L11: Lipid Metabolism Notes

Fatty Acids and Triglycerides

Fatty acids have four major functions:
1. Fuel molecules stored as triglycerides.
2. Components of phospholipids and glycolipids.
3. Attached to proteins to direct proteins to membranes.
4. Function as hormones and intracellular messengers.
98% of ingested lipids are triglycerides (TAGs).
Fatty acids are stored in adipose tissue as TAG in which fatty acids are linked to glycerol with ester linkages.
TAGs are stored in large droplets in the cytoplasm of adipocytes (fat cells).
Adipose tissue is located throughout the body, with subcutaneous (below the skin) and visceral (around the internal organs) deposits being most prominent.

Energy Intake, Utilization, and Storage

Diagram illustrating how carbohydrates, proteins, and lipids are processed for energy.
Carbohydrates are broken down into glucose, proteins into amino acids, and lipids into fatty acids and monoglycerides.
These are then catabolized into $CO2 + H2O$ and energy.
Lipogenesis consists of fatty acid synthesis and subsequent triglyceride synthesis, taking place in the liver and adipose tissue.
Lipolysis is the catabolic process of breaking down triacylglycerols (TAGs) into fatty acids and glycerol.
Fatty acids released into the blood are transported and used as: precursors for synthesis, fuels for energy production, substrates for ketone body synthesis.
Ketone bodies may be exported to other tissues and used for energy production.

Overview: Lipogenesis and Lipolysis

Neutral fats (triglycerides) are stored in adipose tissue.
Dietary fats undergo lipolysis to form fatty acids, which can then undergo beta-oxidation to form acetyl CoA.
Acetyl CoA enters the Krebs cycle to produce $CO2 + H2O$ and ATP.
Alternatively, acetyl CoA can be used in lipogenesis to form fatty acids, cholesterol, and steroids.
Ketogenesis converts fatty acids into ketone bodies in the liver.
Glucose undergoes glycolysis to form glyceraldehyde phosphate and then pyruvic acid, which can also form Acetyl CoA.

Digestion of Lipids

Digestion in the stomach:
- Gastric Lipase hydrolyzes ~10% of TAGs.
- This produces a mixture of fats (TAGs), diacylglycerols, short-chain and medium-chain fatty acids, phospholipids, and cholesterol esters.
Digestion in duodenum:
- Lipids are emulsified by bile salts, synthesized from cholesterol in the liver.
- Bile salts are amphipathic molecules (polar head and non-polar tail).
- They insert into lipid droplets, making the triglycerides more readily digested, resulting in smaller lipid droplets.

Bile Salts

Bile salts are derived from cholesterol.
Cholesterol is hydrophobic, but when converted to bile salts, it gains oxygen-containing polar groups, making it hydrophilic.
All polar groups are on one side of the molecule, making it amphipathic.
Bile salts break up fat globules into smaller hydrophilic-coated droplets that mix more readily with water.

Digestion and Absorption of Lipids

In the small intestine, emulsified fats (TAGs) are hydrolyzed by pancreatic lipase into two fatty acids and monoacylglycerol (MAG).
- Monoacylglycerol = glycerol and 1 fatty acid.
Free fatty acids and monoacylglycerol are carried in micelles to the intestinal epithelium for absorption.
Micelles are globular structures formed by small lipids in aqueous solutions.
In a micelle, polar head groups of fatty acids and MAG are in contact with the aqueous solution, and non-polar hydrocarbon chains are sequestered in the interior.

Absorption of Lipids – Micelle Formation

Micelle formation is facilitated by bile salts, which are on the exterior of the micelle.
Fatty acids and other lipids are incorporated between the bile salts and in the interior, forming mixed micelles.
Mixed micelles contain fatty acids, diacylglycerols (DAG), monoacylglycerols (MAG), phospholipids, cholesterol, vitamins A, D, E, and K, and bile salts.
Free fatty acids and MAG are absorbed by intestinal epithelial cells by diffusion.
In the intestinal cells, triglycerides are re-formed from fatty acids and monoacylglycerol and then packaged into lipoprotein transport particles called chylomicrons.
Chylomicrons are released into the lymph system and then into the blood.
Triglycerides are stored in adipose tissue and muscle.
In muscle, triglycerides can be oxidized to provide energy.

