Lipid Metabolism & Fatty Acids

Lipid Metabolism Objectives

• Discuss the major lipid pathways consisting of biosynthesis, catabolism, storage, and transport
• Describe the biochemistry of diseases associated with abnormalities in lipid metabolism, namely essential fatty acid deficiency, Refsum's disease, carnitine shuttle deficiencies, MCAD, Zellweger syndrome, and Acetyl-CoA carboxylase deficiency

Functions of Lipids

• Lipids provide a way to store energy in the body
• Lipids such as phospholipids are major components of cell membranes
• Sugar residues use lipids at the cell surface
• Cell membranes require lipids to maintain their fluid properties
• Lipids insulate nerve fibers, aiding in electrical signal transmission
• Lipids create an impermeable barrier to water
• Certain lipids are essential for vitamin activity
• Lipids mediate hormonal activity

Fatty Acids

• Fatty acids consist of an alkyl chain with a terminal carboxyl group
• At physiological pH, fatty acids are ionized
• Fatty acids are classified based on chain length: short, medium, long, and very long
• Fatty acids have a nomenclature system for identification
• Most fatty acids are present in triglycerides
• Fatty acids can be saturated or unsaturated
• Fatty acids can be mono- or polyunsaturated, depending on the number of double bonds

Important Fatty Acids in Humans

• Fatty acids like palmitic, palmitoleic, stearic, oleic, linoleic, linolenic and arachidonic acid are very important

Essential Fatty Acids

• Linoleic acid is a parent compound of omega-6 fatty acids, found in sources like corn and sunflower oil
• Linolenic acid is a parent compound of omega-3 fatty acids and is typically sourced from fish oil
• Linoleic acid deficiency can lead to decreased arachidonic acid, leukotrienes, and prostaglandins
• Linoleic deficiency is associated with dermatitis and poor wound healing
• Linolenic acid functions to decrease plasma TAG, protect against thrombosis, and reduce inflammation

Lipid Synthesis Objectives

• Describe fatty acid synthesis pathway, including roles of acetyl CoA carboxylase and fatty acid synthase
• Explain the regulation of fatty acid synthesis
• Discuss the concepts of elongation and desaturation
• Explain the synthesis and transport of triglycerides

Production of Cytosolic Acetyl-CoA (AcCoA)

• The glycolytic pathway produces pyruvate which is the primary source of mitochondrial acetyl CoA for fatty acid synthesis
• The glycolytic pathway also produces cytosolic reducing equivalents of NADH
• Mitochondrial oxaloacetate (OAA) is produced by the first step in the gluconeogenic pathway
• Acetyl CoA, produced in the mitochondria, condenses with OAA to form citrate, the first step in the tricarboxylic acid cycle
• Citrate leaves the mitochondria and is cleaved in the cytosol to produce cytosolic acetyl CoA
• Cytosolic reducing equivalents (NADH) produced during glycolysis contribute to the reduction of NADP+ to NADPH needed for palmitoyl CoA synthesis
• The carbons of cytosolic acetyl CoA are used to synthesize palmitate, with NADPH as the source of reducing equivalents for the pathway

Carboxylation of Acetyl-CoA (AcCoA)

• Citrate, insulin, and a high CHO diet stimulates carboxylation of AcCoA
• In contrast Malonyl CoA, palmitoyl CoA, epinephrine, and glucagon inhibits carboxylation of AcCoA

Carboxylation of AcCoA Details

• The regulatory/rate-limiting step in fatty acid synthesis involves the carboxylation of AcCoA
• Five regulatory subunits of acetyl CoA carboxylase include a biotin carboxylase, a transcarboxylase, a biotin-carboxyl carrier protein, a molecule of biotin, and a regulatory allosteric binding site for citrate/palmitoyl CoA
• The reaction sequence involves three stages of carboxylation of biotin requiring ATP, the transfer of a carboxyl group to acetyl CoA to produce malonyl CoA, and the release of the free enzyme-biotin complex

Acetyl-CoA Carboxylase Regulation

• Acetyl CoA carboxylase is inactive when it is a phosphorylated monomer
• This enzyme is dephosphorylated and activated by protein phosphatase
• The active form of Acetyl CoA carboxylase is when it is polymeric, with three or more dephosphorylated monomers polymerizing to increase activity
• AMP kinase accelerates phosphorylation, leading to enzyme inactivation

Hormonal Regulation of Acetyl-CoA Carboxylase

• Glucagon and epinephrine activate protein kinase A which inhibits protein phosphatase by phosphorylating it
• Insulin stimulates protein phosphatase resulting in an active monomer
• AMP activates AMP kinase, which phosphorylates the monomer, leading to inactivation

