BIOCHEMISTRY Lecture 21. MITOCHONDRIA.

Mitochondrial Structure and Composition.

1. Outer membrane a. Permeable to small metabolites b. Does not serve as a barrier to proton diffusion Inner membrane a. Contains key proteins for energy production (e.g. ATP synthase) and transport systems for necessary metabolites b. High proportion of cardiolipin (diphosphatidyl glycerol) which enhances its lack of permeability to protons Human mitochondrial DNA a. Small, circular maternally-derived DNA (~17 kb) b. Endosymbiotic origin (own system for DNA replication and transcription) mtDNA differs from nuclear DNA a. Contains very little introns b. Genetic code differs also; encodes 37 genes: i. 2 structural ribosomal RNAs ii. 22 transfer RNAs needed for mitochondrial protein synthesis iii. 13 of the 70 proteins that form the ETC machinery

2.

3.

4.

5. Majority of the hundreds of proteins in the mitochondria are imported from cytosol into the mitochondria; energy dependent
a. Presequence (matrix targeting sequence): sequence at the N-terminus that directs precursor proteins to the mitochondria

b. Two paths into the matrix i. Translocases of the outer membrane (TOMs) ii. Translocases of the inner membrane (TIMs) Mitochondrial Respiratory Chain - Structure & Function 1. Substrates for respiratory chain: a. NADH (3 ATP generated) b. Complex I (NADH dehydrogenase) c. FADH2 (2 ATP generated) d. Complex II (succinate, acetyl-CoA, glycerol, phosphate dehydrogenases) e. Prosthetic groups: harbor the transferable electrons
i. Pyridine Nucleotides (NAD+) 1. NAD+ accepts two electrons and a proton to generate NADH 2. Remaining proton enters the aqueous medium of the cell

ii. Flavoproteins 1. Complex II: FAD undergoes reversible oxidation and reduction
a. FAD-linked proteins: i. Succinate dehydrogenase; glycerol-3-phosphate dehydrogenase; Acetyl-CoA dehydrogenase

2. Complex I: NADH dehydrogenase enzyme accepts electrons 1

from NADH and utilizes FMN (flavin mononucleotide)

3. The flavoproteins usually have a more positive reduction potential than NAD+/NADH, making electrons more readily transferred to flavoproteins from NADH, regenerating NAD+

iii. Coenzyme Q (Ubiquinone) 1. Transfers electrons from Complexes I and II to complex III
2. 3. 4. Benzoquinone derivative inserted into inner membrane Fully oxidized: CoQ Two electron reduced form: CoQH2

iv. Cytochromes 1. Proteins containing the prosthetic group heme 2. Transfer electrons sequentially from CoQH2 to molecular oxygen 3. Cytochrome b: component of complex III
a. has the most negative reduction potential; accepts electrons from CoQH2; the protons removed from CoQH2 during this process are released and transported from the mitochondrial matrix into the intermembrane space

4. Cytochrome c1: also component of complex III; transfers electrons to cyto c 5. Cytochrome c: on outer side of the inner membrane (facing the intermembrane space) 6. Cytochrome Oxidase: contains two hemes: Cytochrome a and Cytochrome a3 in addition to copper
a. b. Accepts 4 electrons from cytochrome c and transfers them via cytochrome a3 to a molecule of oxygen As it picks up 4 protons from the medium, it generates two molecules of water

v. Iron-Sulfur proteins (Fe-S*)

1. 2. 3. Proteins containing non-heme iron prosthetic groups Serve as electron carriers Components of Complexs I, II, and III

2. Complex I a. NADH: CoQ reductase 3. Complex II a. Succinate: CoQ reductase 2

4. Complex III a. Reduced CoQ: cyt c reductase 5. Complex IV a. Cytochrome oxidase 6. Electron transfer by respiratory chain is coupled (physically, energetically) to the transfer of proteins
7. Total of 10 protons pumped from matrix a. 4 protons during transfer of 2 electrons through Complexes I and III b. 2 protons during transfer of 2 electrons through Complex IV c. Complex II is not a proton pump

8. Proton-motive force a. pH gradient & membrane potential (m): source of potential energy for ATP synthesis 9. The addition of an inhibitor together with a substrate will cause all components proximal to the block to be fully reduced and components distal to the block to be fully oxidized a. Complex I i. Rotenone (insecticide) binds to Complex I and prevents the reduction of ubiquinone; also Amytal and Piercidin A ii. NADH cannot be used but Electron flow can continue because the oxidation of succinate at Complex II allows electrons to enter the chain b. Complex III i. Antimycin inhibits electron transfer through CIII c. Complex IV i. Cyanide and azide bind tightly to the oxidized (ferric) form of cytochrome a3
ii. Mitochondrial respiration and energy production ceases; cell death rapidly occurs

iii. Carbon monoxide also inhibits CIV, but by binding to the reduced (ferrous) form of cytochrome a3 Transport Systems to Move Biomolecules into and out of the Mitochondrion 1. Sources of substrates (reducing equivalents) for the ETC
a. b. c. Glycolysis: NADH generated in cytosol i. Pyridine nucleotides cannot penetrate the mitochondrial inner membrane Amino Acid Oxidation: generates pyruvate or acetyl-CoA, which can be further oxidized to CO2, generating NADH Fatty Acid Oxidation: generates NADH, reduced flavoprotein, and acetyl-CoA i. NADH: enters ETC ii. Flavoproteins: reduces ubiquinone TCA cycle i. Four of the oxidative reactions of the TCA cycle, that are catalyzed by dehydrogenases, generate NADH; the reaction catalyzed by succinate dehydrogenase transfers electrons directly to ubiquinone

d.