Digestion and Absorption of Lipids

Emulsified fat droplets are broken down by lipase into fatty acids and monoglycerides.
These small lipids, along with cholesterol and vitamins, form micelles.
The small lipids gradually leave the micelles and diffuse into the epithelial cells of the intestine.
In epithelial cells, triglycerides are re-formed and enclosed by a membrane from the SER.
They are coated with proteins to form chylomicrons and enter lacteals to be transported to the blood.

Transport of Lipid - Lipoprotein

Phospholipids, triglycerides, cholesterol, and cholesterol esters are all transported as lipoproteins.
Lipoproteins consist of a protein(s) component and various lipids, depending on the type of particle.
Proteins solubilize the lipids and direct the particles to specific targets.
Lipoprotein particles are classified according to density: the greater the proportion of lipid (TAG), the less dense the particle.
Chylomicrons for exogenous lipids transport lipids from the intestine.
Other lipoproteins for endogenous lipids transport lipids between tissues: Very low-density lipoproteins (VLDL), Intermediate-density lipoproteins (IDL), Low-density lipoproteins (LDL), High-density lipoproteins (HDL).

Transport of Lipid - Lipoprotein Basic Structure

A non-polar lipid core containing (1) triacylglycerols (TAG) and (2) cholesterol esters (CE) surrounded by a polar outer coat of (3) phospholipids (PL), (4) free cholesterol (C), and (5) proteins known as apolipoproteins (apoproteins, P).

Cholesterol and Cholesterol Esters

Illustrates the structure of cholesterol and cholesterol ester, highlighting the difference in their chemical composition.

Properties of Plasma Lipoproteins

A table summarizing the properties of plasma lipoproteins including density, diameter, apolipoprotein composition, physiological role, and percentage composition of TAG, CE, free cholesterol, phospholipid, and protein for chylomicrons, VLDL, IDL, LDL, and HDL.

Size & Compositions of Lipoproteins

A typical lipoprotein contains an interior of triglycerides and cholesterol surrounded by phospholipids.
The phospholipids' fatty acid "tails" point towards the interior, where the lipids are.
Proteins near the outer ends of the phospholipids cover the structure.
This arrangement of hydrophobic molecules on the inside and hydrophilic molecules on the outside allows lipids to travel through the watery fluids of the blood.

Lipoproteins Transport - Exogenous pathway

Lipoprotein lipase (LPL) is found primarily on the surface of cells (endothelium) of capillaries within muscles and in fatty (adipose) tissue.
Lipoprotein lipase breaks down triacylglycerols in chylomicrons and releases fatty acids and MAG into tissue fluid. Muscle cells absorb fatty acids for ATP production or storage as glycogen. Adipocytes absorb fatty acids and use them to synthesize TAG for storage.

Lipoproteins Transport - Endogenous pathway

Cholesterol and TAG are made primarily in the liver, and they are transported in various forms of lipoprotein in blood to other tissues.
LDL is the major carrier of cholesterol in blood.
LDL delivers cholesterol from the liver to peripheral tissues. At the tissues, cholesterol enters the cell by receptor-mediated endocytosis.
High-density lipoprotein (HDL), synthesized in the liver, carries cholesterol released into the blood back to the liver, a process called reverse cholesterol transport.
HDL is often considered “good” cholesterol because it picks up excess cholesterol from blood vessels for excretion.

Degradation of Lipids: Hydrolysis of Lipids

Hormone-sensitive triacylglycerol lipase is involved in hormone-induced fatty acid mobilization in adipocytes; epinephrine activates adenylate cyclase, increasing cAMP levels, which activates PKA, leading to the activation of hormone-sensitive lipase.

