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)

GLUCONEOGENESIS

• Synthesis of glucose from non-carbohydrate precursors
• Occurs mostly in the liver and minor process in kidney
• Kidney produces 10% total glucose during overnight fast

ENZYMES UNIQUE TO GLUCONEOGENESIS

1. Pyruvate carboxylase (mitochondrial matrix)

• Converts pyruvate to oxaloacetate
• Requires 1 ATP, biotin and 1 CO2 (ABC Reaction)

2. Phosphoenol carboxykinase (cytosolic and mitochondrial isozymes)

• Converts oxaloacetate to phosphoenolpyruvate (PEP)
• Consumes 1 GTP and releases 1 CO2

Cytosolic PEPCK

• Used in the malate shuttle: shuttles oxaloacetate from mitochondrion to cytosol via malate
• Pathway dominates when pyruvate is the gluconeogenic substrate
• Mitochondrion: oxaloacetate –> malate (consumes 1 NADH)
• Cytosol: malate –> oxaloacetate (releases 1 NADH)
• Released NADH used in G3P synthesis
• Pyruvate substrate uses cytosolic PEPCK: consumes 2 NADH

Mitochondrial PEPCK

• Pathway dominates when lactate is substrate
• Cytosol: lactate –> pyruvate (releases 1 NADH)
• Released NADH used in G3P synthesis
• Mitochondrial PEPCK: oxaloacetate –> PEP (can cross mitochondrial membranes)
• Lactate substrate uses mitochondrial PEPCK: does NOT consume NADH

PEP converted to glyceraldehyde 3-phosphate in 4 reversible reactions: 1 ATP and 1 NADH consumed (double energy inputs, substrates and products)

Glyceraldehyde 3-phosphate reversibly combines w/ DHAP to form fructose 1,6-bisphosphate

3. Fructose 1,6-bisphosphatase (cytosol)

• Produces fructose 6-phosphate
• Consumes 1 H20 and releases 1 Pi

Fructose 6-phosphate reversibly converts to glucose 6-phosphate

4. Glucose 6-phosphatase (ER membrane-bound)

• Enzyme complex with 4 proteins
  i. Transport protein: G6P from cytosol to ER lumen
  ii. Phosphatase: removes Pi from G6P to form glucose (consumes 1 H2O)
  iii. Transport protein: transports glucose to cytosol
  iv. Transport protein: transports Pi to cytosol

FINAL BOOKKEEPING:
2 Pyruvate + 4ATP + 2GTP + 2NADH + 6H20 –> 1 Glucose + 4ADP + 2GDP + 2NAD+ + 6Pi + 6H+
2 Lactate + 4ATP + 2GTP + 6H2O –> 1 Glucose + 4ADP + 2GDP + 6Pi + 6H+

Lactate substrate lacks net NADH requirement

CLINICAL CORRELATION

Avidin

• Protein in egg whites
• Binds biotin very tightly (biotin required by pyruvate carboxylase)
• Consuming large amounts of raw eggs over an extended period of time can produce biotin deficiency
• Symptoms: CNS problems (lethargy)

MECHANISMS OF REGULATION

• Allosteric regulation
• Hormonal regulation
• Substrate availability

ALLOSTERIC REGULATION

Pyruvate carboxylase

• 2Pyruvate + 2CO2 + 2ATP –> 2Oxaloacetate + 2ADP
• Activated by Acetyl CoA (product of FA breakdown, marker of energy abundance & low blood glucose)

Phosphoenolpyruvate carboxykinase (PEPCK)

• 2Oxaloacetate + 2GTP –> 2Phosphoenolpyruvate (PEP) + 2GDP + 2CO2

Corresponding glycolytic reaction

• Pyruvate kinase: 2PEP + 2ADP –> 2Pyruvate + 2ATP
• Inhibited by Acetyl CoA

Fructose 1,6-bisphosphatase-1 (FBP-1)

• Fructose 1,6-BP + H2O –> Fructose 6-P + Pi
• Activated by Citrate (CAC intermediate & marker of energy abundance)
• Inhibited by AMP (marker of low energy) & fructose 2,6-BP (hormonally regulated)

Corresponding glycolytic reaction

• PFK-1: Fructose 6-P + ATP –> Fructose 1,6-BP + ADP
• Inhibited by Citrate
• Activated by AMP & fructose 2,6-BP

SUBSTRATE AVAILABILITY

Glucose 6-phosphatase

• Glucose-6-phosphate + H2O –> Glucose + Pi
• Not allosterically regulated because Km >>> [glucose 6-phosphate]
• Substrate level control

