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It is important to note that not all diseases associated with lipid metabolism are genetic disorders. The biosynthesis and catabolism of TAG(triacylglycerol), a major storage molecule,excessive triacylglycerol storage leads to obesity.
Discuss major pathways of lipid
Biosynthesis
Catabolism
Storage
Sphingolipids
Cholesterol metabolism
Transport
Describe the biochemistry of diseases associated with abnormalities in lipid metabolism
It is important to understand the precise biochemical reactions where abnormalities occur, including the involved substrates, enzymes, and cofactors.
Deficiencies in these pathways lead to substrate accumulation, which can cause disease.
lipids play crucial roles in the body, the most significant being energy storage in the form of fatty acids, which are stored as triacylglycerols.
Lipids provide 2.5 times more energy than carbohydrates and can be stored in various locations, unlike glycogen, which is limited to the liver and muscles. Triacylglycerols, as neutral lipids, are stored in pure form and must be hydrolyzed when energy is needed.
This process releases fatty acids, which are oxidized in the mitochondria to produce energy. The mitochondrion, often referred to as the powerhouse of the cell, breaks down fatty acids into a two-carbon molecule called acetyl-CoA.
Acetyl-CoA serves as both the endpoint of fatty acid breakdown and the starting point for fatty acid synthesis. Its complete oxidation in the Krebs cycle generates NADH, which donates electrons in the electron transport chain, ultimately producing energy through oxidative phosphorylation.
The storage of energy requires fatty acid synthesis, followed by their esterification to glycerol, forming triacylglycerols. In addition to energy storage, lipids play a critical role in cell membranes.
Phospholipids, a specialized type of lipid, consist of two fatty acids esterified to glycerol-3-phosphate, with a phosphate residue attached to an alcohol. Their amphipathic nature allows them to form the lipid bilayer of cell membranes, with hydrophobic tails facing inward and hydrophilic heads facing outward. This structure facilitates compartmentalization within cells.
Phospholipids also play a role in the respiratory system by contributing to surfactant, a substance that keeps alveoli open for effective gas exchange.
Another class of lipids, glycolipids, contains sugar residues on their surfaces. These molecules can be antigenic and influence membrane fluidity.
Cholesterol, a steroid molecule, modifies membrane fluidity and exists primarily in an esterified form, which affects its solubility. (
It interacts with phospholipids in the membrane by fitting between them, influencing their movement and packing.
At high temperatures: Cholesterol reduces fluidity by preventing phospholipids from moving too freely.
At low temperatures: It prevents membranes from becoming too rigid by disrupting the close packing of phospholipids, acting like a "buffer" to keep the membrane functional.
Cholesterol exists in two forms:
Free (Unesterified) Cholesterol – This form is amphipathic, meaning it has both hydrophilic (hydroxyl group) and hydrophobic regions, making it more compatible with membranes.
Esterified Cholesterol (Cholesteryl Ester) – This occurs when cholesterol's hydroxyl (-OH) group is replaced by a fatty acid, making it completely hydrophobic.
Effect on solubility:
Free cholesterol can integrate into membranes due to its amphipathic nature.
Esterified cholesterol, being entirely hydrophobic, is insoluble in water and is stored in lipid droplets rather than being part of membranes.
This esterification makes cholesterol easier to transport in lipoproteins like LDL and HDL in the bloodstream.)
Lipids are essential for nerve function, particularly in insulating nerve fibers, which enables rapid signal transmission. Proteolipids and sphingolipids contribute to the myelin sheath and overall nervous system efficiency.
Additionally, lipids provide water-impermeable barriers, a function observed in plant waxes but also relevant in certain biological contexts.
Essential fatty acids are crucial because our bodies cannot synthesize them, making their presence in our diet essential. They are considered "good" fatty acids due to their important role in signaling and other physiological processes. One key reason they are beneficial lies in the structure of their double bonds.
Specifically, the hydrogens in these fatty acids exist in a cis formation, meaning they are positioned on the same side of the double bond, creating a kink in the molecule, contributing to its fluidity and function. In contrast, if the hydrogens were in a trans configuration, the result would be trans fatty acids, which are unhealthy. Trans fatty acids are introduced during food processing and cooking, making them prevalent in highly processed foods, where they pose risks to overall health.