Degradation of Lipids: Hormone-Sensitive Triacylglycerol Lipase

Hydrolysis of triacylglycerol by lipase (Lipolysis) occurs in the cytosol of adipose cells. A hormone-sensitive lipase hydrolyzes triacylglycerol at C1 and C3 positions to form monoacylglycerol (MAG).
Subsequent removal of the remaining FA is carried out by monoacylglycerol-specific lipase.
Glycerol is complexed with serum albumin and carried to other sites of utilization.
Epinephrine and glucagon activate PKA, which in turn activates hormone-sensitive lipase, while insulin has the opposite effect.
Breakdown of fatty acids in β-oxidation is controlled by concentration of FA in blood and hormone-sensitive lipase.

Lipoprotein Lipase vs. Hormone-Sensitive Lipase

Lipoprotein lipase and hormone-sensitive lipase are two types of lipases which hydrolyze triglycerides.
The main difference is that lipoprotein lipase is extracellular, while hormone-sensitive lipase is intracellular.

Degradation of Lipids: Hydrolysis of Lipids

Lipolysis occurs in the cell cytosol of adipocytes, resulting in glycerol and free fatty acids.
Fatty acids are used as fuel by many tissues; citric acid cycle is utilized to obtain maximum energy.
Released fatty acids are not soluble in blood plasma, so serum albumin in the bloodstream binds the fatty acids and serves as a carrier.

Degradation of Fatty Acids: Step 1 Synthesis of Acyl CoA

Before entering the mitochondria, fatty acid (FA) is activated by forming a thioester link with CoA, catalyzed by fatty acyl CoA synthetase in the outer membrane (OM) of mitochondria.
$ATP \rightarrow AMP + PPi$ (1)
$Fatty acid + CoA \rightarrow acyl CoA$ (2)
$PPi \rightarrow 2Pi$ (irreversible reaction)
Activation of F.A. consumes 2 ATPs.
F.A.s are non-polar molecules and can diffuse out of cells, but the attachment of polar CoA traps FA inside the cells.
Acyl CoA molecules are transported into the mitochondrial complex by carnitine shuttle, followed by -oxidation (FA breakdown) occurring in the mitochondria matrix.

Fatty Acid Synthesis and Breakdown

A diagram comparing the processes of lipolysis (breakdown of triglycerides) and lipogenesis (synthesis of fatty acids).
Lipolysis involves hormone-sensitive lipase, epinephrine, glucagon, and PKA, breaking down TAG into fatty acids.
Lipogenesis involves acetyl CoA carboxylase, FA synthase, PKA, ATP, and NADPH, synthesizing fatty acids.

Degradation of FA: Step 2 Transport of Acyl CoA

The inner mitochondria membrane is relatively impermeable to long-chain acyl CoA molecules. To transfer the fatty acid across, the carnitine shuttle contains enzymes translocase and two carnitine acyltransferases CAT I and CAT II.
The acyl group is transferred from CoA to carnitine to form acyl-carnitine by carnitine acyltransferase I, which is bound to the outer mitochondrial membrane.
Acyl carnitine is shuttled across the inner mitochondrial membrane by a specific translocase. Acyl carnitine is transformed back to acyl-CoA by carnitine acyltransferase II.
Carnitine is returned to the intermembrane space by translocase (antiport).

Metabolism of the Four Classes of Fatty Acids

The length of the fatty acid dictates where it is activated to CoA:
Short- and medium-chain fatty acids can cross the mitochondrial membrane by passive diffusion and are activated to their CoA derivative within the mitochondrion.
Very long-chain fatty acids from the diet are shortened to long-chain fatty acids in peroxisomes.
Long-chain fatty acids are the major components of storage triglycerides and dietary fats. They are activated to their CoA derivatives in the cytoplasm and are transported into the mitochondrion via the carnitine shuttle.