Acetyl-CoA Carboxylase Deficiency

• Defective metabolic processes due to Acetyl-CoA Carboxylase Deficiency includes impaired malonyl CoA synthesis, impaired fatty acid synthesis and elongation
• Possible causes of Acetyl-CoA Carboxylase Deficiency include biotin deficiency and high CHO diet, which can cause accumulation of acetyl CoA

Fatty Acid Synthase

• Fatty acid synthase has 7 enzymatic centers and a carrier group
• Fatty acid synthase is made of 2 dimers
• One enzyme center consists of phosphopantetheine esterified to SH group of serine and the other is a cysteine SH group
• Fatty acid synthase undergoes regulation

Fatty Acid Synthesis

• In fatty acid synthesis, COO- is added by acetyl CoA carboxylase
• Decarboxylation is followed by reduction, dehydration and another reduction

Details of Fatty Acid Synthesis

• An acetyl group (from AcCoA) is esterified to ACP to activate the fatty acid synthase complex
• The acetyl group is then transferred to the Cys-SH carrier group
• Malonyl CoA is esterified to ACP
• MalonylCoA is decarboxylated and then condenses with the acetyl group from Cys-SH
• Reduction results in a gain of H
• Dehydration adds unsaturation into the molecule
• Another reduction occurs which produces a gain of H
• A 4C molecule generated is transferred to the Cys-SH and the ACP can accept another malonyl group
• The process is repeated until a fatty acid of desired length is formed to create 16C palmitate
• The fatty acid is cleaved from fatty acid synthase

Palmitic Acid Synthesis

• Palmitic acid synthesis: 8 AcCoA + 14 NADPH + 14 H + 7 ATP = Palmitic acid + 8 CoA + 14 NADP+ + 7 ADP + 7Pi + 7 H₂O
• Elongation involves the fatty acid elongase in the ER and mitochondrion
• Desaturation involves the desaturase

Fat Storage

• Most fat storage is in the form of triacylglycerol

Glycerol-3-Phosphate

• Glycerol-3-Phosphate forms the backbone of triacylglycerols
• In the liver, glucose undergoes glycolysis to produce dihydroxyacetone phosphate, converted to glycerol phosphate via glycerol-P dehydrogenase
• In adipose tissue, glycolysis converts glucose to dihydroxyacetone phosphate, then glycerol-P dehydrogenase reduces it to glycerol phosphate

TAG Synthesis

• Fatty acids are esterified through their COO- group, leading to the loss of negative charge and forming of neutral fat
• The glycerol molecule in triacylglycerols is esterified to 3 fatty acid molecules
• Glycerol phosphate is the initial acceptor of fatty acids during TAG synthesis
• Glycerol can be produced from glucose using the reactions of the glycolytic pathway to produce DHAP, next DHAP is reduced by glycerol phosphate dehydrogenase to glycerol phosphate
• Another pathway in the liver but not in adipose tissue uses glycerol kinase to convert free glycerol phosphate

Fate of Phosphatidic Acid

• Cardiolipin, TAG, and phospholipids can be derived from phosphatidic acid

Phospholipids

• Structures of some glycerophospholipids include dipalmitoyl phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine.
• Phosphatidic acid contributes to the structure of pulmonary surfactant

Degradation of Phospholipids

• Phospholipase A2 is present in many mammalian tissues and pancreatic juice, and is also present in snake and bee venoms
• Phospholipase A2, acting on phosphatidyl-inositol, releases arachidonic acid, the precursor of prostaglandins
• Pancreatic secretions contain lots of the phospholipase A2 proenzyme, which gets activated by trypsin and needing bile salts for its activity
• Phospholipase A2 is inhibited by glucocorticoids like cortisol
• Phospholipase A₁ is present in many mammalian tissues
• Phospholipase D is primarily found in plant tissue
• Phospholipase C is found in liver lysosomes and the α-toxin of clostridia and other bacilli
• Membrane-bound phospholipase C is activated by the PIP2 system and therefore plays a role in creating second messengers

Fatty Acid Oxidation

Catabolism of Stored TAG

• Triacylglycerol is catabolized by triacylglycerol lipase into glycerol and FFA
• Glycerol can be reused and FFA undergoes oxidation

Hormone Sensitive Lipase

• Hormone-sensitive lipase is regulated via hormones, such as Epinephrine and Glucagon
• cAMP activates protein kinase A, which results in activation of the Hormone-sensitive lipase

Types of Oxidation

• Alpha-oxidation is one type where a Refsum’s disease can result if impaired
• Mitochondrial Beta-oxidation
• Peroxisomal Beta-oxidation
• Omega-oxidation

