LIPOPROTEIN METABOLISM

• Exogenous pathway: chylomicrons clear dietary lipids
• Endogenous pathway: VLDL and LDL transport/distribute endogenously synthesized lipids
• Reverse cholesterol transport: HDL clears excess plasma cholesterol

Enzymes

• Degrade triacylglycerol to glycerol and free fatty acids

1. Lipoprotein lipase (LPL), bound to the endothelial layer of peripheral capillaries.
2. Hepatic lipase, localizes within hepatic endothelial cells.

CHYLOMICRONS

• Fed state only
• Cleared ~ minutes
• 98% lipids (1.TAG 2. Free/esterified cholesterol)
• ApoB-48 = dietary lipoprotein marker

EXOGENOUS PATHWAY

• Only occurs in the fed state.
• Clears dietary lipids
  Step 1: Dietary lipids are packaged into a chylomicron
  Step 2: Chylomicron enters lymphatic system (empties into circulation via left subclavian vein)
  Step 3: HDL donates ApoE and ApoC-II to chylomicron
  Step 4: ApoC-II binds/activates LPL (peripheral tissues)
  Step 5: LPL degrades TAG into glycerol & FFA
• FA enter tissues & glycerol returns to liver
• Chylomicron = chylomicron remnant (low TAG, high CE and C)
  Step 6: ApoC-II returns to HDL
  Step 7: Apo E binds liver remnant receptor: chylomicron endocytosis
  Step 8: Remnant releases cholesterol in liver
• Remnant degraded by lysosomes

CLINICAL CORRELATION

Familial Hyperchylomicronemia

• Rare hereditary disease
• LPL or ApoC-II deficiency: cannot clear chylomicrons
• Fasting blood plasma: cloudy
• Lipemia retinalis: cloudy appearance of capillaries in the retina

LIPOPROTEIN FUNCTION

• Solubilize cholesterol and triacylglycerol (TAG) & transport them in plasma
• Deliver them to peripheral tissues

Triacylglycerol function

• Energy storage
• Membrane synthesis

Cholesterol function

• Component of cell membranes
• Precursor for steroid hormones and vitamin D

LIPOPROTEIN STRUCTURE

Apolipoprotein

• On surface of lipoprotein
• 3 functions:
  i. Structure: bind and solubilize hydrophobic lipids
  ii. Recognition: contain signals that target lipoproteins to cells (recognized by receptors)
  iii. Enzymatic: activate or act as coenzymes in lipid metabolism

Single layer of phospholipids

• Free cholesterol in layer

Hydrophobic core

• Esterified cholesterol and TAG

CLASSES OF LIPOPROTEINS

1. Chylomicrons (fed state only)

• Size: largest
• Origin: intestine (dietary)
• Composition: dietary lipids (mainly TAG)
• Density: least dense

2. Very low density lipoprotein (VLDL)

• Size: LDL < VLDL < chylomicron
• Origin: liver (endogenous lipids)
• Composition: Endogenously synthesized lipids (mainly TAG & phospholipids)
• Density: LDL > VLDL > chylomicron

3. Low density lipoprotein (LDL)

• Size: VLDL > LDL > HDL
• Origin: VLDL
• Composition: Major cholesterol carrier in body (greatest relative amount)
• Density: VLDL < LDL < HDL
• “Bad cholesterol” (atherosclerosis)
• Regulates de novo cholesterol biosynthesis

4. High density lipoprotein (HDL)

• Size: smallest
• Origin: intestine and liver
• Composition: picks up plasma cholesterol (released due to membrane turnover or cell death; carries less cholesterol than LDL)
• Density: most dense (greatest % of protein)
• Delivers cholesterol to liver for excretion (bile salts) and to tissues (steroid synthesis)
• “Good cholesterol”

CLINICAL CORRELATION

HDL:LDL cholesterol ratio

• Diagnostic used to determine a patient’s risk for developing heart disease
• Healthy ratio ~ 3.5

