β-oxidation, ketogenesis, fatty acid synthesis, lipoprotein metabolism, cholesterol synthesis

β-Oxidation and Ketogenesis

• Refer to D2L for weekly objectives and associated readings.
• Control of Fatty Acid Synthesis or Oxidation
  • High insulin levels promote fatty acid synthesis.
  • High glucagon levels promote lipolysis, fatty acid β-oxidation, and ketogenesis.
  • These processes do not occur simultaneously.

Lipolysis

• Process of releasing free fatty acids from triacylglycerols stored in adipose tissue during the fasted state.
  • Hormone-sensitive lipase: Activated by glucagon and epinephrine.
    • Hydrolyzes triacylglycerols into free fatty acids and glycerol.
    • Inhibited by insulin.
  • Free fatty acids (long-chain fatty acids) travel bound to albumin to peripheral tissues for energy.
  • Glycerol travels to the liver and is used as a substrate for gluconeogenesis.
  • Acyl-CoA synthetase: Can re-convert free fatty acids to acyl-CoA for re-esterification in adipose tissue, creating a continuous cycle of lipolysis and re-esterification.

β-Oxidation (Mitochondrial Pathway)

• Process of generating acetyl-CoA, NADH, and FADH2 from the oxidation of free fatty acids in peripheral tissues (primarily liver and skeletal muscle).
  • Long-chain fatty acids cannot cross the mitochondrial membrane without a transport system.
  • Acyl-CoA synthetase: Adds an acyl-CoA to free fatty acids.
  • Carnitine palmitoyl transferase I (CPTI): Transfers fatty acyl-CoAs to carnitine, forming fatty acyl-carnitine.
    • Inhibited by malonyl-CoA.
    • Not required for short or medium-chain fatty acid transport.
  • Carnitine palmitoyl transferase II and carnitine acylcarnitine translocase: Bind fatty acyl-carnitine, moving it to the inner mitochondrial matrix and recycling carnitine.

β-Oxidation Spiral

• Fatty acid oxidases are in the mitochondrial matrix.
  • Four major enzymes involved:
    • FAD-coupled oxidation
    • Hydration
    • NAD+-coupled oxidation
    • Thiolytic cleavage
  • Products: Acetyl-CoA, NADH, and FADH2
    • Acetyl-CoA: Used primarily for:
      • Ketogenesis
      • Allosteric activation of pyruvate carboxylase
      • To a lesser extent, the TCA cycle
      • Not a substrate for GNG
    • NADH and FADH2: Oxidized to produce ATP

Ketogenesis (Mitochondrial Pathway)

• Process of generating ketones from excess acetyl-CoA produced from β-oxidation.
  • HMG-CoA synthase: Generates HMG-CoA.
  • HMG-CoA lyase: Cleaves HMG-CoA into acetyl-CoA and acetoacetate.
  • Acetoacetate: Can be reduced to D-3 hydroxybutyrate (β-hydroxybutyrate) or spontaneously decarboxylated to acetone.
  • Two ketone bodies: Acetoacetate and D-3 hydroxybutyrate (β-hydroxybutyrate).
  • Ketones can be used as fuel by other tissues but cannot be oxidized by the liver.
    • Skeletal muscle can oxidize ketones.
    • The brain will oxidize ketones during starvation, reducing reliance on glucose, typically associated with decreased GNG activity.
  • Ketogenesis is regulated by:
    • Activity of lipolysis.
    • Cytosolic levels of malonyl-CoA (inhibits CPTI).
    • Flux through the TCA cycle based on NADH levels and concentration of intermediates, particularly OAA.
  • Ketoacidosis: Can occur from prolonged ketogenesis due to starvation or uncontrolled diabetes.

Important Connections Between β-Oxidation and Other Pathways

• Provides high ATP levels in the fasted state.
• ATP needed for:
  • Glucose synthesis via gluconeogenesis.
  • Nitrogen disposal via the urea cycle.
• Deficiencies in β-oxidation can lead to hypoglycemia due to the inability to support glucose synthesis.
  • CPTI deficiency.
  • Medium-chain acyl-dehydrogenase deficiency.