To illustrate the significance of essential fatty acids, it is important to note their role in downstream biochemical functions.
There are two primary essential fatty acids: those from the omega-6 and omega-3 families.
These must undergo elongation, facilitated by elongase enzymes, and desaturation, where additional double bonds are introduced. Through these modifications, the fatty acids can transform into 18- or 20-carbon molecules, which serve as precursors for various bioactive compounds, including prostaglandins, thromboxanes, and leukotrienes.
Arachidonic acid, derived from omega-6 fatty acids, plays a role in producing leukotrienes, prostaglandins, and thromboxanes, which are crucial for inflammation and immune responses.
Meanwhile, omega-3 fatty acids contribute to the synthesis of eicosanoids, which are essential for multiple physiological functions. Notably, leukotrienes have been linked to protective effects against prostate cancer in men.
Moving on to the oxidation of fatty acids, this process primarily involves palmitate, also known as palmitic acid or palmitoyl-CoA.
Palmitate is an acyl unit that undergoes sequential breakdown into two-carbon acetyl-CoA units to generate energy. Fat is stored in the form of triacylglycerols, composed of three fatty acid chains bound to a glycerol molecule.
These triacylglycerols are primarily stored in adipose tissue and must be mobilized before they can be oxidized for energy. The mobilization(Breaking down triacylglycerols into free fatty acids (FFAs) and glycerol.) process is facilitated by an enzyme called triacylglycerol lipase, also known as hormone-sensitive lipase.
The regulation of triacylglycerol lipase is closely tied to energy states in the body.
<Role of Palmitate (Palmitic Acid/Palmitoyl-CoA)
Palmitate (C16:0) is a saturated fatty acid that serves as a key molecule in fatty acid oxidation (β-oxidation).
Before oxidation, palmitate is activated into palmitoyl-CoA, a process that requires Coenzyme A (CoA) and ATP.
Once activated, palmitoyl-CoA undergoes β-oxidation, where it is broken down sequentially into two-carbon acetyl-CoA units.
These acetyl-CoA molecules enter the citric acid cycle (Krebs cycle) to generate ATP, the body's main energy currency.
2. Storage of Fat in Triacylglycerols
The body stores fat primarily as triacylglycerols (TAGs), which consist of three fatty acid chains bound to a glycerol molecule.
These TAGs are stored in adipose tissue (fat cells) as a long-term energy reserve.
Before the fatty acids can be oxidized for energy, they must first be released from triacylglycerols through lipolysis.
3. Mobilization of Stored Fat
The breakdown of triacylglycerols into free fatty acids and glycerol is catalyzed by an enzyme called triacylglycerol lipase, also known as hormone-sensitive lipase (HSL).
HSL is activated when the body needs energy, such as during fasting, exercise, or stress.
Once activated, HSL breaks down triacylglycerols, releasing free fatty acids into the bloodstream, where they bind to albumin for transport to tissues needing energy.
4. Regulation of Triacylglycerol Lipase
The activity of hormone-sensitive lipase is regulated by hormonal signals:
Increased activation:
Glucagon, epinephrine (adrenaline), and norepinephrine signal low energy availability and activate HSL via cAMP signaling.
This promotes fat breakdown to provide energy.
Inhibition:
Insulin signals high energy availability and inhibits HSL, preventing unnecessary fat breakdown.
This promotes fat storage instead of mobilization.>
When the body is in a low-energy state, the hormone glucagon is released, activating triacylglycerol lipase to break down stored fats into glycerol and free fatty acids for oxidation.
Epinephrine can also activate this enzyme during stress, triggering the fight-or-flight response and promoting energy mobilization.
Conversely, in a high-energy state, insulin inhibits triacylglycerol lipase to prevent unnecessary fat breakdown. This regulatory mechanism ensures that the body conserves energy when needed and mobilizes it efficiently during times of demand.
At the molecular level, hormone-sensitive lipase is activated through phosphorylation by protein kinase A. When phosphorylated, the enzyme becomes active and facilitates fat breakdown. In contrast, when the body needs to inactivate this enzyme, a protein phosphatase removes the phosphate group, rendering it inactive.