Hypercholesterolemia (high blood cholesterol)

• Common consequence of low HDL:LDL cholesterol ratio
• Can lead to atherosclerosis & heart attack

KETOGENESIS

• Ketone bodies synthesized in liver only (mitochondrial matrix)
• Occurs under 2 clinical conditions: prolonged starvation & uncontrolled diabetes
• Substrate: excess acetyl CoA (derived from fatty acid oxidation)
• Normal, healthy adult: excess acetyl CoA shunts to citric acid cycle or cholesterol biosynthesis

KETONE BODIES

1. Acetoacetate
2. Acetone

• Volatile
• Breathed out unused

3. Beta-hydroxybutyrate

KETOGENIC PATHWAY

• Fasting conditions (starvation or uncontrolled diabetes)

1. Oxaloacetate shunts into gluconeogenesis: slows down citric acid cycle
2. Acetyl CoA builds up and shunts into ketogenesis
3. 2 Acetyl CoA –> Acetoacetyl CoA (acetoacetyl CoA thiolase, reversible)
4. 1 Acetyl CoA + Acetoacetyl CoA –> HMG CoA (HMG CoA synthase)

• Thiolase & HMG CoA synthase also in cholesterol biosynthesis (ketogenic isozymes in matrix not cytosol)

5. HMG CoA –> Acetyl CoA + Acetoacetate (HMG CoA lyase)
6. Acetoacetate + NAD+ –> beta-hydroxybutyrate + NADH (beta-hydroxybutyrate dehydrogenase, reversible)

KETOSIS

• Spontaneous when [acetoacetate] is high
• Acetoacetate –> Acetone + CO2

RATE LIMITING STEP

• HMG CoA synthase: enzyme localized in liver
• Activated by: fasting, increased cAMP and increased lipolysis
• Inhibited by: feeding & insulin

TARGET CELL KETONE BODY USE

• Cells that can use ketone bodies
• Include: cardiac/skeletal muscle, renal cortex, intestinal mucosa, brain cells in starvation
• Ketone bodies can cross blood brain barrier: do NOT bind albumin (fatty acids do)
• Mobilized in matrix

Enzyme beta-ketoacyl CoA transferase

• NOT in liver (liver cannot mobilize ketone bodies)
• Acetoacetate + succinyl CoA –> acetoacetyl CoA + succinate (reversible)
• Remaining reactions are the reverse of ketogenesis

CLINICAL CORRELATION

Untreated diabetics

• Have fruity breath due to exhalation of acetone (ketosis)
• Decreased cellular glucose and CAC intermediates leads to inc. FA mobilization & acetyl CoA
• Excess acetyl CoA shunts into ketogenesis

BETA OXIDATION

• Occurs in liver and peripheral tissues (mitochondrial matrix)
• Occurs under low energy conditions (starvation, insulin:glucagon ratio is low)
• NOT reverse of fatty acid synthesis

TRIACYLGLYCEROL (TAG)

• Stored in adipose and liver (circulates as lipoprotein)
• TAG breaks down to glycerol and fatty acids
• Fatty acids exit adipose and travel to muscle for degradation (provide ATP when energy is low)

TYPES OF FATTY ACIDS

• Degradation pathways differ depending on chain length and saturation

Saturated long chain fatty acids

• Model pathway in diagram

Unsaturated fatty acids

• Already partially oxidized: yield less FADH2 and ATP
• Req. different enzymes to work with additional double bonds

Branched chain fatty acids

• Alpha-oxidation: produce acetyl CoA and propionyl CoA

Medium and short chain fatty acids

• Can be 6-12C long
• Do NOT need carnitine shuttle to enter matrix
• Req. medium-chain acyl CoA dehydrogenase (MCAD) for oxidation step

FATTY ACIDS DELIVERY (ADIPOSE TO MUSCLE)