Malate-Aspartate shuttle

2. Malate-Asparate Shuttle
a. b. Malate can serve as an electron carrier between the cytoplasm and mitochondria Depends on presence of i. Two enzymes: 1. Malate dehydrogenase (MDH) 2. Glutamate-oxaloacetate transaminase (GOT) This shuttle is reversible ii. And two transporters: 1. -Ketoglutarate transporter NADH electrons from the cytosol are shuttled to NADH in the matrix for use by complex I 2. Glutamate-asparate transporter Cytoplasm: NADH is used to reduce OAA to malate i. Enters mitochondria via -Ketoglutarate transporter Mitochondria: Malate reoxidized to OAA, regenerating NADH (to be used for oxidative phosphorylation) i. OAA is used to form glutamate by GOT, generate asparate which is transported back to cytoplasm Cytoplasmic NADH is used to reduce dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G-3-P) G3P is reoxidized by a FAD-linked glycerol phosphate dehydrogenase present on surface of mitochondrial inner membrane, which transfers electrons to ubiquinone Unlike malate-asparate shuttle, no compounds, only electrons, are transferred through mitochondrial inner membrane
The electrons from NADH in the cytosol are carried across the inner membrane employing two different shuttles.

c. d.

3. Glyerol Phosphate Shuttle

a. b.

glycerol phosphate shuttle (non reversible)

start here

c.

d. Since the electrons from mitochondrial FADH2 feed into the oxidative phosphorylation pathway at coenzyme Q (as opposed to NADH-ubiquinone oxidoreductase [complex I]) only 2 moles of ATP will be generated from glycolysis rather than 3 1. ATP Synthase (a.k.a. ATPase or Complex V)
a. b. Dissipates the proton gradient, using the energy to synthesize ATP Inhibitor of ATP synthase: Oligomycin

glycerol phosphate shuttle

CoQH2

CoQ

IMM

FADH2 transfers electrons to Q Thus, no compounds move through the inner membrane

c. Complex consists of two components with different functions i. F0 component: forms transmembrane channel ii. F1 component: contains 3 identical active sites in which synthesis of ATP from ADP and
Pi occurs 1. The conformational changes cause the dissociation of the product ATP and promotes the ADP phosphorylation reaction Active center of ATP synthase is accessible only from the matrix side of the inner membrane i. ADP and Pi have to enter the mitochondria, and ATP has to leave it; all via two transporters:

d.

1. Pi/OH- exchanger (Pi in; OH- out of matrix and into cytosol) 2. ATP/ADP exchanger (inhibited by atractyloside) 2. In the mitochondrial OXPHOS system electrons flow to complexes of a lower reduction potential to an oxidized molecule of higher reduction potential 3. In coupled mitochondria change in the rate of respiration leads to the change in the rate of ATP synthesis and vice versa
a. Respiratory control ratio (RCR): ratio of the rates of both respiration and oxidative phosphorylation, with and without ADP, including how tightly couples the mitochondria are

4. Uncouplers disrupt the interdependence of respiration and ATP synthesis by allowing protons to bypass the ATP synthase (dissipate the H+ gradient)
a. Causes respiration to proceed at maximal rate, no longer constrained by the increase in membrane potential; The energy goes to heat

b. Uncouplers: FCC; dinitrophenol; termogenin or UCP1 (brown fat- burning calories to generate heat) c. If the ETC is interrupted at a step after formation of the electrochemical 4

proton gradient, an uncoupler will restore respiration but not ATP synthesis d. Possible causes for this situation: i. Lack of ADP or Pi ii. Inhibition of ADP/ATP translocator (e.g. by atractyloside) iii. Inhibition of phosphate transporter iv. Inhibition of ATP synthase (e.g. by oligomycin) 5. Uncouplers will not restore respiration if the ETC itself is inhibited (e.g. by cyanide) or if there is a lack of respiratory substrates within the matric (e.g. steps before the formation of the proton-motive force) Mitochondria and human disease 1. Mitochondrial Permeability Transition (MPT) is the loss of the inner mitochondrial membrane impermeability to solutes caused by extended opening of the Permeability Transition Pore (PTP)
a. b. c. No longer a barrier against protons re-entering matrix Leads to the cessation of ATP synthesis and swelling of the mitochondrial matrix apoptosis and necrosis CypD-deficient Mice are Resistant to Ischemia/Reperfusion-induced Cardiac Injury
D

MPT and stroke

Calcium Glut-R Glutamate O2

MPT

Ym loss, loss of OMM, cell death 2. Reactive Oxygen Species a. Incorrect or overactive functioning of the respiratory chain (e.g. in presence of inhibitors or when excess NADH is present) leads to an increase in ROS synthesis b. ROS can damage DNA, fatty acids, triglycerides and proteins i. Detoxification of ROS occurs in pentose phosphate pathway c. Are implicated in many pathological processes such as diabetes, Alzheimers, Parkinsons, and aging

Lecture 21b. Mitochondrial Myopathies. Mitochondrial Myopathies 1. Mitochondrial Disease

a. Multi-system involvement b. Progressive and variable course c. Easy fatigability d. Unusually severe reactions to infectious illnesses e. Autonomic symptoms f. Family history of similar symptoms g. Even dysmorphic features Mitochondrial Myopathies can result from defects in nuclear DNA or in mitochondrial DNA a. Majority of mitochondrial proteins are coded for by nuclear DNA, translated in the cytosol and imported into the mitochondrion; allows the possibility to have a mitochondrial myopathy inherited from the father Defects in mitochondrial genome (Maternal inheritance) a. Account for majority of the disorders

2.

3.