This dynamic regulation ensures that fat metabolism is tightly controlled according to the body's energy needs.
The activation sequence begins at the cell membrane, where the presence of glucagon signals a low-energy state, triggering a cascade of biochemical events that ultimately lead to the activation of hormone-sensitive lipase and the mobilization of stored fats.
When we are in a stressful situation, epinephrine levels are high. When glucagon or epinephrine binds to their cognate receptors, they stimulate and activate the enzyme adenylyl cyclase, which converts ATP to cyclic AMP. Cyclic AMP subsequently activates protein kinase A, which facilitates the phosphorylation of hormone-sensitive lipase. In its active form, hormone-sensitive lipase hydrolyzes triacylglycerol by removing a fatty acid, leaving behind diacylglycerol.
The freed fatty acid can then be activated to undergo beta oxidation, ultimately providing energy.
In a high-energy state, the breakdown of triacylglycerol is unnecessary, and hormone-sensitive lipase needs to be inactivated. Insulin is responsible for this process by stimulating protein phosphatase, which dephosphorylates hormone-sensitive lipase, thereby stopping triacylglycerol hydrolysis.
Additionally, insulin plays a crucial role in inactivating cyclic AMP by stimulating phosphodiesterase, which converts cyclic AMP to AMP.
As cyclic AMP levels decrease, protein kinase A becomes inactive, further inhibiting lipase activity. However, this inactivation can be prevented by caffeine, a stimulant that inhibits phosphodiesterase, maintaining cyclic AMP levels and stimulating fat breakdown for energy. Another molecule, theophylline, also prevents the conversion of cyclic AMP to AMP, leading to similar effects.
There are various types of fatty acid oxidation. Alpha oxidation involves the alpha carbon and does not directly provide energy, whereas beta oxidation occurs at the beta carbon and is essential for energy production.
Short, medium, and long-chain fatty acids undergo complete oxidation in the mitochondrion, while very long-chain fatty acids are first shortened in the peroxisome before being transported to the mitochondrion for complete oxidation.
Omega oxidation, which occurs at the omega end, does not generate energy but instead produces dicarboxylic acids, which can lead to acidosis and even death.
Alpha oxidation is necessary for branched-chain fatty acids, such as phytanic acid, which is a 20-carbon branched-chain fatty acid. Due to the presence of a methyl group on the beta carbon, beta oxidation is not initially possible.
To address this, alpha oxidation occurs in the peroxisome, where phytanic acid is first activated to phytenoyl-CoA.
The key step in alpha oxidation is hydroxylation at the alpha carbon by phytenoyl-CoA hydroxylase (also referred to as fatty acid hydroxylase).
This hydroxylation allows for the subsequent decarboxylation of the molecule, effectively removing one carbon.
As a result, the new beta carbon is no longer obstructed by a methyl group, enabling beta oxidation to proceed. After undergoing several rounds of beta oxidation in the peroxisome, the fatty acid reaches medium-chain length and is transferred to the mitochondrion for complete oxidation into carbon dioxide and water.
Within the peroxisome, fatty acids containing between 6 and 12 carbons undergo oxidation before being transferred to the mitochondrion for further oxidation. Alpha oxidation is critical because if the enzyme fatty acid hydroxylase is nonfunctional or absent, the necessary hydroxylation does not occur, preventing subsequent beta oxidation.
As a result, the molecule phytanic acid accumulates in the body, since it is not broken down.
The accumulation of phytanic acid due to a deficiency in alpha hydroxylase causes a rare autosomal recessive disorder. Like other fatty acid disorders associated with peroxisomal dysfunction, this condition presents with neurological symptoms.
As discussed in the inborn errors of metabolism lectures, the treatment for this disorder involves dietary restriction of phytanic acid—specifically, avoiding foods high in branched-chain fatty acids—to reduce the symptoms associated with Refsum's disease.
Within the mitochondria, beta oxidation occurs for short-, medium-, and long-chain fatty acids. Short- and medium-chain fatty acids can freely cross both mitochondrial membranes and are activated in the matrix for beta oxidation.
However, long-chain fatty acids must first be activated—a process known as mobilization—by converting them into their CoA derivatives. Because the inner mitochondrial membrane is impermeable to CoA derivatives, the activated long-chain fatty acids must be shuttled across the membrane using the carnitine shuttle system.