Corresponding glycolytic reaction

• Glucokinase: Glucose + ATP –> Glucose 6-phosphate + ADP

HORMONAL REGULATION

• FBP-2 & PFK-2 are hormonally regulated (PFK-2 inactive when phosphorylated)
• High blood glucose = increased Insulin: glucagon ratio = PFK-2 active
  = increased fructose 2,6-BP = promote glycolysis and inhibits gluconeogenesis
• Low blood glucose = decreased insulin: glucagon ratio = PFK-2 phosphorylated & inactive
  = decreased fructose 2,6-BP = slows glycolysis and removes inhibition from gluconeogenesis
• INSULIN: promotes glycolysis
• GLUCAGON: promotes gluconeogenesis

• Synthesis of glucose from non-carbohydrate precursors
• Occurs mostly in the liver and minor process in kidney
• Kidney produces 10% total glucose during overnight fast

THREE KEY SUBSTRATES

1. Lactate: enters pathway via pyruvate

• Produced by exercising muscle and red blood cells
• Reconverted to pyruvate in liver

2. Glycerol: enters via DHAP

• TAG hydrolyzes to glycerol in adipose tissue
• Liver converts glycerol to DHAP in 2 step reaction

3. Amino acids: enter via pyruvate or citric acid cycle intermediates

• Major source of glucose during extended fast
• Muscle tissue hydrolysis releases glucogenic AA during fast
• AA produce alpha-ketoacids in the liver: enter CAC or gluconeogenesis

ESSENTIAL FUNCTIONS OF GLUCONEOGENESIS

• Clears blood lactate from red blood cells and exercising muscle
• Maintains blood glucose during high fat diet or fast

ENZYMES UNIQUE TO GLUCONEOGENESIS

1. Pyruvate carboxylase (mitochondrial matrix)

• converts pyruvate to oxaloacetate
• Requires 1 ATP, biotin and 1 CO2

2. Phosphoenol carboxykinase (cytosol)

• Preceded by malate shuttle (1 NADH consumed and 1 NADH produced)
• Converts oxaloacetate to phosphoenolpyruvate (PEP)
• Consumes 1 GTP and releases 1 CO2

PEP converted to glyceraldehyde 3-phosphate in 4 reversible reactions: 1 ATP and 1 NADH consumed (double energy inputs, substrates and products)

Glyceraldehyde 3-phosphate reversibly combines w/ DHAP to form fructose 1,6-bisphosphate

3. Fructose 1,6-bisphosphatase (cytosol)

• Produces fructose 6-phosphate
• Consumes 1 H20 and releases 1 Pi

Fructose 6-phosphate reversibly converts to glucose 6-phosphate

4. Glucose 6-phosphatase (ER membrane-bound)

• Translocates glucose 6-phosphate to the ER lumen and removes Pi
• Consumes 1 H2O

ENERGY REQUIREMENTS:
(1 ATP + 1 GTP + 1 NADH + 1 ATP) x 2 = 4 ATP + 2 GTP + 2NADH

INNER MITOCHONDRIAL MEMBRANE

• Contains the ETC, ATP synthase, ADP-ATP transporter, phosphate translocase and more
• Impermeable to small molecules (H+, ATP, ADP & Pi)
• ETC pumps protons across impermeable inner membrane: generates chemiosmotic gradient (proton-motive force)

ATP SYNTHASE STRUCTURE

F0 (c, gamma, and epsilon subunits)

• Cylindrical structure embedded in membrane
• Channel through which H+ flows down gradient

F1 (alpha and beta subunits)

• Sits on top of F0 on matrix side

Stator (a, b, and delta subunits)

• Prevents F1 from rotating as F0 does

ATP SYNTHESIS

1. H+ from intermembrane space enters F0
2. H+ protonates asparagine residue within channel
3. Induces rotation of c-ring

• Electrochemical energy (H+ gradient) converted to mechanical energy (rotation)
• On the matrix side, ADP & Pi bind F1 beta subunit

4. Beta subunit interacts with rotating F0: activates and catalyzes formation of ATP

• ATP released into matrix along with H+ that passes through channel

ADP-ATP TRANSPORTER

• Antiporter
• Driven by the electrical potential across membrane
• Intermembrane space more positive than matrix
• ATP has -4 charge while ADP has -3 charge
• Charge difference favors movement of ATP OUT of negatively charged matrix