4. Each cell contains hundreds of mitochondria, thus thousands of copies of mitochondrial DNA a. Heteroplasmy: cell may contain populations of mutant and of normal mitochondrial DNAs, which may not be distributed equally to daughter cells after meiosis and mitosis. This would allow the mitochondrial DNA 5

populations to become successively more homogeneous (Homoplasmic), yielding cells with predominantly normal or predominantly mutant mitochondrial DNA 5. Threshold effect: severity of symptoms is a combination of the degree of impairment of function caused by the mutation, the proportion of mutant mitochondrial DNA, and the dependence of the particular tissue on oxidative phosphorylation
6. Lebers Hereditary Optic Neuropathy (LHON) a. Acute adult onset blindness with rapid loss of vision b. Maternal mode of inheritance; males affected more than females c. Due to missense mutation causing a single amino acid substitution in Complex I peptide Kearns-Sayre Syndrome (KSS) a. Ocular myopathy; retinitis pigmentosa and lactic acidosis b. Single mtDNA deletion, but size and position of deletion differs among patients c. Rate of replication of mtDNA is proportional to the molecules length i. The shorter, deleted molecules have a replicative advantage over the longer, normal molecules leads to an increasing proportion of mtDNA molecules with the deletion d. Symptoms show age-related progression Myoclonus Epilepsy with Ragged Red Fibers (MERF) a. Ragged red muscle fibers and abnormal mitochondria b. Point mutation in a loop of mitochondrial tRNA, causing inhibition of mitochondrial protein synthesis; has a larger effect on Complexes I and IV Diagnosis: a. The only way to be 100% sure of the diagnosis is to identify a DNA mutation b. Biochemical features: Abnormalities in blood, urine, spinal fluid c. Radiologic features: MRI scan; MR spectroscopy d. Physiologic testing: resting energy expenditure; exercise testing e. Tissue biopsy: Abnormalities in size, shape, or number of the mitochondria, or their structure f. Organ inventory: Testing for multiple organ dysfunction: Determination: a. Experiment 1: Is the problem with a protein coded for by N-DNA or Mt-DNA? i. Fuse cells from: 1. Patient (with destroyed mtDNA) and 2. Normal individual (with nucleus removed) ii. if mitochondria are normal, problem resulted from a defect in the patients mitochondrial DNA iii. if the mitochondria are not normal, the problem resulted from a defect in the patients nuclear DNA b. Experiment 2: Reverse situation Enzyme activity a. Can assay each segment of the ETC separately by selective use of inhibitors and artificial electron donors and acceptors b. Can also determine whether mitochondria are tightly coupled by measuring respiratory control ratio Level of components a. Determination of CoQ levels or presence or absence of different peptides Determine whether mitochondrial function has been compromised by an external agent a. Promote energy production b. Reduce energy loss Procedure/ Treatment a. KSS treatment: giving the patient the cofactors (such as coQ) and oxidizable substrates to keep the residual ETC functioning at its full capacity b. To circumvent defects in Complexes I and II: use electron donors to reduce Complex III or IV directly c. Fusing the DNA sequence that would encode the normal protein in the cytosol to a sequence that would encode a matrix targeting sequence at the N-terminus of a protein

7.

8.

9.

10.

11.

12. 13.

14.

Lecture 22. HEME

1. Porphyrins are important prosthetic groups of many proteins; all have the same porphin ring; sidechains on pyrrole rings distinguish prophyrins a. Type III: physiologic form of porphyrins (ring D) b. A porphyrinogen spontaneously oxidizes to a porphyrin i. porphyrinOGENS (reduced porphyrins) are intermediates in heme biosynthesis
A PorphyrinOGEN oxidizes spontaneously to a Porphyrin
Type I & Type III porphyrins differ in order of substituents on ring D
A M
A C H C C N P A P C C N C C NH HN C N C H C C P C A C C H C H C C P C A C P A H C

P V C C N H2 C

A = Acetyl
C C H C

C H2 C C

M A
C H C C N

V P
C C H C

P = Propionyl
H C C

P A

C C

A P

C C

C C NH HN C N C C H C

A M
A P

C C

C NH HN

C C C

A M P V

A M P

C C

C NH HN

C C C

A M P V

D
C

D
C

P P

C N C H2 C C P P C A M C

C C H2

C N C H C C P P C A M C C H

ProtoporphyrinOGEN III
UROPORPHYRIN TYPE I UROPORPHYRIN TYPE III (~physiological)

Protoporphyrin III
Conjugated double-bond system Colored red-brown

Type III is a physiologic form of protoporphyrin

PorphyrinOGENS are intermediates in heme biosynthesis

2.

All cells synthesize heme; Most is synthesized in bone marrow (presence of RBCs); rest in liver

Regulation of heme synthesis: completing the cycle

3. Heme biosynthesis: Proto porphyrin a. SuccinylCoA and Glycine are combined by Proto SuccinylCoA III porphyrinogen + Glycine aminolevulinaste synthase (ALAS) to form a III Heme delta-aminolevulinate Copro Delta-aminoporphyrinogen mitochondrion levulinate i. Occurs in the mitochondria; product is III transported into cytoplasm Copro Delta-aminoii. Slowest reaction; key (only) regulated porphyrinogen levulinate Uro Porpho III step porphyrinogen bilinogen III (pyrrole) Lead Copro (tetrapyrrole) b. -ALA dehydratase condenses two molecules of porphyrinogen Uroporphyrinogen I I -ALA to form porphobilinogen i. Enzyme is a target for lead poisoning, where lead displaces the zinc atoms c. Condensation of four molecules of porphobilinogen to form uroporphyrinogen, with the loss of 4 molecules of ammonia i. Enzyme: Porphobilinogen deaminase (uroporphyrinogen synthase)
ii. Uroporphyrinogen co-synthase assures closure in type III form 1. Without co-synthase, make type I which can be converted to coproporphyrinogen but not protoporphyrinogen; leads to deposits in skin & photosensitivity

d. Decarboxylation forms Coproporphyrinogen i. Molecule now more hydrophobic; diffuses back into mitochondria e. Decarboxylation and oxidation forms Protoporphyrinogen f. Protoporphyrinogen is rapidly oxidized to form protoporphyrin g. Protoporphyrin captures iron to form Heme i. Catalyzed by Ferrochelatase 7