Once they have completed the four steps of beta oxidation, the resulting acetyl-CoA units enter the Krebs cycle, leading to the production of NADH and FADH₂, which donate electrons to the electron transport chain to produce ATP.
For long-chain fatty acids, activation occurs in the cytoplasm through the action of the enzyme thiokinase.
Thiokinase facilitates a two-step reaction: it first removes two phosphate groups from ATP to form adenosine and condenses the fatty acid acyl unit with adenosine to form an acyladenylate intermediate.
This intermediate then reacts with coenzyme A (CoA), with the sulfhydryl group of CoA attaching to the fatty acid and releasing AMP. Because this process consumes ATP, it is an energy-requiring reaction equivalent to using two ATP molecules. Activation by thiokinase produces an activated fatty acyl-CoA molecule that can traverse the outer mitochondrial membrane.
However, the bulky CoA residue prevents fatty acyl-CoA from crossing the inner mitochondrial membrane. This is where the carnitine shuttle comes into play.
Carnitine palmitoyltransferase 1 (CPT1), located on the outer mitochondrial membrane, catalyzes a transesterification reaction that removes the CoA group from fatty acyl-CoA and replaces it with a carnitine molecule, forming fatty acyl-carnitine.
This molecule is then transported across the inner mitochondrial membrane by carnitine-acylcarnitine translocase, which exchanges one carnitine residue out of the matrix for each fatty acyl-carnitine molecule that enters.
Once inside the matrix, carnitine palmitoyltransferase 2 (CPT2) removes carnitine and reattaches CoA, regenerating fatty acyl-CoA for beta oxidation.
Before discussing the four steps of beta oxidation, it is important to review the metabolic defects associated with the carnitine shuttle. As shown on the previous slide, the shuttle requires the addition or removal of a carnitine molecule from the fatty acyl unit.
Three key enzymes are involved in this process: carnitine acyltransferase, carnitine palmitoyltransferase 1 (CPT1), and carnitine palmitoyltransferase 2 (CPT2).
Defects in any of these enzymes can lead to specific conditions. For example, a congenital absence of carnitine acyltransferase in skeletal muscle, or deficiencies of CPT1 or CPT2, can result in the buildup of long-chain fatty acids in muscle tissue.
This accumulation impairs energy production—since muscles rely on fatty acid oxidation—and leads to symptoms such as muscle pain, easy fatigability, and even muscle breakdown that may cause reddish-brown urine.
Patients with these enzyme deficiencies are advised to avoid fasting.
Fasting induces a low-energy state that activates glucagon, which in turn activates hormone-sensitive lipase to break down triacylglycerol, releasing long-chain fatty acids. In individuals with these defects, this leads to further accumulation of fatty acids. Instead, they should consume more frequent meals and follow a low-fat, low-protein, high-carbohydrate diet to maintain their energy levels. Notably, CPT2 deficiency is an autosomal recessive disorder.
Carnitine itself is also crucial. When its deficiency is limited to muscle, it is known as myopathic carnitine deficiency; when generalized, it is termed systemic carnitine deficiency.
Both conditions are autosomal recessive. A lack of carnitine in muscle results in generalized weakness and the accumulation of long-chain fatty acids, which can also affect cardiac muscle, given its reliance on fatty acids for energy. In such cases, carnitine supplementation is recommended. In generalized carnitine deficiency, the problem is often linked to a defect in the OCTN2 transporter, encoded by the SLC22A5 gene on chromosome 5, which impairs carnitine uptake in muscle, liver, kidney, and heart, leading to lipid accumulation despite high circulating carnitine levels.
Once the activated fatty acyl-CoA unit reaches the mitochondrial matrix, it undergoes the four steps of beta oxidation to remove one acetyl-CoA unit from the fatty acyl-CoA chain. The process begins with an oxidation reaction catalyzed by acyl-CoA dehydrogenase, which uses FAD as a cofactor and reduces it to FADH₂ while forming a trans-enoyl-CoA intermediate. Next, enoyl-CoA hydratase hydrates the unsaturated bond, adding a hydroxyl group to the beta carbon and forming beta-hydroxyacyl-CoA.