Degradation of FA: Step 3 Fatty Acid Oxidation

β-oxidation of fatty acids in mitochondria is the main pathway to generate energy.
Unsaturated fatty acids also undergo β-oxidation, but the ATP yield is less due to the presence of double bonds (unsaturated fats are “less reduced”).
FAs are converted into their acyl-CoA derivatives in the mitochondria matrix and metabolized by sequential removal of 2-carbon acetyl CoA units from the carboxyl end of the acyl-chain (occurs mainly in the liver and muscle but NOT in brain and RBC).
-oxidation is a repeat of 4 reaction steps processes and regulated by hormonal and allosteric controls.

Activation of FA and B-oxidation of FA

A diagram illustrating the overview of the fatty acid oxidation pathway, including activation and transport, and β-oxidation.

Coenzyme A (COA-SH)

The structure of Coenzyme A (CoA-SH) showing the reactive thiol group that attaches to the acyl group being transferred to form a thioester linkage.

-Oxidation of Saturated Fatty Acids

Fatty acids are oxidized, FAD is reduced, donating 2e- to oxidative phosphorylation.
Oxidation/dehydrogenation, hydration, thiolysis/thiolytic cleavage occur.
The citric acid cycle also takes place.
The fatty acid is shortened by 2 carbon atoms, and the cycle through -oxidation occurs again.
NAD+ is reduced, donating 2e- to oxidative phosphorylation.

-Oxidation of Saturated Fatty Acids

Mitochondrial oxidation of fatty acids takes place in three stages:
1. β-oxidation: Fatty acids undergo oxidative removal of successive two-carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acid.
2. Acetyl-CoA is oxidized to carbon dioxide in the citric acid cycle, which also takes place in the mitochondrial matrix.
3. Electrons produced from the 2 oxidative processes (NADH, FADH2) go into the mitochondrial electron transport chain (ETC) to produce ATP.
$1 NADH \rightarrow 2.5 ATP$
$1 FADH_2 \rightarrow 1.5 ATP$
-oxidation occurs in the matrix of mitochondria where the TCA cycle and ETC take place.

Energy Yield by -Oxidation of Saturated Fatty Acid – Palmitate (C16)

The degradation of palmitoyl-CoA (16C) requires seven reaction cycles.
Palmitate + ATP + CoA → Palmitoyl-CoA + AMP + PPi (or 2 Pi) − 2 ATP (activation)
Palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H2O → 8 Acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+
7 FADH2 → 7 × 1.5 ATP = +10.5 ATP
7 NADH → 7 × 2.5 ATP = +17.5 ATP
8 Acetyl-CoA → TCA → 8 × 10 ATP = +80 ATP
Net = 108 – 2 = 106 ATP (Each TCA cycle → 1 GTP + 3 NADH + 1 FADH2 = 10 ATP)
Complete oxidation of one palmitate molecule yields TOTAL 106 ATP molecules.

β-oxidation of Palmitate

16-carbon fatty acid yields a total of 8 acetyl-CoA (2C carboxyl end + 7 acetyl CoA).
The net ATP production is 106.

Class Practice - Arachidic Acid

ẞ-oxidation of arachidic acid produces how many acetyl CoA, NADH, FADH2 and ATPs?
Arachidic acid is a C20 saturated fatty acid found in peanuts.

Class Practice - Arachidic Acid Calculation

The degradation of arachidic acid (20C) requires NINE reaction cycles.
Arachidic acid + ATP + CoA → Arachidate-CoA + AMP + PPi (or 2 P) − 2 ATP (β-oxidation)
Arachidate -CoA + 9 CoA + 9 FAD + 9 NAD+ + 9 H2O → 10 Acetyl-CoA + 9 FADH2 + 9 NADH + 9 H+
9 FADH2 → 9 × 1.5 ATP = +13.5 ATP
9 NADH → 9 × 2.5 ATP = +22.5 ATP
10 Acetyl-CoA → TCA → 10 × 10 ATP = +100 ATP
Net = 136 – 2 = 134 ATP (Each TCA cycle → 1 GTP + 3 NADH + 1 FADH2 = 10 ATP)
Complete oxidation of one palmitate molecule yields TOTAL 134 ATP molecules.