Summary of Pathway Regulation

Fatty Acid Synthesis

• Refer to D2L for weekly objectives and associated readings.
• Fatty acid synthesis (cytosolic process)
  • Activated by insulin and inhibited by glucagon
  • Substrates need to be shuttled out of the mitochondria
  • In the mitochondria:
    • Acetyl-CoA + OAA in the mitochondria → Citrate
    • Tricarboxylate transporter → removes excess citrate from the mitochondria
  • In the cytosol:
    • Citrate lyase cleaves citrate producing → OAA and acetyl-CoA
    • OAA is converted to pyruvate in a two-step process:
      • Malate dehydrogenase: OAA is reduced to malate
      • Malic enzyme: decarboxylates malate → pyruvate
        • NAPDH is produced and required for FA synthesis
    • Acetyl-CoA carboxylase: converts acetyl-CoA → malonyl-CoA
      • Regulatory enzyme for the process
      • Activated by:
        • Insulin, citrate, dephosphorylation
      • Inhibited by:
        • Glucagon, palmitoyl-CoA (palmitate), phosphorylation
    • Fatty acid synthase: multimeric enzyme that synthesizes C-16 palmitate
      • Acyl-carrier protein (ACP): This subunit is initially primed by acetyl-CoA and requires pantothenic acid as a cofactor.
        • All other carbons are added as malonyl-CoA iteratively after this priming.
        • Malonyl-CoA inhibits CPT1 such that oxidation is NOT occurring at the same time as synthesis
      • Requires NADPH
      • Palmitate (final product) is released from the enzyme by thioesterase
      • Elongation
        • Also requires malonyl-CoA and NADPH

Connection to the PPP

• The pentose phosphate pathway provides glycerol (for TAG synthesis) and NADPH (for fatty acid synthesis)

Fate of Newly Synthesized Fatty Acids

• Fatty acids are not stored in the liver
• They are packaged into VLDL particles as TAGs (with cholesterol) and are transported to adipose for storage

Summary of Pathway Regulation

Lipoprotein Metabolism

• Refer to D2L for weekly objectives and associated readings

Overview

• C : cholesterol ;
• CE: cholesteryl ester;
• TAG: triacylglycerol

Glossary of terms:

• ACAT (acyl-CoA‒cholesterol acyl transferase): catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group on carbon 3 of cholesterol
• ApoE: Apoprotein on chylomicrons and VLDL used for uptake by the liver
• ApoCII: interacts with LPL to activate the enzyme.
• ABCG1/ABCA1 transporter: on the cell surface and is responsible for active transport of cholesterol and lipids out of the cell into the HDL particle
• CETP (Cholesteryl ester transfer protein): transfers cholesteryl ester from HDL to VLDL and transfers TG from VLDL to HDL
• Phosphatidylcholine:cholesterol acyltransferase (PCAT, aka LCAT lecithin-cholesterol acyltransferase (LCAT)): esterifies cholesterol in the plasma when moving in and out of HDL particles
• LDL Receptor: Binds ApoB100 on LDL particles and facilitates the uptake of LDL particles
• LPL (lipoprotein lipase): On vascular epithelium cleaves triacylglycerols into glycerol and free fatty acids to be stored in the adipose after being reformed into triacylglycerols
• SR1 (Scavenger receptor): on liver cells and function in HDL particle uptake
• Microsomal transfer protein (MTP): Involved in the loading of ApoB proteins on to both chylomicrons (in the intestine) and VLDL in the liver.

Chylomicrons

• Transport dietary lipids and fat-soluble vitamins
  • Synthesized in the intestinal epithelial cell → released into circulation through lymph (chyle)
    • Microsomal transfer protein (MTP) essential for the loading of ApoB48 on to the chylomicron
    • TAG + Phospholipids + dietary cholesterol + ApoB48 = nascent chylomicron
      • Secreted into the thoracic duct → circulation
    • Nascent chylomicron interacts with HDL in circulation → get a full complement of ApoE / ApoCII
      • Apo CII on chylomicron interacts with Lipoprotein lipase (LPL)
        • This hydrolyses TAGs → FFA
          • FAA are taken up by muscle (directly oxidized) or adipose (stored as TAGs)
          • Insulin enhances the activity of LPL
      • Apo E on chylomicron remnants → facilitates uptake by the liver SR-B1 receptor → broken down into cholesterol, amino acids and glycerol