In the third step, beta-hydroxyacyl-CoA dehydrogenase oxidizes beta-hydroxyacyl-CoA to beta-ketoacyl-CoA using NAD⁺ as a cofactor, reducing it to NADH. Finally, thiolytic cleavage occurs, in which the addition of CoA releases one acetyl-CoA molecule and shortens the fatty acyl-CoA by two carbons.
For instance, starting with 16-carbon palmitoyl-CoA, repeated cycles eventually yield eight acetyl-CoA units that enter the TCA cycle.
During beta oxidation, the reducing equivalents FADH₂ and NADH generated donate electrons to the electron transport chain, producing approximately two ATP molecules per FADH₂ and three ATP molecules per NADH.
Therefore, each cycle of beta oxidation contributes about five ATP molecules from these reducing equivalents, in addition to further ATP produced when acetyl-CoA enters the TCA cycle. It is essential to note that different isoforms of acyl-CoA dehydrogenase exist for short-, medium-, and long-chain fatty acids, and deficiencies in these enzymes can lead to metabolic disorders.
Before discussing the four steps of beta oxidation, it is important to review the metabolic defects associated with the carnitine shuttle. The shuttle requires the reversible addition or removal of a carnitine molecule from the fatty acyl unit and involves three key enzymes: carnitine acyltransferase, carnitine palmitoyltransferase 1 (CPT1), and carnitine palmitoyltransferase 2 (CPT2).
Deficiencies in any of these enzymes—for example, a congenital absence of carnitine acyltransferase in skeletal muscle or deficiencies of CPT1 or CPT2—lead to the accumulation of long-chain fatty acids in muscle tissue.
This buildup impairs fatty acid oxidation, resulting in muscle pain, easy fatigability, and even muscle breakdown that may manifest as reddish-brown urine.
Patients with these deficiencies are advised to avoid fasting, which induces a low-energy state that activates glucagon and hormone-sensitive lipase, thereby increasing the release and accumulation of long-chain fatty acids. Instead, they should consume frequent, high-carbohydrate meals while following a low-fat, low-protein diet. Notably, CPT2 deficiency is an autosomal recessive disorder.
In addition, carnitine itself is essential for fatty acid transport. When carnitine deficiency is limited to muscle, it is termed myopathic carnitine deficiency; when generalized, it is called systemic carnitine deficiency. Both forms are autosomal recessive. Inadequate carnitine levels lead to lipid accumulation in muscle and even in cardiac tissue, which relies on fatty acids for energy, causing generalized weakness.
In such cases, carnitine supplementation is recommended. In generalized deficiency, the defect is often due to mutations affecting the OCTN2 transporter (encoded by the SLC22A5 gene on chromosome 5), which impairs carnitine uptake in multiple tissues.
Once the activated fatty acyl-CoA enters the mitochondrial matrix, it undergoes beta oxidation through four major steps that sequentially remove two-carbon acetyl-CoA units. It is crucial to remember the very first reaction of beta oxidation, where palmitoyl-CoA (a 16-carbon fatty acid) is oxidized by acyl-CoA dehydrogenase using FAD as a reducing equivalent, forming a trans-enoyl-CoA molecule.
This reaction is a key point because deficiencies in the various isoforms of acyl-CoA dehydrogenase (specific for short-, medium-, or long-chain fatty acids) can lead to metabolic diseases. Regardless of chain length, this oxidation reaction consistently produces a trans-enoyl-CoA molecule by utilizing FAD.
Complete oxidation of a 16-carbon palmitoyl-CoA molecule requires that the beta oxidation cycle be repeated seven times, yielding eight acetyl-CoA units. For each cycle, one NADH (worth 3 ATP) and one FADH₂ (worth 2 ATP) are produced, amounting to 5 ATP per cycle; over seven cycles, this yields 35 ATP. Each acetyl-CoA entering the TCA cycle produces approximately 12 ATP (2 from FADH₂, 9 from NADH, and 1 from GTP), so eight acetyl-CoA molecules produce 96 ATP. In total, the complete oxidation of one palmitoyl-CoA molecule yields 131 ATP, but since activation of the fatty acid consumes 2 ATP, the net gain is 129 ATP.