Alpha-Oxidation

• Branched-chain fatty acids such as phytanic acid oxidation involves Alpha-Oxidation
• Methyl group presence is localized on its third (β) carbon
• Phytanic acid activates to a CoA derivative
• The alpha-carbon is hydroxylated by fatty acid hydroxylase
• Production proceeds then decarboxylated to Beta-oxidation, a common theme
• Refsum's disease can result, a rare, autosomal recessive disorder from deficiency of α-hydroxylase accumulating phytanic acid in the plasma and tissue
• Neurological symptoms manifest from such and halting disease progression requires restriction

Mitochondrial Beta-Oxidation

• Transport of fatty acids into the mitochondrion includes Mobilisation and Shuttling
• Reactions of Beta-oxidation occurs in the matrix
• Energy yield comes from such Beta-oxidation and ensuing reactions in the Krebs Cycle

Fatty Acid Activation

• Long chain fatty acids, major components of storage triglycerides and dietary fats, are activated to their CoA derivatives in the cytoplasm via thiokinase, which requires ATP
• The activated CoA derivatives are transported into the mitochondria via the carnitine shuttle

Carnitine Shuttle

• The function of the shuttle brings the fatty acyl group across the IMM to the site of β-oxidation
• Carnitine parmatoyl transferase I (CPT I) - Catalyzes the transfer of the fatty acyl group from CoA to carnitine
• Carnitine acyl carnitine translocase - Translocates the acyl group across the IMM as acyl carnitine ester
• CPTII - Catalyzes the transfer of the fatty acyl group from carnitine to CoA in the mitochondrial matrix.

Genetic Defects in Carnitine Shuttle

• Genetic defects include congenital absence of carnitine acyltransferase in skeletal muscle, CPT-I deficiency, CPT-II deficiency, myopathic carnitine deficiency, and systemic carnitine deficiency

Beta-Oxidation

• Oxidation reactions produces FADH2 – acyl CoA dehydrogenase
• Hydration of fatty acids
• Another Oxidation produces NADH
• Thiolytic cleavage (thiolase) releases AcCoA and an acyl CoA that is 2 carbons shorter than the one that entered the cycle

Oxidation of Palmitate

• 14C palmitate oxidized in the mitochondria generates 7AcCoA, 6 NADH, 6 FADH2, and 112 ATP

Regulation of Fatty Acid Metabolism

• Malonyl CoA inhibits Beta-oxidation
• Fatty acyl carnitine and Acetyl CoA inhibits Pyruvate Dehydrogenase

Fatty Acid Diseases

• Acyl CoA dehydrogenase deficiency arises from three different isozymes

• Omega oxidation producing dicarboxylic acid leads to Acidaemia & metabolic acidosis causing death

• Medium chain acyl CoA dehydrogenase (MCAD), an autosomal recessive disorder with births of 1:10000. The enzyme mutation being 985A>G, 583G>A gives Inability to carry out the first step of Beta-oxidation, diminishing oxidation and leading to hypoglycaemia. Associated with SIDS

• The B-oxidation of peroxisomes can occur with with Long chain, branched chain, and hydroxylated fatty acids without any carnitine required

Metabolism of Propionyl CoA

• Important vitamins: Biotin and B12 aids metabolism
• Inheritable diseases can result such as Mutase deficiency and with Inability to convert VitB12 to its co-enzyme, deoxyadenosylcobalamin
• Rare to have a VitB12 deficiency

β-oxidation in peroxisomes summary

• It is a long chain, branched chain, and hydroxylated fatty acid degradation pathway in which no carnitine is required. It has FAD and FADH2 that oxidases fatty acids
• The genetic defects can result in lethal conditions

Zellweger Syndrome

• Zellweger syndrome is an autosomal recessive disorder resulting in peroxisome biogenesis issues and deficient peroxisomal functions. Enzyme deficiencies occur leading to buildup of very long chain fatty acids

Ketogenesis

• Balance of CHO and fat metabolism: Fatty acids go through ketogenesis

Ketogenesis Details

• fatty acid oxidation results in acetoacetyl-CoA being converted to acetoacetate which can be released as the ketone body, acetone
• acetoacetate can also be converted to β-hydroxybutyrate, another ketone body

Utilization of Ketone Bodies

• The heart preferentially uses fatty acids for energy production
• The brain requires glucose for energy production
• Both organs can reconvert ketone bodies to acetyl CoA for energy production

Metabolism of Sphingolipids

• Sphingolipids are a diverse class of lipids with roles in various biological processes
• Sphingolipids consist of a sphingosine backbone modified with different functional groups via the amino group
• A fatty acid derivative creates a ceramide
• With phosphocholine, it creates sphingomyelin
• Attachment of a sugar creates a cerebroside
• If X = complex oligosaccharide, globoside results containing N-acetyl galactosamine
• Adding an N-acetyl neuraminic acid results in a ganglioside

