BIOCHEM 4.5 - BIOSYNTHESIS & STORAGE OF FATTY ACIDS

Biosynthesis and Storage of Fatty Acids

• The lecture covers the biosynthesis and storage of fatty acids.
• The key questions addressed are the conditions under which lipogenesis occurs and when fatty acid oxidation happens.

Objectives

• Evaluation of fatty acids role in energy homeostasis, assessing synthesis/degradation, considering substrates/enzymes in fed, fasting, and starvation.
• Compare and contrast fatty acid metabolism in different biological tissues, cellular compartments, and metabolic states.
• Explain the role of shuttles in fatty acid oxidation and lipogenesis, evaluating each pathway from enzyme and substrate availability.
• Summarize the activity, location, and regulation of Citrate Lyase, Acetyl-CoA Carboxylase, Fatty Acid Synthase, and Lipoprotein lipase.
• Explain the role of citrate lyase in chronic diseases and why it's a good therapeutic target.
• Compare and contrast saturated and unsaturated lipids, their synthesis, and metabolism.
• Identify common features between lipid synthesis pathways, list lipogenesis steps, explain fatty acid chain length's role, and desaturation importance.

Fatty Acid Metabolism Overview

• Fatty acid metabolism involves dietary fat, synthesis from non-lipid precursors, and endogenous NEFA(non-esterified fatty acids).
• Fatty acids can be used for synthesis, esterification for storage as triglycerides, or transported into the mitochondria for oxidation.
• Synthesis occurs from non-lipid dietary precursors that enter a fatty acid pool.
• Fatty acids in the pool can be esterified into cytosolic triglyceride stores or ER secretory triglycerides.
• Oxidation occurs in the mitochondria, resulting in CO2 and ketone bodies.

Lipogenesis Introduction

• Lipogenesis refers to both fatty acid synthesis and triacylglycerol (triglyceride) synthesis.
• Lipogenesis happens in the cytosol and needs NADPH.
• Glucose metabolism through the Pentose Phosphate Pathway provides NADPH.
• Acetyl-CoA is required for fatty acid synthesis.
• Pyruvate decarboxylation to form Acetyl-CoA is done in the mitochondria.
• Acetyl-CoA can't cross the inner mitochondrial membrane so transport of precursors occurs via the Malate Shuttle.

Malate Shuttle

• Pyruvate translocates to mitochondria where it is then oxidized into Acetyl-CoA.
• Acetyl-CoA combines with Oxaloacetate in the TCA cycle to form Citrate.
• The movement of Citrate from the mitochondria to the cytosol ensures cytosolic Acetyl-CoA generation.
• In the presence of ATP and CoA, cytosolic Citrate is cleaved into acetyl-CoA and Oxaloacetate by Citrate Lyase.
• This provides Acetyl-CoA for fatty acid synthesis.

ATP-Citrate Lyase (ACLY) and Disease

• ACLY is a critical link between carbohydrate and lipid metabolism.
• ACLY generates acetyl-CoA for fatty acid and cholesterol synthesis.
• SNPs of ACLY are linked to cancer outcomes.
• Overexpression is linked to increased fatty acid synthesis and development/progression of chronic diseases.
• Inhibitors reduce both symptoms and disease presentation.
• Chronic diseases include non-alcoholic fatty liver disease, diabetes mellitus, obesity, cardiovascular and neurodegenerative diseases.

Lipogenesis Introduction Continued

• Lipogenesis is active during excess energy intake, mainly from carbohydrates and amino acid precursors.
• The liver and adipose tissue are primary sites; adipose tissue can store large amounts of triacylglycerols.

Steps of Lipogenesis

• The two stage process involves the formation of Malonyl-CoA and elongation of the fatty acid chain.
• Formation of Malonyl-CoA from acetyl-CoA is done by acetyl-CoA carboxylase.
• Elongation of the fatty acid chain is done by fatty acid synthase, adding 2 carbons at a time.
• Carboxylation of acetyl-CoA to malonyl-CoA commits to fatty acid synthesis.
• Biotin is carboxylated by Acetyl co-A carboxylase using ATP; the carboxyl is then transferred to Acetyl-CoA.
• Free enzyme biotin is released.

Acetyl-CoA Carboxylase (ACC)

• ACC1 is located in the cytoplasm and is involved in fatty acid synthesis.
• ACC2 is located in the mitochondria and regulates fatty acid oxidation.
• Both ACC1 and ACC2 are biotin-dependent.
• ACC1 has enzymatic and carrier protein functions.
• Blocking ACC2 reduces malonyl-CoA.
• Malonyl-CoA inhibits fatty acid oxidation via direct allosteric inhibition of Carnitine palmitoyl transferase I (CPTI).
• When malonyl-CoA is reduced, CPTI allows fatty acids uptake by mitochondria.

Acetyl-CoA Carboxylase Regulation

• Acetyl-CoA Carboxylase is strictly regulated.
• It is inhibited by palmitoyl-CoA, indicating sufficient product accumulation.
• It is activated in the presence of citrate or isocitrate (energy storage).
• Phosphorylation of the enzyme inhibits it.
• In the presence of glucagon, it will be phosphorylated and inactive.
• In the presence of insulin, it will be dephosphorylated and active.
• Long-term regulation is via synthesis.
• Upregulation occurs with a high carb/low fat diet, and downregulation occurs in starvation or high fat/low carb diet.