4. Liver, and other non-erthroid cells a. Can synthesize heme for cytochromes, particularly cytochrome P450 enzymes, which metabolize drugs and other endogenous intermediates b. -amino levulinaste synthase (ALAS) is regulated by free heme c. Accumulation of heme causes: i. Inhibition of gene transcription, 1. Inhibition of synthesis of ALAS1: a. Decreased mRNA stability (heme) b. Decreased transcription of ALAS1 gene (insulin) c. Steroid hormones increase ALAS1 synthesis ii. Inhibition of transport of newly synthesized ALAS1 enzyme into mitochondria iii. Allosteric inhibition of ALAS1 by heme in mitochondria d. Drugs upregulating CYPP450 (barbiturates) increase heme synthesis i. Any enzyme that uses heme will conjugate to it and decrease the level of free heme in mitochondria and thus increase ALAS1 5. Erythroid cells a. Heme is synthesized in immature erythrocytes
b. Mature erythrocytes do not contain mitochondria and do not synthesize heme.

c. Express a gene for ALAS-2 (different from liver) d. Regulation: i. Iron 1. Increases translation of ALAS2 (displaces inhibitor from mRNA) 2. Increases ferrochelatase activity ii. Erythrocyte-specific transcription factors act on ALAS2 gene promoter iii. Inhibition of ALAS2 transport into mitochondria (heme feedback inhibition) 6. None of the intermediates of heme biosynthesis accumulate because after the first step, all the steps are extremely fast; also have negative feedback control 7. Porphyrias: diseases associated with defects in heme synthesis a. Levels of free heme decrease, which causes the first steps activity to increase (partial release from feedback inhibition), resulting in an even larger increase in amount of intermediates up to the defect
b. Porphyrias are associated with abnormally high levels of amino levulinaste acid synthase activity and an overproduction of the intermediates up to the step at which the deficiency occurs

c. Therapies try to decrease activity of the first step: give hemin; glucose d. e.g. patients cannot use any drugs that will interact CYPP450 or steroids
e. Diagnosis: i. Urine PBG test ii. Analysis of excreted (ALA, iii. PBG, Uroporphyrinogen urine; iv. Uroporphyrinogen, Coproporphyrinogen, Protoporphyrinogen stool sample)

Heme breakdown Heme Biliverdin Bilirubin Conjugated Bilirubin (liver)

macrophages

8. Steps in heme degradation Jaundice a. A cytochrome P450 mixed function oxidase converts heme to biliverdin (a green compound) b. Biliverdin reduced to yellow-brown compound bilirubin c. Free bilirubin is insoluble; bilirubin is bound to serum albumin for transport to the liver i. Bilirubin is very insoluble & forms deposits; takes a while for macrophages to get rid of it (e.g. extended presence of bruise) d. Bilirubin is conjugated to glucuronic acid in the liver i. Conjugated bilirubin is water-soluble and is excreted into bile ii. Bile is collected into the gallbladder, and drains via the common bile duct into the small intestine e. Intestinal bacteria metabolize bilirubin to fecal and urinary pigments
9. 10. 11. 12.

Conjugated bilirubin exceretion through bile ducts

Bilirubin breakdown (intestine)

Liver disease: accumulation of bilirubin Excessive hemolysis: accumulation of bilirubin Occlusion of bile duct: accumulation of conjugated bilirubin Jaundice in newborns due to defects in heme breakdown a. Bilirubin can accumulate in fenestrae of the brain if not properly excreted b. Light therapy can help remove bilirubin; isomerizes the h-bonds and breaks them; molecule becomes a lot more hydrophilic, dissolves, and is easily excreted

LECTURE 23: INTRODUCTION TO LIPID METABOLISM

1. Lipid: a. b.

Isoprenoids i. Membranes, signals, cofactors, fat soluble vitamins Fatty acid bases i. Energy storage, fat deposits (fat is the most efficient way of storing energy), membranes, signals

The alpha and omega of fatty acids

a = 1st carbon after carboxyl (C2), b = 2nd carbon after carboxyl (C3), etc. w = last carbon after carboxyl (terminal methyl) regardless of chain length.
aLinolenic Acid
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 CH3-CH2-CH=CH-CH 2-CH=CH-CH2-CH=CH-CH2-CH2-CH2-CH2-CH2-CH2-CH2-COOH

2. Fatty acids a. Numbering: carboxyl carbon =1 b. FA symbol: # before colon = total length of chain (how many carbons); number after colon = how many double bonds; Superscript = list of double bond positions with regard to carboxyl group c. Unsaturated have double bonds
i. Saturated: Stearic Acid 18:0 ii. Monounsaturated: Oleic Acid 18:19 iii. Polyunsaturated Fatty Acid: aLinolenic Acid 18:3
9,12,15

w Omega number

b a

Number of carbons from the terminal methyl (w) carbon to the nearest double bond w# = (# of carbons) - (highest # for double bond position).

aLinolenic Acid

18:3D9,12,15

18-15 = w-3

3. Triacylglycerols (TAGs) = dietary and storage fats a. Fully hydrophobic b. Formed by dehydration between 3 FAs and glycerol c. Ester linkage (carbon through oxygen to carbonyl carbon of FA) d. Glycerol is symmetric (at the 1 & 3 position)
e. Melting point of pure triacylglycerols or mixtures of TAGs (e.g. liquid vegetable oils versus solid animal fats)

i. Melting points of uncharged FAs (R-COOH) increase with chain length 9

4. Almost all naturally occurring double bonds are cis; forms a kink in the double bond and decrease in melting point
a. b. Can convert cis to trans (=processed foods); Melting point is almost the same as the unsaturated molecule Saturated FA (or trans-unsaturated FA) = high MP
Digestion & Transport of Dietary Fat
Dietary Fat Droplets (TAGs) Various tissues for energy.

c. As number of double bonds increase, melting point decreases (solid liquid)

5. Digestion: 6. Emulsification increases fats accessibility to digestive enzymes

a. a. b. Bile salts & phospholipids are good emulsifiers Overall reaction: TAG 2x FA + 2-MAG Pancreatic lipase doesnt recognize olestra (FAs in ester linkages to hydroxyl groups on sucrose)

7. Hydrolysis of TAGs by Pancreatic Lipase

Intestinal Lumen (Emulsication & digestion) Blood (Lipoprotein transport)