1. Fatty acids travel from adipose to muscle cell
2. Fatty acid transporter transports fatty acids into cytosol

• Carnitine transporter moves carnitine into cytosol

3. Fatty acyl CoA synthetase: Fatty acid + CoA + ATP –> Fatty acyl CoA + AMP

Carnitine

• Specialized carrier that transports fatty acids within cell
• “Car” in carnitine ~ shuttle

CARNITINE SHUTTLE

1. Carnetine acyl-transferase I (CAT-1): Fatty acyl CoA + Carnitine –> Fatty acyl-carnitine + CoA
2. Fatty acyl-carnitine enters matrix
3. CAT-2 (embedded in inner membrane): Fatty acyl-carnitine –> Carnitine + Fatty acyl CoA

BETA-OXIDATION

• Breaks down fatty acids in 2C increments (acetyl CoA) + shorter chain fatty acid
• Req. four reactions per 2C increment
• All reactions occur at beta-carbon (beta-oxidation)

Each 2C increment

i. Oxidation

• Acyl CoA dehydrogenases (ACAD): family of chain-length specific enzymes
• Releases FADH2 (~ 2 ATP via oxidative phosphorylation)
  ii. Hydration
  iii. Another oxidation
• Releases NADH (~ 3 ATP via oxidative phosphorylation)
  iv. Release of 2C (acetyl CoA)
• Acetyl CoA can enter fatty acid synthesis or citric acid cycle
• Citric acid cycle: 1 ATP, 1 FADH2 & 3NADH (HIGH ENERGY YIELD!)

ODD CHAIN FATTY ACIDS

• Final products: 2C Acetyl CoA + 3C propionyl CoA

Propionyl CoA Breakdown

• Propionyl CoA carboxylase (ABC-carboxylase reaction):
  Propionyl CoA + ATP + biotin + CO2 –> Methylmalonyl CoA (4C)
• Methyl-malonyl CoA mutase (req. Vit B12 cobalamin): Methylmalonyl CoA –> Succinyl CoA

Product: Succinyl CoA (4C)

• Citric acid cycle intermediate
• Gluconeogenic intermediate: only fatty acid that can be converted to glucose

• ccurs in the liver and adipose tissue (cytosol)
• After a carbohydrate-rich meal (high insulin:glucagon ratio)
• Not just reverse of beta-oxidation: distinct enzymes and compartments

Reactions

1. Citrate Shuttle

• Acetyl CoA in mitochondrial matrix transported to cytosol as citrate
• Citrate synthase: citrate from oxaloacetate and acetyl CoA (also first CAC rxn)
• Citrate a marker of high intracellular energy (CAC intermediate)

2. Malonyl CoA Formation

• Acetyl CoA carboxylase: adds 1 carbon to acetyl CoA to form malonyl CoA
• ABC carboxylase reaction
• Citrate activates rxn
• Long-chain fatty acyl CoA (intermed. of FA breakdown): inhibits rxn

ABC carboxylase reactions: consume ATP, require biotin, consume CO2

• Malonyl CoA synthesis in fatty acid biosynthesis
• Gluconeogenesis: pyruvate carboxylase
• Odd chain fatty acid oxidation: propionyl carboxylase

3. Palmitate Synthesis

• Palmitate: 16-carbon fatty acid
• Catalyzed by fatty acid synthase
• ACP is carrier protein component of fatty acid synthase
• Series of 4 reactions:
  i. Condensation: acetyl-ACP + malonyl-ACP = 4-C intermediate + CO2
  ii. Reduction (NADPH)
  iii. Dehydration: water molecule released
  iv. Reduction (NADPH)
• First condensation = 4C molecule
• 6 increments x 2C (malonyl-ACP) = 12 C
• Total = 16 C palmitate

4. Palmitate Modification
   i. Elongation: in smooth ER or mitochondria

• 2-carbon increments using malonyl CoA (NOT malonyl-ACP)
• Each increment includes four rxn’s from palmitate synthesis
  ii. Desaturation: in smooth ER or peroxisomes
• Fatty acyl-CoA desaturase
• Short electron transport chain (requires O2 and NADPH)

CLINICAL CORRELATION
Essential fatty acids: linoleic acid and linolenic acid cannot be synthesized endogenously.