For a 14-carbon fatty acid, beta oxidation is repeated six times to yield seven acetyl-CoA units. Here, the cycles produce a total of 30 ATP from the oxidation steps (6 cycles × 5 ATP each) and 84 ATP from the TCA cycle (7 × 12 ATP), summing to 114 ATP. Subtracting the 2 ATP required for activation gives a net yield of 112 ATP. Note that for medium-chain fatty acids (e.g., 10 or 6 carbons), activation occurs within the mitochondrial matrix, so the extra ATP cost for activation is not incurred.
Although beta oxidation itself is not directly regulated, several mechanisms prevent the wasteful expenditure of substrates and energy. A key regulatory point is the pyruvate dehydrogenase complex. Pyruvate, derived from lactate or alanine transamination, has two fates: it can be decarboxylated by pyruvate dehydrogenase to form acetyl-CoA or carboxylated by pyruvate carboxylase to form oxaloacetate. Fatty acyl-carnitine, generated during beta oxidation, inhibits pyruvate dehydrogenase to prevent the simultaneous production of acetyl-CoA from carbohydrates, ensuring that acetyl-CoA originates exclusively from beta oxidation. Additionally, acetyl-CoA itself feeds back to inhibit pyruvate dehydrogenase. During the fed state, acetyl-CoA is converted to malonyl-CoA—the committed step in fatty acid synthesis—which further inhibits CPT1 to prevent concurrent fatty acid oxidation and synthesis.
Deficiencies in acyl-CoA dehydrogenase isoforms can lead to an accumulation of acyl-CoA residues. When beta oxidation is impaired, an alternative pathway known as omega oxidation occurs. Omega oxidation adds a carboxyl group at the omega end of the fatty acid, producing dicarboxylic acids, which can result in acidemia and metabolic acidosis. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, an autosomal recessive disorder, is associated with point mutations (e.g., 985A>G and 583G>A) that impair beta oxidation and can lead to severe hyperglycemia and energy deficits; MCAD deficiency has even been linked to sudden infant death syndrome.
For fatty acids with an odd number of carbons, complete oxidation yields acetyl-CoA and a three-carbon propionyl-CoA. Propionyl-CoA is carboxylated by propionyl-CoA carboxylase (requiring biotin and bicarbonate) to form methylmalonyl-CoA. Methylmalonyl-CoA is then isomerized to succinyl-CoA by methylmalonyl-CoA mutase, an enzyme that requires deoxyadenosylcobalamin (the coenzyme form of vitamin B12). Deficiencies in vitamin B12 or in its conversion to its active form can lead to methylmalonic aciduria, characterized by an accumulation of methylmalonyl-CoA in the blood and urine. Succinyl-CoA then enters the TCA cycle to contribute to energy production.
In contrast to mitochondrial beta oxidation, peroxisomal beta oxidation handles very-long-chain and branched-chain fatty acids, as seen in Refsum's disease. Peroxisomes have a single-layer membrane, so they do not require a shuttle system; however, activation still occurs. In the peroxisome, acyl-CoA oxidase catalyzes the first oxidation step, generating FADH₂ that is reoxidized by oxygen to produce hydrogen peroxide—a reactive oxygen species detoxified by catalase. The resulting enoyl-CoA is further processed by hydration and oxidation (via a bifunctional protein) to form ketoacyl-CoA, which then undergoes thiolytic cleavage to produce acetyl-CoA and a shortened acyl-CoA. This cycle continues until the fatty acid is reduced to a chain length that mitochondria can fully oxidize. The energy yield from peroxisomal oxidation is lower than that from mitochondrial oxidation due to differences in enzyme systems.
Genetic defects in peroxisomal biogenesis, such as those seen in Zellweger syndrome (also known as Zalweger syndrome), are autosomal recessive disorders caused by mutations in genes responsible for peroxisome formation (for example, in the PEX genes or related receptors). These defects impair the import of essential proteins into the peroxisome, leading to deficient oxidation of very-long-chain fatty acids. The resulting accumulation of these fatty acids disrupts neurological development, particularly myelination, and manifests as severe neurological deficits and mental retardation. Zellweger syndrome typically presents in the fetal or neonatal period, and treatment is limited to supportive care.