8. Absorption: a. To enter intestinal epithelial FAs must be monomers Intestinal Cell (Absorption, preparation i. FA salt solubility decreases as alkyl chain length for transport) increases ii. Short to Medium Chain FA Salts (6-12C): Diffuse easily (simple diffusion) iii. Long Chain FA Salts (14C or longer): Most dietary FAs. Solubility too low to facilitate diffusion into cells b. Micelles: self-association of amphipathic molecules in water; facilitate absorption i. Form at the Critical Micelle Concentration (CMC) c. FA activation & reformation of TAGs drives absorption i. Used to keep fatty acids inside the cell ii. Inorganic Pyrophosphatase Rapidly and irreversibly converts the covalently linked phosphates to free phosphates
iii. Linking the low G of fatty acyl CoA synthetase to the high G of inorganic Pyrophosphatase makes the reaction irreversible so it doesnt go backwards in the cell

Adipose cells (storage)

9. Intestinal Cell Resynthesis of TAGs a. Activated FA cant diffuse out of epithelial cell b. Transport to body starts with TAG re-synthesis c. Enzymes: i. Fatty acyl-CoA synthetase ii. Triacylglycerol synthase 10. Digestion Disorders
Lipid Digestion Disorders Lead to Steatorrhea (Lipid in the Stool)
Dietary Fat (TAGs) Bile Salts released through Bile Duct from Gall Bladder Release of Pancreatic Lipase Biliary Obstruction Emulsication Pancreatic Disease Lipolysis Celiac Disease (Low Intestinal Surface Area) Absorption by Intestinal Epithelia Intestinal discomfort, loss of essential fatty acids and fat soluble vitamins

10

11. Similar story for dietary cholesterol a. Hydrolysis of cholesterol esters (CEs) to allow absorption b. Resynthesis of CEs to drive absorption & prepare for transport
c. Plant sterols, sitosterols, cannot be used and are actively pumped out of epithelia into intestinal lumen

12. Chylomicrons

Apoprotein Class Lipoprotein Class A-I B-48 B-100 C-II E CETP LCAT HDL Chylomicrons VLDL, IDL, LDL VLDL, Chylomicrons, HDL VLDL, IDL, HDL, Chylomicrons (serum protein) (serum prot., ApoAI-activated)

Function Structural, LCAT Activator Structural, Remn.-R interaction Structural, LDL-R interaction LPL Activator Facilitates LDL-R recognition. Exchanges HDL CE for TAG Cholesterol Ester Formation

LECTURE 24: LIPID TRANSPORT AND FATTY ACID CATABOLISM 1. Very Low Density Lipoprotein (VLDL) a. Very similar to Chylomicrons (but made in the liver) b. Characteristic Apoprotein: ApoB100 not ApoB48 i. ApoB100 is derived from same gene as ApoB48 by RNA editing c. ApoC and E are transferred to it from HDL after release from hepatocytes 2. Intermediate Density & Low Density Lipoproteins (IDL & LDL) a. When cells need cholesterol they will express the LDL receptor on their surface b. Internalization into an endosome, fusion with a lysosome, release of contents

11

LDL-receptors on cells needing cholesterol bind ApoB100 on LDL, resulting in endocytosis & hydrolysis of CEs and apoB100. Normal half life for LDL ~3 days
Lysosomal acid lipase has cholesterol exsterase activity: hydrolyses CEs to release FA & free cholesterol (also a TAG lipase) Esterase deciency -> CE storage disease. Complete deciency -> childhood lethal accumulation of CEs & TAGs (Wolmans Syndrome )

Same process mediates degradation of chylomicron remnants bound by LRP (liver cells only).

3. LDL as the bad cholesterol

a. b. c. d. e. LDL, if not taken up rapidly, becomes oxidized and initiates a cascading inflammatory reaction Oxidized B100 no longer functions and LDL can no longer be taken up Modified LDL stimulates endothelial cells to recruit Monocytes and promotes differentiation into Macrophages which release Cytokines Macrophages ingest modified LDL, become Foam Cells & begin to die Dying foam cells form fatty streak & release metalloproteinases & growth factors that promote smooth muscle cell growth Can protect against CHD by promoting efflux of excess cholesterol HDL is anti-inflammatory; protects against the development of atherosclerosis Efflux of cholesterol from foam cells leads to a reduction in foam cell formation i. Released as an empty shell by the liver & intestine (contains only membrane components & Apoproteins A, C and E). ii. Transfers Apos C-II & E to other lipoproteins

4. HDL as the good cholesterol

a. b. c.

d. How HDL works

iii. Removes excess cholesterol from cell surfaces (via serum Lecithin Cholesterol Acyl Transferase (LCAT)) and packs into hydrophobic core
iv. Binds to Scavenger Receptor-BI (HDL receptor) on liver & adrenals for endocytosis & degradation

e. Lecithin cholesterol acyl transferase (LCAT) i. LCAT needs an activated FA to add to membrane cholesterol to turn it into a cholesterol ester
ii. Lectin + Cholesterol Lysolecithin + Cholesterol Ester 1. Takes a FA from a membrane phospholipid; converts it into something that can act as a detergent iii. ApoA-I on HDL stimulates this process

f. Cholesterol Ester Transfer Protein: HDL can also pick up triglycerols by exchanging them for cholesterol esters with chylomicrons, VLDL, and IDL
5. Characterizing lipoproteins a. Lipoproteins with a small volume (HDL) have more protein versus lipid high density b. Large complexes slowed by electrophoresis gel matrix

6. Heart Disease a. Saturated fat increases heart disease risk, but trans fats are worse i. Trans fat does increase LDL, but more importantly, lowers HDL 12

7. Genetic Defects in Lipid Transport a. Hyperlipoproteinemias: Overproduction of one or more lipoprotein Condition Frequency Lipoprot. Causes Increased Familial 1:500 LDL Defective Hypercholesterolemia (VLDL) LDL-R Fam. Combined 1:500 LDL ApoB100 hyperlipidemia overproduction Fam. Ligand-defective 1:1000 LDL ApoB binds apoB100 poorly to R 8. Fatty Acid Catabolism
a. b. c. Most dietary fat (from chylomicrons) and synthesized fat (from VLDL) is stored in adipose cells Serum albumin allows free FAs to be transported in the blood without allowing them to act as detergents Acyl-CoA activation keeps FA in the cell i. Want to be able to control when you oxidize FA
Release and Transport of FAs stored in Adipose TAGs. 1) Glucagon or Epinephrine activate cAMP-dependent protein kinase*. * Same mechanism used to activate glycogen
phosphorylase to release glucose from glycogen.