• Mammals cannot induce double bonds beyond C9

Blood cholesterol levels (average healthy adult)

< 200 mg/dL (cholesterol levels vary more than tightly controlled glucose levels)

• 2 sources of cholesterol: biosynthesis and dietary

REGULATORY POINTS

1. Cholesterol Biosynthesis

• Occurs in most tissues, but primarily in liver

2. LDL receptors

• LDL transports cholesterol throughout body

3. Bile acid biosynthesis

• Sole mechanism for cholesterol clearance

1. CHOLESTEROL BIOSYNTHESIS

HMG CoA Reductase Regulation

i. Transcription

• SREBP-2 binds sterol regulatory element (SRE) and enhances transcription
• SREBP-2/SCAP release from Golgi inhibited by excess intracellular cholesterol
  ii. Translation
• Mevalonate (biosynthetic intermediate) produces non-sterol metabolites that block RNA translation in the cytosol
  iii. Proteolysis
• Excess cholesterol stimulates reductase proteolysis by ubiquitination
  iv. Covalent modification
• Insulin promotes dephosphorylation and activation of reductase
• Glucagon promotes phosphorylation and inactivation of reductase (AMP-dependent pathway)

Acyl CoA cholesterol acyltransferase (ACAT)

Esterifies cholesterol in hepatic cells: “There is A CAT in the liver”

2. LDL RECEPTORS

LDL: main cholesterol carrier in the body

• Excess cholesterol inhibits LDL-receptor synthesis

3. BILE ACID BIOSYNTHESIS

HDL: picks up plasma cholesterol released by cell death or membrane turnover

• Contains lecithin cholesterol acyl transferase (LCAT): esterifies cholesterol
• Delivers esterified cholesterol to liver cell
• Liver converts esterified cholesterol to bile salts: secreted into intestine

CLINICAL CORRELATIONS

Diabetics taking exogenous insulin

Have high levels of HMG CoA reductase in hepatocytes

Statins

Class of drugs that inhibit HMG CoA reductase: combat high cholesterol

Cholestyramine

Bile acid binding resin: binds bile salts in intestine and prevents reabsorption

• Initiates feedback mechanism: increases bile production, cholesterol and LDL receptor synthesis
• Combats high cholesterol

CHOLESTEROL

• Maintains membrane fluidity
• Precursor for bile acids and salts
• Sterol with a double bond between C5 and C6
• Obtained from diet or de novo synthesis

Sterol

Any steroid that has a side chain at C17 with 8-10 carbons and a hydroxyl group at C3.

DE NOVO CHOLESTEROL SYNTHESIS

• All tissues can synthesize cholesterol — mostly from liver, intestines, adrenal cortex and reproductive tissues
• Cellular level: occurs in the cytosol

Reactions

1. HMG-CoA formation:
   Acetyl CoA + Acetoacetyl CoA + H2O –> HMG CoA + CoA

2. Committed step: HMG CoA reductase (rate-limiting enzyme)
   HMG CoA + 2NADPH –> Mevalonate + 2NADP+

3. Phosphorylation
   Mevalonate + 3ATP –> Isopentenyl pyrophosphate (IPP) + Pi + 3ADP + CO2

4. Condensation reactions
   IPP + 2IPP –> Farnesyl pyrophosphate (FPP) + 2PPi
   FPP + FPP + NADPH –> Squalene + 2PPi + NADP+

5. Cholesterol formation
   Squalene + O2 + NADPH –> Cholesterol + H2O + NADP+

CHOLESTEROL FUNCTION

1. Bile acids and salts: emulsify fats and facilitate digestion in small intestine

• Only mechanism for cholesterol excretion

2. Steroid hormones: homeostatic regulators in the body
3. Vitamin D: synthesized in skin upon light exposure

CHOLESTEROL CIRCULATION

Lipoproteins

Transport lipids in circulation; contain lipids, proteins, triacylglycerol, free and esterified cholesterol

