Fatty Acid Metabolism I and II

Martin Kaczocha
Health Sciences Center, Level 4, L4-077
Phone: (631)444-6077
E-mail: [email protected]

Office hours 12-1pm

 Outline
• Adipose tissue function
• Physiology of triglyceride absorption and delivery to target tissues for
storage and utilization
• Liberation of fatty acids from adipose tissue
• β-oxidation of fatty acids (Fatty acid oxidation)
• Ketone body biosynthesis and utilization
 Objectives
• Understand how lipids are absorbed and
transported
• Know steps in β-oxidation
• Understand how ketone bodies are synthesized
and utilized
• Understand basic regulation of lipid metabolism
 Classes of Lipids

(FFA)
Nonesterified fatty acid (NEFA)

Triglyceride: Major lipid in diet

Fatty Acids:
Long alkyl chains with carboxylic acid at
one end (head). α
β
General forumula: γ
CH3-(CH2)n-COO-
(number from carboxylate)

Stearic acid: saturated C18

18:0

Oleic acid: unsaturated C18

18:1 ∆9

Linoleic acid: C18, two unsaturations

18:2 ∆9 ∆12 ω
Linolenic acid: C18, three unsaturations
18:3 ∆9 ∆12 ∆15
 Adipose Tissue
• Major storage site for lipids: Triglycerides
• Adipocytes: Major cell-type of adipose tissue (lipid storage)
• Endocrine organ

Coelho et al. Arch Med Sci. 2013 Apr 20; 9(2):191–200

 Fasting and Fed States
Glucagon Insulin
Glycogenolysis Glycolysis
Gluconeogenesis Glycogenesis
Fatty acid oxidation Fatty acid biosynthesis

Glycolysis in liver Glycogenolysis

Glycogenesis Gluconeogenesis
Fatty acid biosynthesis Fatty acid oxidation

“Fasting State” “Fed State”

Control of Food Intake (Satiety/Hunger)

OEA

Endocannabinoids
 Ghrelin
• Peptide hormone released by the
stomach
• Acts in the hypothalamus of the
brain to stimulate food intake

G Natalucci et al. Eur J Endocrinol 2005;152:845-850

Lipid Digestion
Lipid Digestion
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
 Triglycerides/Triacylglycerol (TAG)

• Highly hydrophobic!!!
• Pack into large lipid globules
 Bile Salts Emulsify Lipids
• Synthesized by liver, stored in gall bladder,
and released into the small intestine to aid
in lipid digestion (increase surface area)
 Triglyceride Hydrolysis by Pancreatic Lipase
• Pancreatic lipase is an enzyme that is secreted by the pancreas
• Hydrolyzes triglycerides into 2 fatty acids + 2-monoacylglycerol (MAG) in the small
intestine
• Cleaves triglycerides at the 1 and 3 positions
Summary of Triglyceride Metabolism and Absorption
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
Triglyceride and Cholesteryl Ester Synthesis in Intestinal
Cells

• Triglycerides and cholesteryl esters are highly hydrophobic!!!

 Chylomicron Synthesis

• Triglycerides are too hydrophobic to be released into bloodstream

• Triglycerides are packaged alongside cholesterol and cholesteryl esters into chylomicrons
• Chylomicron surface is decorated with proteins (e.g., apoC-II)
Chylomicrons Are Released Into Systemic Circulation
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
 Lipoprotein Lipase (LPL)

• Enzyme that lines the endothelium of capillaries surrounding adipose and muscle tissue
• Cleaves triglycerides within chylomicrons at the 1 and 3 positions to generate free fatty acids
(FFA) and 2-monoacylglycerols
 GPIHBP1 Anchors Lipoprotein Lipase to the Endothelium of
Capillaries Surrounding Adipose and Muscle Tissue

• Apolipoprotein C-II (apoC-II) activates LPL to increase triglyceride hydrolysis

GPIHBP1 Anchors Lipoprotein Lipase to the Endothelium of
Capillaries Surrounding Adipose and Muscle Tissue

Proc Natl Acad Sci U S A. 2022 Sep 6;119(36):e2211136119.

Chylomicron Remnants Are Taken Up By The Liver

Clin Lipidol. 2013 Aug 1;8(4)

What would you expect to occur in individuals
deficient in lipoprotein lipase?
 Lipoprotein Lipase Deficiency
• Rare: 1-2 cases/million
• Elevated chylomicrons
– Elevated triglycerides
– Elevated cholesterol

• Symptoms
– Abdominal pain (may appear as colic in infancy)
– Loss of appetite
– Nausea
– Vomiting
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
Fatty Acids and Cholesterol are Stored in Intracellular Lipid
Droplets
• Storage depots for cellular lipids
– Triglycerides and cholesteryl esters
 Lipid Droplets Are Dynamic
• Lipid droplets grow and shrink depending upon the rate of lipid turnover

Jungst et al. J. Lipid Res. 2013. 54: 3419–3429.