VLDL

• Transports fatty acids synthesized in the liver
  • Review fatty acid synthesis newly synthesized fatty acids are packaged into VLDLs and released into circulation
    • Synthesized in the hepatocyte → released into circulation
      • Microsomal transfer protein (MTP) essential for the loading of ApoB100 on to the VLDL
        • Process is very similar to chylomicron synthesis
        • TAG + Phospholipids + de novo cholesterol + ApoB100 = VLDL
          • Secreted directly into→ circulation
        • VLDLs interact with HDL in circulation → get a full complement of ApoE / ApoCII
          • ApoCII on VLDL interacts with Lipoprotein lipase (LPL)
            • VLDL receptor enhances this interaction
              • This hydrolyses TAGs → FFA
                • FAA are taken up by muscle (directly oxidized) or adipose (stored as TAGs)
                • Insulin enhances the activity of LPL
            • ApoE on IDL → facilitates uptake by the liver SR-B1 receptor → broken down into cholesterol, amino acids and glycerol

LDL

• Maturation product of VLDL that retains ApoB100
  • LDL is largely filled with cholesterol ester
  • Extrahepatic tissues and the liver expresses LDL-receptor
  • Uptake of LDL particles by peripheral tissues increases intracellular concentration of cholesterol

HDL

• reverse cholesterol transport
  • HDLs originate from the liver and intestine; ApoA present on HDL participles
  • Acts as a repository for ApoC and ApoE
  • ABC transporters: assist in the transport of cholesterol / lipid from the cells → HDL
  • Cholesterol esterase transfer protein
    • Associated with HDL
    • Exchanges TAGs from VLDL with cholesterol ester from HDL

Clinical correlates

• Abetalipoproteinemia: Loss of the ability to form lipoproteins containing ApoB → loss of chylomicrons and VLDL
• Familial hypercholesterolemia Loss of LDL receptor → increases LDLs in circulation → elevated cholesterol
• Total cholesterol is a measurement of: LDL + HDL + 20% of TAGS
• TAG measurement is a proxy for VLDLs as they carry to greatest amount of TAG in circulation.

Cholesterol Synthesis

• Refer to D2L for weekly objectives and associated readings

Cholesterol synthesis (cytosolic process):

• Cholesterol is present in all tissues as cholesterol or cholesterol ester
• Used as a substrate for the synthesis of:
  • Steroid hormones
  • Sex hormones
  • Bile acids
  • Vitamin D
• Transported in lipoproteins
• Acetyl-CoA is the source of all carbons in cholesterol synthesis
• Synthesis can be divided into 4 stages (only the first is regulated and will be the focus):
  • Stage 1: Synthesis of mevalonate
    • Most important → Regulatory enzyme HMG-CoA reductase

Regulation of HMG-CoA reductase

• Activated by:
  • Sterol response element-binding protein (SREBP) mediated transcription
  • Insulin mediated dephosphorylation
• Inhibited by:
  • Insig which binds SREBP and retains it in the ER
  • Elevated levels of sterols/cholesterol → enhance the → degradation of the enzyme
  • AMPKinase mediated phosphorylation
  • Glucagon
• As LDL binds LDL Receptor and is taken up by the cell→ this decreases the activity of HMG- CoA by increasing intracellular levels of cholesterol
• LDL receptor expression is regulated by:
  • PCSK9 mediated degradation
  • Elevated intracellular cholesterol inhibits LDL receptor transcription
• Statins → class of drugs that inhibit HMG-CoA reductase
  • For management of elevated cholesterol

Cholesteryl esterase transfer protein

• Associated with HDL
• Exchanges TAGs from VLDL with cholesteryl ester from HDL

Excretion of excess cholesterol

• Cholesterol is excreted primarily as →
  • Unesterified cholesterol
  • Bile acids
• Primary bile acids are →
  • Cholic acid and chenodeoxycholic acid
• Cholesterol 7 α-hydroxylase: regulatory step in bile acid synthesis
  • Inhibited by: bile acids
• The majority of bile acids are reabsorbed in the ileum
  • Secondary bile salts → less soluble due to changes in pKa
    • Largely excreted