• Chylomicron: only transport dietary lipids
• LDL (low density lipoprotein): carries most esterified cholesterol
• HDL (high density lipoprotein): carries 2nd most esterified cholesterol
• VLDL (very low density lipoprotein)
  Total fasting cholesterol = LDL +HDL + VLDL

CLINICAL CORRELATIONS

Statins (HMG CoA reductase inhibitors)

Cholesterol-lowering medications

FPP

Chemotherapeutic target that links Ras (small GTP-binding protein) to the membrane.

• Ras mutations ~ 1/3 human cancers

Atherosclerosis

Narrowing of blood vessels due to plaque formation

• Vessel walls become leaky and vulnerable: LDL’s accumulate
• Vessels become more vulnerable with age, smoking, poor diet and lack of exercise

REGULATED ENZYMES

Glycogen synthase: glucose 1P polymerization to glycogen

• Catalyzes rate-limiting step in glycogen synthesis
• Active form: dephosphorylated
• Inactive form: phosphorylated

Glycogen phosphorylase: releases glucose 1P residues from glycogen

• Catalyzes rate-limiting step in glycogenolysis
• Active form: phosphorylated
• Inactive form: dephosphorylated
• Activated by phosphorylase kinase

Phosphorylase kinase: phosphorylates glycogen phosphorylase

• Activate form: phosphorylated
• Inactive form: dephosphorylated

KEY ORGANS

Liver

• Regulates blood glucose, responds to needs of all organs
• Insulin, glucagon & epinephrine receptors

Skeletal muscle

• Synthesizes/breaks down glycogen based on own metabolic needs
• Only insulin and epinephrine receptors (NOT glucagon)

REGULATORY MECHANISMS

1. Hormonal: insulin (high glucose), glucagon (low glucose) & epinephrine (stress)

Glucagon (low glucose) & epinephrine (stress):

• Activate protein kinase A (PKA) in cAMP-dependent manner
• PKA phosphorylates glycogen synthase (inactivates it)
• PKA phosphorylates phosphorylase kinase (activates it)
• Phosphorylase kinase phosphorylates glycogen phosphorylase (activates it)
• Upregulates glycogenolysis, down regulates glycogen synthesis

Insulin (elevated glucose):

• Activates phosphoprotein phosphatase (PPP)
• PPP dephosphorylates glycogen synthase (activates it)
• PPP dephosphorylates phosphorylase kinase (inactivates it)
• Upregulates glycogen synthesis, down regulates glycogenolysis

Glucagon receptor NOT in muscle: muscle only responds to stress (epinephrine)

2. Allosteric regulation

Both hepatic and muscle cells

• Glycogen synthase activation: glucose 6P (glucose 1P –> glucose 6P)
• Glycogen phosphorylase inhibition: glucose 6P, ATP (energy abundance)

Liver only

• Glycogen phosphorylase inhibition: glucose

Muscle only

• Glycogen phosphorylase activation: Ca2+, AMP (low energy)
• Muscle contraction: Ca2+ released from sarcolemma & allosterically activates phosphorylase kinase –> activates glycogen phosphorylase

CLINICAL CORRELATION

McArdle’s Disease (Type V glycogen storage disease)

• Muscle glycogen phosphorylase deficiency
• Glycogen accumulates in muscle
• Muscle cramps & decreased exercise tolerance

GLYCOGEN
• Body’s glucose reserve
• Can be mobilized more quickly/efficiently than fats
• Stored in liver & muscle
• Mobilized during fast (low insulin: glucagon)