d. Activated FAs

ii. Carnitine acyltransferase I is 3) Free FAs are transported on Serum Albumin** & passively transported inhibited by Malonyl-CoA into cells for oxidation. e. Oxidation Occurs on the Beta Carbon (C3) **Limited capacity (cant handle load after fat-rich meal). Also, cant transport i. Dehydrogenase cholesterol or fat-soluble vitamins. ii. Enoyl Hydratase iii. L-3-OH dehydrogenase iv. B-keto thiolase fatty acyl CoA + acetyl CoA
1.

2) Phosphorylated Hormone Sensitive Lipase (or TAG Lipase) hydrolyzes Adipose TAGs to FAs.

Acyl-CoA activation by Fatty acyl-CoA Sythetase

Brings CoA in and bumps 2 carbons off, making acetyl CoA to feed into TCA cycle

v. repeats until FA is all AcetylCoA: [(# of carbons in FA)/2 1]cycles

9. Fatty acids are the most efficient means of energy storage a. ~4x more ATP stored per gram of fat versus glucose storage b. A 70 kg man could survive ~12 hours on glycogen reserves, ~two weeks on amino acids from muscle protein & ~80 days on fat reserves

10. Oxidation of Unsaturated and Nonsaturated FAs 11. Oxidation of branched chain FA (phytanic acids, from plants) a. Cant form the double bond if the B carbon has a methyl group Must move it to the a-carbon

13

12. Ketone bodies: soluble energy from fats that can travel freely a. The blood-brain barrier prevents albumin carrying FAs from entering b. To deal with this, the liver converts FAs to ketone bodie c. There is no human enzyme to convert Acetyl-CoA to pyruvate. Thus FAs cannot be used for gluconeogenesis
Ketone Body Formation
When carbohydrate is scarce, TCA cycle slows (due to use of OAA in gluconeogenesis). Acetyl CoA from b-oxidation accumulates in mitochondria & thiolase works in reverse.
O

Ketone Body Metabolism

(Extra-hepatic Tissues Only)
O H3C C CH2
acetoacetate

O CO O 3-keto-acyl CoA transferase

Normal TCA Rxn.

CH 2 CO O -

Thiolase CoASH

O O C C C H H S C o A 3 C 2

CoA S C CH2

C C H C o A 3 S

succinyl coenzym eA

GDP GTP
-O O C CH2 CH2 CO O succinate

2 Acetyl CoA

Acetoacetyl CoA

HMG CoAsynt synthetase HM G CoA hase Acetyl CoA + H20

Not present in liver O

KETONE BODIES
O

H3C C CH2 C S CoA

acetoacetyl coenzym eA

acetyl CoA

C C O O C H H 3 C 2

acetoacetate NADH +H+ NAD+

O H O C H C O C H H 3 C 2

HMG CoA lyase

O CoASH C H 3 C O C C H S C o A C C H 2 2 O O H

CC

O C S CoA CH 3
acetyl coenzym eA

! -hydroxy-! -methylglutaryl CoA (HMG CoA) CO2

O(Spontaneous

! -hydroxybutyrate

C C H H 3 C 3

breakdown product)

CoA SH O C S CoA CH3

acetone

(Readily transported in blood)

13. Ketoacidosis a. Ketone bodies build up in the blood

i. Occurs in extreme starvation (due to extreme depletion of OAA, arresting TCA cycle) ii. Also occurs in diabetes due to uncontrolled signal to release unneeded FAs (glucagon uncontrolled by insulin)

b. Loss of ketone bodies in the urine causes loss of associated Na+ and K+ c. This reduces the buffering capacity of blood, causing a drop in blood pH, or ketoacidosis 14. Respiratory quotient: a measure of how much CO2 is released by a given tissues per O2 consumed a. Low RQ (0.7 to 0.8): tissue is primarily using FAs and/or proteins for energy (Palmitic Acid) b. High RQ (1.0): tissues primarily using glucose or ketone bodies LECTURE 25: FATTY ACID BIOSYNTHESIS AND MODIFICATION 1. The liver can efficiently synthesize fatty acids in the cytoplasm, de novo, from carbohydrate breakdown products
Fatty Acid Synthesis Step 1: Raw Materials
FA synthesis requires Acetyl-CoA but occurs in the cytoplasm. When energy charge is high (ATP>>ADP), isocitrate dehydrogenase is inhibited, citric acid builds up and is passively transported to cytoplasm.
2 P a lm ita te

G lu c o se

P y r u v a te 3 M a lo n a te 1 N A D H O A A A c e ty lC o A 4 O A A N A D P H 5 A T P

P y r u v a te

Pyruvate Carboxylase 6
A T P

Pyruvate Dehydrogenase

A c e ty lC o A

C Y T O S O L

A T P

Citrate Lyase
C itr a te

M IT O C H O N D R IA

C itr a te

Citrate Transporter (passive)

14
Isocitrate Dehydrogenase [ATP]>>>[ADP]

Note: Pyruvate carboxylase ensures that mitochondrial OAA is not depleted.