II. Synthesis of Fatty Acids: De Novo Lipogenesis (DNL)

De novo pathway – synthesis of complex molecules from simple molecules.
Fatty acids can be synthesized from carbohydrates, via acetyl CoA, under circumstances of energy excess.
The process of de novo lipogenesis occurs in several tissues: liver, adipose tissue, brain, lung, mammary gland – cytosolic.
Requires NADPH, ATP, biotin, and bicarbonate as a source of $CO_2$
Palmitate (16:0) is the end product.

Fatty Acid Synthesis vs Degradation Diagram

A diagram comparing the processes of lipolysis (breakdown of triglycerides) and lipogenesis (synthesis of fatty acids).
Lipolysis involves hormone-sensitive lipase, epinephrine, glucagon, and PKA, breaking down TAG into fatty acids.
Lipogenesis involves acetyl CoA carboxylase, FA synthase, PKA, ATP, and NADPH, synthesizing fatty acids.

Synthesis of Fatty Acids

FA synthetic (lipogenesis) process is NOT the reverse of F.A. β-oxidation
Key differences:
- Synthesis of fatty acids (Lipogenesis) occurs in the cytosol, while lipolysis (β-oxidation) occurs in mitochondria.
- Lipogenesis is a series of reactions by sequential addition of 2 carbon units derived from 3C malonyl CoA (after activation of acetyl CoA).
- Lipogenesis uses NADPH as a reducing agent, while β-oxidation produces NADH.
- Lipogenesis involves covalently linked acyl carrier protein (ACP), while acyl groups are attached to CoA in β-oxidation.
- Lipogenesis is carried out by a single, multifunctional polypeptide chain called fatty acid synthase, while β-oxidation involves many individual enzymes.

Synthesis of Fatty Acids: Step 1 Citric Acid Shuttle

FAs are synthesized in the cytosol, but acetyl CoA is produced from pyruvate in the mitochondria, and acetyl CoA cannot pass through the inner mitochondria membrane freely.
Acetyl CoA combines with oxaloacetate (OAA) to form citrate, resulting in a citrate shuttle.
Citrate is transported into the cytosol and cleaved to regenerate acetyl CoA and oxaloacetate by ATP-citrate lyase in an ATP requiring process.
OAA returns to the mitochondrial matrix through conversion to malate by MDH, then to pyruvate by NADP+-linked malic enzyme, generating NADPH for FA synthesis.
Pyruvate is carboxylated to form OAA in the matrix of mitochondria.

Synthesis of Fatty Acids: Sources of NADPH

Two routes to NADPH, catalyzed by (a) malic enzyme and (b) the pentose phosphate pathway.

Synthesis of Fatty Acids: Step 2 Formation of Malonyl CoA

The first irreversible, rate-limiting step of fatty acid synthesis is carboxylation of acetyl CoA (2C) to form malonyl CoA (3C) by acetyl CoA carboxylase with biotin as a prosthetic group.
One molecule of ATP is hydrolyzed in the reaction.
Similar to the synthesis of all biopolymers, fatty acid synthesis requires an activation step.
Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA, which is the activated form of acetyl CoA.

Synthesis of Fatty Acids: Formation of Saturated Long-Chain Fatty Acids

Fatty acid synthase catalyzes the synthesis of saturated long-chain fatty acids from acetyl CoA, malonyl CoA, and NADPH.
This enzyme is a complex of distinct enzymes, each of which has a different function in fatty acid synthesis.

Synthesis of Fatty Acids: Elongation Cycle

On fatty acid synthase:
- Acetyl-CoA serves as a starting unit (not converted to malonyl CoA).
- Addition of 2C unit from malonyl-CoA.
- Each 2C unit added must be reduced by 2 NADPH.
- Use 2 NADPH in elongation by 2C.

Synthesis of Fatty Acids: Repeat Elongation Cycles

Growing FA chain:
- Addition of 2C unit from malonyl-CoA.
- Produce 1 $CO_2$
- Direction of FA formation: from methyl end to carboxyl end → reverse of that of β-oxidation which starts from the carboxyl end.