ENZYMES OF GLYCOGENOLYSIS

Glycogen phosphorylase

• Breaks alpha (1,4) bonds

Debranching enzyme

• Breaks alpha (1,6) bonds
• Aka alpha 1,6 glucosidase

Glucose 6-phosphatase

• Tissue specific: liver only

GLYCOGENOLYSIS

1. Glycogen phosphorylase removes terminal residues of glycogen branches
   • Cleaves alpha (1,4) glycosidic bonds until 4 glucose residues remain per branch
   • Cleaves 1 glucose residue at a time
   • Glycogen + Pi –> Glucose 1-phosphate
2. Debranching enzymes transfers 3 residues of shortest branches to longer ones
   • One glucose residue remains per branch
   • Creates more alpha (1,4) linkages for glycogen phosphorylase to hydrolyze
3. Debranching enzyme cleaves last glucose residue from short branches
   • Cleaves alpha (1,6) bond
   • Glycogen + H2O –> Glucose
   • Releases residue as glucose NOT glucose 1P
4. Repeat: glycogen phosphorylase & debranching enzyme degrade glycogen to glucose & glucose 1P

LIVER VERSUS MUSCLE: FATES OF GLUCOSE & GLUCOSE 1P
• Both organs: glucose 1P reversibly converts to glucose 6P

Muscle

• Glucose 6P enters glycolysis –> Pyruvate + ATP
• If O2 is present: pyruvate decarboxylation –> acetyl CoA –> aerobic respiration
• If O2 is absent (exercising muscle): anaerobic glycolysis –> lactate
• Both pyruvate fates produce ATP: fuel for muscle cells
• Stores glycogen for its own use

Liver

• Glucose 6-phosphatase: Glucose 6P –> Glucose + Pi (enzyme NOT in muscle)
• Hepatic glucose released into circulation: fuels peripheral tissues (brain & rbc’s)

CLINICAL CORRELATION

Von Gierke’s Disease (Type I glycogen storage disease)

• Glucose 6-phosphatase deficiency in liver/kidney
• Frequent hypoglycemia: cannot mobilize glycogen during fast
• Treatment: frequent feedings with slowly digested carbohydrates (i.e. uncooked starch) to maintain blood glucose

ENDOGENOUS GLUCOSE POLYMERS

Glycogen

• Synthesized in liver & muscle (insulin: glucagon is high)
• Branched structure
• Linear segments: glucose monomers linked with alpha (1,4) glycosidic bonds
• Branch points: alpha (1,6) glycosidic bonds
• Branch point functions: i. solubilize glycogen ii. create terminal sugars for release
EXOGENOUS GLUCOSE POLYMERS
• Dietary

Amylopectin (starch)

• Fewer branches than glycogen
• Obtained from: potatoes, rice, etc.

Cellulose

• No branches
• Obtained from plants
• Glucose monomers linked with beta (1,4) glycosidic bonds
• Humans lack enzymes to break beta (1,4) glycosidic bonds

GLYCOGEN SYNTHESIS

1. Glucose + ATP –> Glucose 6P + ADP
   • Hexokinase (M) and Glucokinse (L)
2. Glucose 6P –> Glucose 1P (reversible)
   • Phosphoglucomutase
3. Glucose 1P + UTP –> UDP-glucose + PPi
   • PPi + H2O –> 2Pi (drives reaction forward)
   • UDP-glucose = substrate for glycogen synthesis
4. UDP-glucose + glycogen polymer –> glycogen polymer (+1 glucose residue) + UDP
   • Glycogen synthase: alpha (1,4) glycosidic bonds (adds 1 glucose-residue/rxn)
5. Branching enzyme adds branches
   • Breaks off (at least) 6 terminal residues from linear portion to make branch
   • Catalyzes alpha (1,6) linkage

Glycogenin: primer for glycogen chain

• Catalyzes first 4-8 glucose residues
• First glucose binds tyrosine residue in glycogenin
• Glycogen synthase adds glucose residues to preexisting glucose polymer

CLINICAL CORRELATION

Type IV Glycogen Storage Disease: Anderson’s Disease

• Branching-enzyme deficiency
• Presents as long, linear polymers of glucose
• Visible at very young age, produces cell damage
• Aka amylopectosis (amylopectin ~ glycogen w/ less branching)