Isocitrate

a. Citrate lyase generates cytoplasmic acetyl-CoA b. Acetyl CoA carboxylase makes MalonylCoA, a committed building block of FAs i. Active only as a polymer and only when NOT phosphorylated 1. High [citrate] indicates high [acetyl-CoA] ii. Inactive when as a monomer and when phosphorylated 1. Product inhibition (high Malonyl-CoA) 2. High Palmitoyl CoA (which Malonyl CoA is used to make) 3. Glucagon 2. Fatty Acid Synthase (FAS) a. FAS uses Malonyl-CoA, Acetyl-CoA and reduced NADPH to form FA chains b. After meal rich in fat, hormones stimulate FAS c. Enzyme exists as a head to tail dimer d. Growing FA chain is held alternatively by -SH of FAS-Cysteine residue or by FAS-linked phosphopantetheine Fatty Acid Synthase Chemistry i. Attach one molecule of Saturated acetyl CoA to FASbutyryl Acyl transferase (AT) Cysteine residue # ' Enoyl-reductase ii. Then attach a molecule of (ER) malonyl CoA to FASlinked # 2,3 trans enoyl Acyl transferase (AT) phosphopantetheine " Acyl Transferase iii. Transferase activity can (AT) & move something from the FAS-linked Dehydratase $ (DH) phosphopantetheine to Malonyl b-hydroxyl transferase the FAS-Cysteine residue (MT) Methyl from ( ! acetyl CoA iv. Heal to tail orientation is always ! the terminal % lines the molecules up end of the ! -keto reductase (KR) growing FA for transfer and chain Condensing Enzyme (COND) b-ketone condensation e. All condensation adds to the carboxyl end elongating the chain doesnt change the omega number f. Synthesis proceeds by two carbon units until 16 carbons (five more times for a total of 7 times) i. Thioesterase cleaves off C16 palmitate leaving FAS ready for another cycle g. Sources of NADPH for FA Synthesis i. Pentosephosphate pathway ii. NADPH-linked malate dehydrogenase (malic enzyme): OAA malate Pyruvate (pyruvate feeds back to mitochondria)
Dehydratase (DH)

1. Way of regenerating NADPH unique to FA synthesis 2. Have effectively converted NADH to NADPH

15

3.

Overall Requirements for FA synthesis (16:0 palmitate) a. 8 Acetyl CoA b. 7 ATP (to convert 7 Acetyl CoA to 7 Malonyl CoA) c. 14 NADPH (7 b-keto and 7 enoyl reductions)

4. Carbohydrates are converted efficiently to FAs a. Efficiency = [100%](129 + 9)/(4.5)(38) = 81% Thus, you can easily gain weight on low fat, high carbohydrate diets
5. Modifications of Fatty Acids a. Elongases: Add 2 carbon units (from Acetyl CoA or Malonyl-CoA) to carboxyl end, followed by reduction & dehydration b. Desaturases: Add cis double bonds, generally spaced by 3 carbons, in mammals never closer than w-7 c. Oxygenases: Oxidize polyunsaturated FAs to create intercellular messengers d. Hydroxylases: Add -OH to alpha carbon (C2) of some FAs used in nervous tissues

6. Fatty Acid Elongation (similar to synthesis but with different enzymes) a. Formation of the b-ketone: i. On smooth endoplasmic reticulum (SER) 2 carbons from MalonylCoA
1. Palmitoyl-CoA+Malonyl-CoAbketostearoylCoA+CO2+ CoA

ii. In mitochondrial matrix 2 carbons from AcetylCoA

1. Palmitoyl-CoA + Acetyl-CoA b-keto-stearoyl-CoA + CoA

b. Reduction to b-OH using NADPH c. Dehydration to 2,3 enoyl d. Reduction to stearoyl-CoA using NADPH e. Elongation adds to carboxyl end- does not change the omega number 7. Fatty Acid Desaturase Mechanism
a. b.

c. Double bonds generally 3-carbons apart (e.g. 20:5D5,8,11,14,17, EPA) d. Human enzymes can only desaturate at w-7 or greater (further from terminal methyl) Summary of Fatty Acid Modications 8. Essential Fatty Acids a. Precursors for all -3 and -6 FAs must be gotten in diet: i. Linolenic Acid 18:3D9,12,15 -3 ii. Linoleic Acid 18:2D9,12 -6
BIOSYNTHESIZED Palmitic 16:0 DIETARY SOURCES BIOSYNTHESIZED Palmitic Stearic 16:0 18:0 Palmitoleic 16:19 Stearic 18:0 Oleic 18:19 Oleic 18:19 18:26,9 -9 18:26,9 -9 DIETARY Linoleic 18:29,12 SOURCES

Takes molecule from saturated to cis-unsaturated double bonds in SER On SER: uses molecular oxygen, NADH and cytochrome b5 to yield a double bond, H2O and NAD+

! -Linolenic

18:39,12,15 ! -Linolenic

b.

Sources: i. Linoleic acid - 20-80% of almost all plant oils ii. Linolenic acid - Linseed (61%), canola, soybean, walnut (~10%); Other -3s - Herring oil (~20%)

Palmitoleic 16:19

Linoleic 18:29,12 #-Linolenic 18:36,9,12 #-Linolenic

18:39,12,15

PG 1 16:2 6,9 PG1 -9 6,9 16:2 PG 2

6,9,12 18:3 Dihomo#-linolenic 20:38,11,14 Dihomo-#-linolenic 20:38,11,14 Arachidonic 20:45,8,11,14 Arachidonic -6 20:45,8,11,14 Eicosapentaenoic 20:55,8,11,14,17 Eicosapentaenoic 20:55,8,11,14,17 -3 PG3
Prostaglandin class created PG from it 3

9. Formation of Triacylglycerol (Neutral Fat) a. Triacylglycerol synthases: glycerol-3-P + 2 -9 acetyl-CoA phosphatidic acid diacylglycerol + acetyl-CoA triacylglycerol b. Can also turn dietary fat into 2-Monoacylglycerol via pancreatic lipase, and that into triacylglycerol
10. Low carbohydrate, high protein (LCHP) diets a. Why it might work: i. Glucagon stimulates FA released from adipose ii. Ketone bodies from FAs increase mild ketosis may curb appetite iii. Protein intake may provide enough AAs for gluconeogensis, no need for muscle breakdown iv. Without DHAP from glycolysis, TAG synthesis in adipose is low

-9 -7

PG2

-6

-3

16

11. Hormonal Appetite Controls: a. Leptin, released by adipose tissue, suppresses food intake by altering the hormones released from the hypothalamus; downregulates FA synthase b. In mice, absence of leptin production leads to extreme adiposity (obesity)
12. Fat free diets didnt produce fat free people a. Obesity: BMI of 30 or higher b. The relative risk of diabetes increases by ~25% for each additional unit of BMI over 22 kg/m2 c. The risk of CHD doubles at BMIs of 25 to 28.9, and triples at BMIs of 29 or greater (relative to BMI of <21) d. High BMIs correlate with i. Increased cholesterol levels; Decreased HDL levels; Hypertension (high blood pressure)

LECTURE 26.