Energy Required for Palmitate Synthesis

In each elongation cycle, 1 $H2O$ is produced, so 7 $H2O$ are produced after 7 cycles.
Hydrolysis of thioester bond by thioesterase  7 $H2O$ – 1 $H2O$ = 6 $H_2O$

Regulation of Fatty Acid Synthesis

Main control at acetyl CoA carboxylase (ACC) reaction (catalyzes the synthesis of malonyl CoA).
ACC is switched off by phosphorylation (at serine) by AMP-dependent protein kinase (AMPK) and is activated by dephosphorylation by protein phosphatase 2A.
When energy is required, glucagon and epinephrine inhibit protein phosphatase 2A, ACC remains off.
When blood glucose levels are high, insulin will turn on ACC.
When the energy charge is low, i.e., low ATP content but high AMP content, AMP-dependent protein kinase is activated, which converts active carboxylase into inactive carboxylase.

Glucagon Effect on Lipid Metabolism

Activation of the glucagon receptor results in adenylate cyclase-mediated cAMP formation.
cAMP accumulation activates cAMP-dependent protein kinase, which leads to inactivation of acetyl-CoA carboxylase and thus suppression of malonyl-CoA (regulation by phosphorylation).
cAMP accumulation activates cAMP-responsible binding-protein (CREB), inducing transcription of carnitine acyl transferase-1 (CAT-1) and other genes required for β-oxidation.
Function of glucagon decreases fatty acid synthesis (inactivation by phosphorylation) and promotes FA breakdown by β-oxidation (gene activation).

Comparison of Fatty Acid Synthesis and Degradation

A table summarizing the comparison of fatty acid synthesis and degradation in terms of active state, main tissues involved, site, 2C donor/product, active fatty acid carrier, enzymes, oxidant/reductant, allosteric control, hormonal control, and product.

Ketone Body – Another Fuel Source Derived from Fats

Most acetyl CoA produced by FA degradation enters the TCA (Krebs) cycle.
However, some acetyl CoA is diverted to an alternative pathway to form ketone bodies (acetone, acetoacetate, and β-hydroxybutyrate).
Ketone body synthesis occurs in the mitochondria of liver cells (hepatocytes).
Ketone bodies are used as fuel by the brain and some tissues, such as heart muscle.
After 2-3 days of starvation, the body will shift the fuel being used from glucose to FA and ketone bodies.
Ketone bodies are water-soluble and are exported into the blood and other tissues.
After their uptake, ketone bodies are converted into acetyl CoA and processed by the TCA cycle.
The liver lacks the converting enzyme (e.g., CoA transferase), so ketone bodies, after being produced, are not metabolized in the liver but are transported out to other tissues.

Ketone Body - Another Fuel Source Derived from Fats

Ketone bodies are an alternative fuel source produced by the liver mitochondria using acetyl-CoA.

Ketone Body Production Pathway

A diagram illustrating the ketone body production pathway showing the conversion of acetyl-CoA to acetoacetate, D-B-hydroxybutyrate, and acetone in liver cells. These ketone bodies are exported as an energy source for other tissues.

Ketone Body Production During Starvation

After gluconeogenesis, OAA are exhausted.
No OAA (4C) is available to accept acetyl CoA (2C) to enter the TCA cycle, so acetyl CoA can’t enter the TCA cycle to produce energy.

Oxaloacetate (OAA) in Mitochondria Gluconeogenesis

Pyruvate enters gluconeogenesis via conversion to oxaloacetate (OAA) and then to phosphoenolpyruvate (PEP).

Oxaloacetate in Mitochondria

When gluconeogenesis is active, pyruvate is transported into mitochondria to enter gluconeogenesis. Pyruvate is converted to OAA.
OAA is a metabolite in both gluconeogenesis and the citric acid cycle, occurring in mitochondria.
In starvation, most OAA is converted to PEP, with no OAA available to accept acetyl CoA to enter the citric acid cycle.