Fatty Acid Synthesis and FA Synthase

• Fatty acid synthesis is high under high calorie, high carb/low fat conditions and low during fasting/starvation or high fat diets.
• Fatty Acid Synthase is a dimer of 2 complex molecules.
• Each molecule contains 7 different enzymatic activities, an Acyl carrier protein (ACP) with a Pantetheine Group with SH.
• It builds the fatty acid up to 16 carbons and is regulated by phosphorylated sugars and synthesis of the enzyme.

Fatty Acid Synthase Activity

• Process uses palmitate.
• Cysteine (Cys-SH) is primed with Acetyl-CoA via Acetyl-CoA Transacylase.
• Malonyl-CoA is transferred to SH residue via Malonyl Transacylase.
• The fragments are brought together by 3-Ketoacyl Synthase.
• CO2 and the Cysteine residue are freed generating the 3-Ketoacyl enzyme complex.
• The 3-Ketoacyl group is reduced, dehydrated, and reduced again to form a saturated acyl-enzyme complex.
• The acyl group is displaced from the pantetheine-SH group by another malonyl-CoA molecule, moving it to the free cysteine group.
• The previous steps are repeated for 6 more cycles, which make up 7 total adding to a 16 carbon chain (palmitate).
• Thioesterase activation releases the final palmitate molecule.

Summary of Palmitate Synthesis

• The reaction requires 8 Acetyl-CoA, 7 ATP, 14 NADPH, and 14 H+.
• The reaction produces Palmitate, 14 NAD+, 8 CoA, 6 H2O, 7 ADP, 7 Pi, and 7 CO2.

Fatty Acid Elongation

• Chain elongation requires another unique set of enzymes.

• Malonyl-CoA provides the 2 carbon fragments that create longer chains.

• Elongation occurs on the ER via fatty acid elongase with 7 enzymes with tissue and substrate specificity.

• Enzymes facilitate elongation of very long chain fatty acids(ELOVL 1 to 7).

• Substrates include saturated and unsaturated fatty acids with chain lengths of 10 carbons or more.

• Chain elongation can also occur in mitochondria using a different system.

• This process requires NADH and Acetyl-CoA as its' 2 carbon sources and acts on shorter chains with less than 16 carbons.

• The brain produces very long chain fatty acids (22 to 24 carbons) from stearoyl-CoA (C18).

• It creates the fatty acids needed for sphingolipids.

Desaturation of Fatty Acids

• Not all required fatty acids are fully saturated requiring enzymatic desaturation.
• Most desaturations occur between carbons 9 and 10 using Δ9 desaturases.
• To create precursors for phospholipids and eicosanoids, fatty acids with desaturation at carbons 3 (ω-3) and 6 (ω-6) from the methyl end are needed.
• These fatty acids must be supplied by diet via essential fatty acids like Linoleic (ω-6) and Linolenic acid (ω-3).

Lipoprotein Lipase

• Lipoprotein lipase processes VLDL in the bloodstream.
• It is located in the basement membrane glycoproteins of capillary endothelial cells and acts on chylomicrons and VLDL.
• In adipose capillaries, lipoprotein lipase is stimulated by insulin and facilitates triacylglycerol synthesis.
• In muscle capillaries, lipoprotein lipase is inhibited by insulin but activated by contractions or adrenergic stimulation.

Disorders of Lipid Metabolism

• Objectives explore the importance of proper lipid metabolism using Gaucher and Tay-Sachs disease as examples.
• The main focuses are explaining the features, causes, and progression of Tay-Sachs and Gaucher diseases.
• In addition, the importance of genetic screening for these diseases is explored.

Gaucher Disease

• A rare, autosomal recessive condition with increased incidence in Ashkenazi Jewish individuals.
• It is caused by mutations in the GBA1 gene, leading to deficient glucocerebrosidase/glucosylceramidase activity in lysosomes.
• This results in toxic accumulation of glucocerebroside lipids in multiple organs.
• Mutations can also be linked to Parkinson’s disease.
• Symptom severity varies widely; symptoms start in childhood and can include delayed growth and puberty, and weak bones.
• The 3 types are Type 1 (most common/ nonneuronopathic), Type 2 (acute infantile neuropathic), and Type 3 (chronic neuronopathic)

Tay-Sachs

• An autosomal recessive disease caused by a loss of function mutation in the HEXA gene located on chromosome 15q23.
• The gene encodes one subunit of β-hexosaminidase A protein.
• β-hexosaminidase A functions within lysosomes to breakdown the GM2 ganglioside sphingolipids which are important in neuronal cell membranes.
• Functional loss leads to progressive neurological issues derived from toxic buildup.
• The disease can be identified by a large accumulation of complex lipids in neuronal macrophages.
• Symptoms are often the direct result of neuronal death.

Tay-Sachs and Genetic Screening

• The general population has few recorded cases while the Ashkenazi Jewish population has much more.
• The general population incidence rate is 1 in 100,000 in the US and 1 in 320,000 worldwide.
• The Ashkenazi Jewish population has an incidence rate of approximately 1 in 3,500 live births US
• As high as 1 in 27 though the generally accepted carrier frequency is 1 in 30.
• Due to the genetic basis of the disease, genetic counseling is commonly standardized.
• If a couple are found to be carriers, testing is highly advised.
• Pre-implantation genetic diagnosis and IVF with non-carrier donors are available.