetyl-CoA + CoA-SH -> 2 Acetyl-CoA.

Under what conditions does fatty acid synthesis occur?

In the well-fed state with high glucose and insulin signaling.

Where does Acetyl-CoA for fatty acid synthesis originate?

From glucose breakdown via glycolysis.

What downstream molecules can be synthesized from long-chain fatty acids?

Triacylglycerols, membrane phospholipids, cholesterol, and steroid hormones.

Which essential fatty acid must be obtained from the diet?

Linoleate.

What are the requirements for cytosolic fatty acid synthesis?

Malonyl-CoA and NADPH.

What is the primary source of NADPH for fatty acid synthesis?

The Pentose Phosphate Pathway (PPP).

How is mitochondrial Acetyl-CoA transported to the cytosol?

It is converted to Citrate, exported, and then cleaved back to Acetyl-CoA.

What happens to cytosolic Oxaloacetate produced during the citrate shuttle?

It is reduced to Malate.

What are the two fates of cytosolic Malate?

1. Imported into mitochondria; 2. Converted to Pyruvate in the cytosol.

What enzyme catalyzes the rate-limiting step of fatty acid synthesis?

Acetyl-CoA Carboxylase (ACC).

What reaction does Acetyl-CoA Carboxylase catalyze?

Acetyl-CoA + HCO3- + ATP -> Malonyl-CoA + ADP + Pi.

What are the functional domains of Acetyl-CoA Carboxylase?

Biotin carboxylase domain, biotin carrier protein domain, transcarboxylase domain.

What is the two-step mechanism of Acetyl-CoA Carboxylase?

1. Carboxylation of biotin; 2. Transfer of CO2 to Acetyl-CoA.

What are the functional domains of the Fatty Acid Synthase complex?

KS, MAT, DH, ER, KR, ACP, Thioesterase.

Which components of the Fatty Acid Synthase complex contain reactive thiol groups?

KS and ACP.

What is the role of ACP during fatty acid synthesis?

It tethers the growing fatty acyl chain and channels substrates.

What is the priming step of Fatty Acid Synthase?

Loading the acetyl group onto KS and the malonyl group onto ACP.

What are the repeating steps of beta-oxidation?

Oxidation, Hydration, Oxidation, Cleavage.

What are the repeating steps of fatty acid synthesis?

Condensation, Reduction, Dehydration, Reduction.

What occurs during Step 1 of Fatty Acid Synthase?

Condensation catalyzed by KS, forming a 4-carbon beta-ketoacyl group.

What catalyzes the decarboxylation of the malonyl group on ACP?

KS (synthase)