Glycosphingolipid Structure

• Glycosphingolipids get classified as neutral or acidic consisting of ceramide
• Neutral get linked to one or more sugar residues
• Acidic gets linked to one or more sugar residues plus N-Acetyl-neuraminic acid

Sphingomyelin Synthesis & Degradation

• Sphingomyelin is in the a sphingomyelin pathway where ceramide is substrate. SMase converts it back and forth
• A De novo biosynthesis pathway results too

Sphingolipid Synthesis

• Ceramide forms the base
• Phosphatidylcholine or UOD-galactose/glucose or glucose can be added

Sphingolipidoses: Genetic Disorders

• Sphingolipidoses are genetic disorders associated with impaired sphingolipid metabolism because of deficient lysosomal hydrolytic enzymes, leading to substrate buildup
• Autosomal recessive diseases, with exception of Fabry disease, which is X-linked
• There is a lysosomal pathway for GM, degradation that is Sequential
• Lysosmal storage results also yielding a variety of enzyme and Irreversible outcomes

Lipid Storage Disease Categories by Accumulation

• Sphingomyelin accumulates, yielding the class: Niemann-Pick where the specific action is the sphingomyelinase action
• Glucosyl ceramide is associated with Gaucher
• Galactosyl ceramide is linked to Krabbe
• A build-up of sulfatides yields a class: Metachromatic leukodystrophy
• Fabry arises with a build up of a-galactosidase
• Gangliosides with a high build up then forms Tay-Sachs

Sphingolipidoses Frequencies

• Gaucher's disease is the most common genetic deficiency with 166 births per 100,000
• Tay-Sachs disease happens 33/100,000
• Farber disease, Krabbe and Hunter syndrome happen on the order of 10^-4

Niemann-Pick Disease

• Niemann-Pick disease leads to a Sphingomyelinase deficiency from a missing SMPD1 gene and is Autosomal recessive
• Result leads to Hepatosplenomegaly
• Can manifest as Type A or B where Type A is identified as an infantile neuronal involvement where as Type B (less severe, non-neuronal).
• Occurrence is Greater frequency in Ashkenazi Jews with No specific treatment available

Gaucher's Disease

• Gaucher's disease has an Autosomal recessive (1:500 to 1,000 - Ashkenazi Jews) pattern
• Defective Beta-glucosidase, glucocerebrosidase, means Glc-Cer can not be processed as ceramide resulting in Hepatosplenomegaly & osteoporosis
• Type 1 is the most common form, that is non-neuronopathic

Tay-Sachs

• Tay-Sachs is an Autosomal recessive (1:30 AJ) disease presenting Beta-hexosaminidase A deficiency. Therefore Ganglioside GM2 can not be converted and accumulates
• The Rapid and progressive neurodegeneration, Blindness, Cherry-red macula, Muscular weakness, Seizures ensue as symptoms

Fabry

• Fabry's disease affects 1 Male per every 40,000 cases with redish skin
• a- galactosidase mutation causes a backlog of GM2 and globosides
• Kidney and heart failure can occur
• There is associated Burning pain in lower extremities

Farber

• Farber disease involves Autosomal recessive deficiency of Ceramidase , yielding a Ceramdie and sphingosine buildup
• Lymphadenopathy, painful and progressive joint deformity, Subcutaneous nodules of lipid-laden cells can accumulate and grow, and a Tissues can reveal granulomas

Cholesterol Metabolism

Sources of Liver Cholesterol

• Major sources of liver cholesterol include dietary cholesterol (from chylomicron remnants), de novo synthesis in the liver, and cholesterol synthesized in extrahepatic tissues (from HDL)
• Cholesterol is depleted by Secretion of HDL & VLDL, conversion to bile acids/salts, and from free cholesterol secreted in bile

Cholesterol Structure

• At site of attachment is Hydrocarbon 'tail' consisting of fatty acid cholesteryl ester

Biosynthesis of Cholesterol

• The committed step and therefore regulated step include insulin, fasting, glucagon, dietary
• The biosynthesis occurs from mevalonic acid forward

Steps of Cholesterol Biosynthesis

• Biosynthesis happens in the sequential pattern: Acetyl CoA + Acetoacetyl CoA, HMG-CoA, Mevalonate
• Mevalonate is converted to isoprenoids then to:
• Geranyl/farnesyl pyrophosphates forward to Presqualene pyrophosphate, then a Squalene Synthase aids in making Squalene, then to Cholesterol

HMG-CoA Reductase

• High Cholesteral levels activates SREBP to make translation happen resulting in HMG CoA reductase to kick start cholesterol

Regulation

• HMG-CoA Regulation occurs using Phospho-protein Phosphatase that helps translation

Statins

• OH group binds to HMG blocking further cholesterol production and inhibiting the HMG-CoA