Fasting State: Low Blood Glucose
Glucagon Insulin
Glycogenolysis Glycolysis
Gluconeogenesis Glycogenesis
Fatty acid oxidation Fatty acid biosynthesis

Glycolysis in liver Glycogenolysis

Glycogenesis Gluconeogenesis
Fatty acid biosynthesis Fatty acid oxidation

“Fasting State” “Fed State”

 Stimulation of Lipolysis
• Fasting (glucagon)
or
• “Fight or Flight” (epinephrine/adrenaline)

• Effect: Hydrolysis of stored triglycerides into fatty acids within adipose

tissue, which are subsequently released for use by other tissues (e.g.,
muscle)
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
Enzymatic Conversion of Triglycerides Into Fatty Acids

ATGL = Adipocyte triglyceride lipase

HSL = Hormone sensitive lipase
MAGL/MGL = Monoacylglycerol lipase
 Regulation of Lipolysis:
Protein Kinase A (PKA) phosphorylates proteins involved in lipolysis

P
Regulation of Lipolysis: ATGL and ABHD5

P
 ABHD5/CGI-58
• Sequestered by perilipin under “basal”
state
• Phosphorylation of perilipin releases
ABHD5, which then activates ATGL by
recruiting it to the lipid droplet surface to
stimulate triglyceride hydrolysis

McMahon D et al. J. Lipid Res. 2014;55:1750-1761

 Chanarin–Dorfman Syndrome
• Rare autosomal recessive neutral lipid storage
disease
• Mutations resulting in nonfunctional ABHD5

Bruno et al. Biochemical and Biophysical Research Communications 369 (2008) 1125–1128
Regulation of Lipolysis: HSL

P
Enzymatic Conversion of Triglycerides Into Fatty Acids

ATGL = Adipocyte triglyceride lipase

HSL = Hormone sensitive lipase
MAGL/MGL = Monoacylglycerol lipase
 Glycerol is Converted Into Glycolytic Intermediates

• Glycerol is released into blood, taken up by the liver, and is used for gluconeogenesis
 Small Intestine Intestinal Cells Blood Adipocyte
Lipid Droplet = Storage
TAG
Pancreatic
Lipase
TAG + Chol
TAG
ATGL
MAG + FFA
Lipoprotein DAG
Lipase HSL
TAG + Chol MAG + FFA
MAG
MAGL
Chol Chylomicron
FFA
Muscle Blood

Lipoprotein
Lipase FFA + Albumin
Β-oxidation
MAG + FFA
Target
Tissue
FFA

Β-oxidation
 Fatty Acid Oxidation/β-Oxidation
• Mechanism through which cells utilize energy stored in fatty acids
(carbons of fatty acyl chains)
• Series of enzymatic reactions within the mitochondrial matrix:
– Fatty Acids -> Acetyl-CoA
 Fatty acid Lactate
Glucose
Acyl-CoA Synthetase
Malate Pyruvate
Acyl-CoA
Carnitine Cytosol
Mitochondrial
Matrix
Acyl-CoA Malate
Β-oxidation
Pyruvate

Β-oxidation Propionyl-CoA
Pyruvate Dehydrogenase
Acetyl-CoA
Acetyl-CoA Acetyl-CoA
Acetyl-CoA
NADH Acetyl-CoA
FADH2 (LIVER)

Ketone Bodies Krebs Cycle

Cytosol

Blood
Ketone Bodies
Brain/Heart/Muscle
Ketone Bodies Acetyl-CoA
 Fatty acid Lactate
Glucose
Acyl-CoA Synthetase
Malate Pyruvate
Acyl-CoA
Carnitine Cytosol
Mitochondrial
Matrix
Acyl-CoA Malate
Β-oxidation
Pyruvate

Β-oxidation Propionyl-CoA
Pyruvate Dehydrogenase
Acetyl-CoA
Acetyl-CoA Acetyl-CoA
Acetyl-CoA
NADH Acetyl-CoA
FADH2 (LIVER)

Ketone Bodies Krebs Cycle

Cytosol

Blood
Ketone Bodies
Brain/Heart/Muscle
Ketone Bodies Acetyl-CoA
Acyl-CoA Synthetase Enzymes (ACS) Covert Fatty Acids
into Acyl-CoAs

Chylomicron
TAG Fatty Acid Capillary
LPL

Fatty Acid
ACS
Acyl-CoA Cell
 Activation of Fatty Acids

Acyl-CoA Synthetase Mechanism:

1. Formation of acyladenylate intermediate
2. 2 ATP equivalents used
H-SCoA
 Transport of Long Chain Fatty Acids Into Mitochondria

-Inner mitochondrial membrane is not permeable to long chain Acyl-CoAs

-Carnitine palmitoyltransferase is also known as carnitine acyltansferase
Transport of Long Chain Fatty Acids Into Mitochondria

Carnitine/Acyl-carnitine translocase
 Fatty acid Lactate
Glucose
Acyl-CoA Synthetase
Malate Pyruvate
Acyl-CoA
Carnitine Cytosol
Mitochondrial
Matrix
Acyl-CoA Malate
Β-oxidation
Pyruvate

Β-oxidation Propionyl-CoA
Pyruvate Dehydrogenase
Acetyl-CoA
Acetyl-CoA Acetyl-CoA
Acetyl-CoA
NADH Acetyl-CoA
FADH2 (LIVER)

Ketone Bodies Krebs Cycle

Cytosol

Blood
Ketone Bodies
Brain/Heart/Muscle
Ketone Bodies Acetyl-CoA
 Fatty Acid Oxidation/β-Oxidation
• Series of enzymatic reactions within the mitochondrial matrix:
– Acyl-CoA -> Acetyl-CoA
Fatty Acid Oxidation/β-Oxidation

Products of each round of β-oxidation:

1 FADH2
1 NADH
1 Acetyl-CoA
 Reaction 1: Acyl-CoA Dehydrogenase

Hydride transferred to FAD

Glutamate abstracts proton

Formation of a trans-α,β double bond through

dehydrogenation by acyl-CoA dehydrogenase (AD)

Mitochondria possess four acyl-CoA dehydrogenases:

VLCAD: very long-chain acyl-CoA DH (12-20C)
LCAD: long chain acyl-CoA DH (8-20C)
MCAD: medium-chain acyl-CoA DH (6-10C)
SCAD: short-chain acyl-CoA DH (4C)
 Reaction 2: Enoyl-CoA Hydratase

2 Glu

Multiple forms for different chain lengths

The water to be added is coordinated

by 2 Glu via H-bonds
Reaction 3: NAD+-dependent Dehydrogenation

Oxidizes a 2o alcohol using NAD+

Multiple forms for different chain lengths

 Reaction 4: Cα-Cβ cleavage in a Thiolysis Reaction

Generates Acetyl-CoA and Acyl-CoA 2C shorter.

Multiple forms for different chain lengths.

 Trifunctional Protein (TFP)
• Single enzyme that contains EH, HAD, and KT activities
• Essential for the metabolism of highly hydrophobic long chain fatty acyl-CoAs

Proc Natl Acad Sci U S A. 2019 Mar 26;116(13):6069-6074.

Products of ß-oxidation are Shuttled to the Citric Acid Cycle
and Oxidative Phosphorylation
 Palmitate (16:0) requires 7 rounds of ß-oxidation

How many ATP from ß-oxidation of palmitoyl-CoA (16:0)?

7 rounds of ß-oxidation:
7 FADH2 (1.5 ATP/FADH2) = 10.5 ATP
7 NADH (2.5 ATP/NADH) = 17.5
8 Acetyl-CoA
(10 ATP/Acetyl-CoA) = 80
108
- 2 ATP for activation -2
106 ATPs/Fatty acid molecule
β-oxidation of Unsaturated Fatty Acids

(18:1 ∆9)

(18:2 ∆9 ∆12)
GOAL: Shift double bonds to
generate trans-Δ2-Enoyl-CoA

Not a substrate for enoyl-

CoA hydratase (EH)

Poor substrate for enoyl-

CoA hydratase (EH)
 ß-oxidation of Odd Chain Fatty Acids

15:0

6 rounds of
ß-oxidation

Propionyl-CoA
(not a substrate for AD)
 Genetic Disorders of Beta Oxidation
• Acyl-CoA Dehydrogenases
– VLCAD: Cardiomyopathy and muscle weakness
– LCAD: Pulmonary surfactant dysfunction
– MCAD: Most common beta-oxidation defect (1:15000). Hypoketotic hypoglycemia with lethargy
that develops into coma
– SCAD: Relatively mild. Leads to elevated levels of butyrate
• HAD
– Lethal cardiomyopathy, infant-onset hepatic form (lethargy), OR peripheral neuropathy

Mitochondria possess four acyl-CoA dehydrogenases:

VLCAD: very long-chain acyl-CoA DH (12-20C)
LCAD: long chain acyl-CoA DH (8-20C)
MCAD: medium-chain acyl-CoA DH (6-10C)
SCAD: short-chain acyl-CoA DH (4C)
Summary of β-oxidation
 Fatty Acids and the Brain
• Fatty acids do not readily diffuse into the brain
– During fasting, the brain cannot use fatty acids released by adipose tissue as a source of
energy
• Alternate source is needed to transfer energy stored in fatty acids (i.e., in adipose
tissue or liver) to the brain

ADIPOSE TISSUE

FATTY ACIDS
X
BRAIN
 Fatty acid Lactate
Glucose
Acyl-CoA Synthetase
Malate Pyruvate
Acyl-CoA
Carnitine Cytosol
Mitochondrial
Matrix
Acyl-CoA Malate
Β-oxidation
Pyruvate

Β-oxidation Propionyl-CoA
Pyruvate Dehydrogenase
Acetyl-CoA
Acetyl-CoA Acetyl-CoA
Acetyl-CoA
NADH Acetyl-CoA
FADH2 (LIVER)

Ketone Bodies Krebs Cycle

Cytosol

Blood
Ketone Bodies
Brain/Heart/Muscle
Ketone Bodies Acetyl-CoA
 Ketone Bodies

Released by liver into the bloodstream

Used for energy during fasting/low blood glucose
Can diffuse into the brain
 Ketone Bodies
• Produced from acetyl-CoA in the liver
• Produced at low levels under normal conditions
• Ketone body levels increase when blood fatty acid concentrations are
high:
– Starvation conditions (>24 hour fast, long-term low blood glucose)
– Untreated diabetes
• Fatty acid metabolism is a major source of acetyl-CoA for ketone body
biosynthesis
 Energy Use by the Brain
• Brain comprises ~2% of body weight but uses ~20% of glucose
• Brain (i.e., neurons) is heavily reliant on glucose metabolism
– This can be supplemented by ketone bodies during fasting/starvation
• During prolonged fasting/starvation, it is imperative that the brain continues
to receive glucose as an energy source
– Liver and muscle shift to fatty acid metabolism to preserve glucose
 Fatty acid Lactate
Glucose
Acyl-CoA Synthetase
Malate Pyruvate
Acyl-CoA
Carnitine Cytosol
Mitochondrial
Matrix
Acyl-CoA Malate
Β-oxidation
Pyruvate

Β-oxidation Propionyl-CoA
Pyruvate Dehydrogenase
Acetyl-CoA
Acetyl-CoA Acetyl-CoA
Acetyl-CoA
NADH Acetyl-CoA
FADH2 (LIVER)

Ketone Bodies Krebs Cycle

Cytosol

Blood
Ketone Bodies
Brain/Heart/Muscle
Ketone Bodies Acetyl-CoA
Products of Beta Oxidation Inhibit Glycolysis in Liver and Muscle
[NADH]
[Acetyl-CoA]

Acetyl-CoA also stimulates

pyruvate carboxylase to increase
gluconeogenesis in the liver
 Ketogenesis

Liver-specific enzyme
 Fatty acid Lactate
Glucose
Acyl-CoA Synthetase
Malate Pyruvate
Acyl-CoA
Carnitine Cytosol
Mitochondrial
Matrix
Acyl-CoA Malate
Β-oxidation
Pyruvate

Β-oxidation Propionyl-CoA
Pyruvate Dehydrogenase
Acetyl-CoA
Acetyl-CoA Acetyl-CoA
Acetyl-CoA
NADH Acetyl-CoA
FADH2 (LIVER)

Ketone Bodies Krebs Cycle

Cytosol

Blood
Ketone Bodies
Brain/Heart/Muscle
Ketone Bodies Acetyl-CoA
Metabolism of Ketone Bodies
 Advantages of Ketone Bodies
• Ketone bodies provide energy to other tissues
– Provide a way to transport acetyl-CoA between tissues
• Lower demand for glucose by brain during starvation
• Reduce amount of protein (i.e., amino acids) that must be broken down
for gluconeogenesis
 Excess of Ketone Bodies
• Diabetic ketoacidosis
– Tissues unable to take up and utilize glucose
– Excess ketone body production
– Acetone can be smelled in breath
– Blood pH
• Alcoholic ketoacidosis
– Found in alcoholics
– High [NADH] and [lactate], depletion of pyruvate
required for gluconeogenesis
– Elevated ketone body production
– Blood pH
 Glut1 Deficiency and the Ketogenic Diet
• Glut1 is a glucose transporter at the blood brain barrier
• Mutations in Glut1 reduce glucose uptake by the brain
• Children with Glut1 deficiency (heterozygous) frequently develop seizures that are
poorly controlled by anti-epileptic medications
• Ketogenic diet reduces seizures in these patients