PHOSPHATE TRANSLOCASE

• Symporter: pumps H+ & Pi from intermembrane space into matrix
• Driven by pH gradient across inner membrane
• pH greater in matrix (more basic, less H+) and lower in intermembrane space (more acidic, more H)
• Protons & Pi move from intermembrane space into matrix
• For every 4 H+ pumped into matrix: 3 drive ATP synthase & 1 drives Pi transport

CLINICAL CORRELATIONS

Uncouplers

• Proteins that make inner mitochondrial membrane permeable to H+
• Example: 2,4 dinitrophenol (DNP)

Brown adipose tissue (BAT)

• Specialized adipose tissue that facilitates non-shivering thermogenesis
• Contains many mitochondria & uncouplers

Complex I: NADH dehydrogenase

• aka NADH-CoQ reductase
• NADH delivers 2 electrons to the complex I and is oxidized to NAD+

CoQ (aka Q10 and ubiquinone)

• Lipid-soluble
• Mobile carrier
• NOT a protein

Complex II: succinate dehydrogenase

• aka Succinate-CoQ reductase
• Also part of citric acid cycle
• FADH2 delivers two electrons to complex II

Complex III: Cytochrome bc1 complex

• aka CoQ-cytochrome c reductase

Cytochrome C

• Water-soluble
• Mobile carrier

Complex IV: cytochrome c oxidase

• Produces one H2O from 2 H+ plus ½ O2 + 2e-
• Complexes I, III & IV pump H+ from matrix to intermembrane space
  (NOT complex II, cyt. C or CoQ)

COFACTORS

• Complex I – flavin mononucleotide (FMN).
• Complex II – FAD & FeS
• Complex III – Heme (Cyt. B & Cyt. C1) & FeS
• Complex IV – heme (Cyt. A & Cyt. A3) & Cu

REDOX REACTIONS

• NADH (2e-) reduces complex I
• FADH (2e-) reduces complex II
• CoQ (mobile carrier) transports 2e- from complex I & II to complex III
• Complex III (simplified diagram): Cyt. B & Cyt. C1 are e- transport proteins
• Cyt. C1 donates 2e- (1e- at a time) to Cyt. C (mobile-carrier)
• Cyt. C transports 4e- to complex IV (2 molecules of Cyt. C deliver 2e- each)
• Complex IV (simplified diagram w/o Cu centers): Cyt. A (4e-) to Cyt. A3 (4e-) to oxygen
• Oxygen (final e- acceptor) 4e- + 4H+ (from matrix) + O2 –> H2O

3 BYPASS REACTIONS

• Bypass complex I & produce less ATP

1. Succinate delivers e- to complex II via FADH2
2. Acyl CoA dehydrogenase (on matrix side) oxidizes fatty acyl CoA & produces FADH2
3. Cytosolic NADH delivers e- via glycerol-3-phosphate

• Enzyme glycerol-3-phosphate dehydrogenase (on intermembrane side) produces FADH2

CHEMIOSMOTIC GRADIENT

• Electrical gradient: from less positive in the matrix to more positive in the intermembrane space.
• pH gradient: from a lower pH in the intermembrane space to a higher pH in the matrix.

Electron transport chain

• Series of controlled redox reactions; pumps H+ into inter-membrane space

Chemiosmosis

• Couples e- transport w/ ATP synthesis

ELECTRON TRANSPORT CHAIN

Complex I: NADH dehydrogenase

• aka NADH-CoQ reductase
• NADH delivers 2 electrons to the complex I and is oxidized to NAD+

CoQ (aka Q10 and ubiquinone)

• Lipid-soluble
• Mobile carrier
• NOT a protein

Complex II: succinate dehydrogenase

• aka Succinate-CoQ reductase
• Also part of citric acid cycle
• FADH2 delivers two electrons to complex II

Complex III: Cytochrome bc1 complex

• aka CoQ-cytochrome c reductase

Cytochrome C

• Water-soluble
• Mobile carrier

Complex IV: cytochrome c oxidase

• Produces one H2O from 2 H+ plus ½ O2 + 2e-
• Complexes I, III & IV pump H+ from matrix to inter-membrane space
  (NOT complex II, cyt. C or CoQ)

CHEMIOSMOSIS

Complex V: ATP synthase

• Inner mitochondrial membrane IMPERMEABLE to most small molecules
• H+ that is pumped across membrane cannot diffuse back through the bilayer
• H+ diffuses down gradient through ATP synthase into the matrix
• Produces 30-34 ATP per glucose molecule (NADH = 3 ATP, FADH2 = 2 ATP