SYNTHESIS AND FUNCTION OF MEMBRANE LIPIDS

1. Lipids are the basis for biological membranes: Hydrophobic core does not allow charged protons to get through; maintains concentration gradient necessary for ETC 2. Glycerophospholipids a. Spontaneously form bilayer vesicles in aqueous solution

3. Cardiolipin: A double phospholipid common on mitochondrial membranes; two phosphatidyl groups linked by a glycerol 4. Nature of attached FAs important for PL function a. Common Lecithin: 1 stearoyl, 2 oleoyl-phosphatidyl choline i. Most abundant human membrane lipid ii. Unsaturated FA on C2 lowers MP fluid membranes b. Lung Surfactant: 1,2 distearoyl-phosphatidyl choline (2 saturated FAs) i. Reduced fluidity important for coating air-water interface, preventing alveolar collapse. A deficiency can lead to respiratory distress syndrome in premature infants. 17

5. Formation of Glycerophospholipids Starts with Phosphatidic acid (PA) or DAG a. Use some of the same intermediates used in PG synthesis b. DAG vs. Head Group Activation i. DAG used for: Inositol (PI), Glycerol@C1(PG), PG@C3 (cardiolipin) ii. Head group used for: Ethanolamine (PE), Choline (PC)
General Mechanism for Head Group Addition
OH
DAG

Glycerophospholipid Interconversions
SAM (S-adenosyl methionine) is used as a methyl donor in several processes.
Methylation 3xSAM

HO-Alc.
Head Group

OO-P-O-Alc. O
Phospholipid

Step 1: CDP activation of one hydroxyl.

R1-OH ATP ADP kinase OR1-O-P-OH O

O- OR1-O-P-O-P-O-Cytidine O O
(R1-CDP)
exchanges with ethanolamine

CTP PPi cytidyltransferase

Step 2: Second -OH displaces CMP to give phosphodiester O R1-O-P-O-R2 O

.. R2-OH OO-P-O-Cytidine O
(CMP)

Only mechanism in humans to form PS.

6. Plasmalogens: Glycerophospholipids with Fatty Alcohol in Ether Linkage at C1 (instead of an ester-linkage) a. Synthetic pathway differs from that for ester-linked PLs b. Abundant plasmalogens in mitochondrial inner membrane may resist oxidative damage that would hydrolyze the ester bonds in normal PLs c. E.g. Activates platelet secretion & alters membrane permeability 7. Phospholipases (PLs) Phospholipases a. PLAs in many venoms: lysophospholipids (missing one FA) act as hemolytic 1) Release PUFAs PLA PLA for prostanoid formation detergents O C H O C R O 2) Rearrange FAs (e.g. to give 8. Modified Fatty Acids as Intracellular Signals RC O C H 1-palmitoyl, 2-oleoyl in O + a. Certain stresses induce Phospholipase A2s lecithin) C HO PO C H C H N ( C H ) O to release long-chain PUFAs from 3) Lysophospholipids act phosphatidyl choline as detergents (venoms) membrane phospholipids PLC PLD
2 1 2 1 2 2 2 3 3 -

i. E.g. Arachidonic acid & other long-chain 3 or 6 PUFAs, such as EPA -eicosopentaenoic acid

b. Cyclooxygenase gives rise to Intracellular 2nd messengers. prostaglandins & thromboxanes (shortrange intercellular signals to promote pain, fever, thrombosis, inflammation) i. Cyclooxygenase removes 2 double bonds, & that subscript shows # of double bonds remaining (is always 2 less than the original PG) ii. 20:4 arachidonate gives rise to PG2 (endothelium) & TX2 (platelets) series; 20:5 eicosopentaenoate gives rise to PG3 series iii. Aspirin & other anti-inflamatory agents block activity of Cyclooxygenase c. Lipoxygenases generate leukotrienes, important for immune responses i. # of double bonds is unchanged by 5-lipoxygenase reaction (does not reduce double bonds) 18

d. DAG and IP3 released from PIP2 act as intracellular signals/second messengers i. Kinases phosphorylate PI to PIP and PIP2 ii. Extracellular Signal binds membrane-spanning receptor to activate Phospholipase C enzyme which cuts PIP2 into: 1. DAG Fatty acid chains in inner lipid monolayer of plasma membrane and activation of protein kinase A 2. IP3 activation of Calmodulin Kinase 9. Sphingolipids: membrane lipids based on spingosine, not glycerol a. FA attached through an amide (rather than an ester) forms a ceramide b. Sphingomyelin in myelin sheath with C24 FAs provides electrical insulation i. Only sphingolipid that is a phospholipid (contains phosphate) c. Glycolipids: Glucocerebroside (or Galactocerebroside) i. Addition of more sugars or sulfate makes Sulfatides, Globosides & Gangliosides 1. e.g. GM2, Tay Sachs ganglioside 2. Gangliosides and globosides are markers used in cellular recognition (such as the blood group antigens) d. Normal Sphingolipid Breakdown Pathways
i. Membrane lipids are constantly pulled from the surface into endosomes ii. Some are reused, others are broken down in lysosomes & their components recycled

iii. Each sugar linkage in the glycosphingolipids (cerebrosides, globosides and gangliosides) requires a separate enzyme for removal e. Lysosomal storage diseases
i. Many sphingolipids are most abundant in the brain ii. Genetic defect in sphingolipid breakdown enzyme leads to slow accumulation of lysosomes full of partial breakdown products iii. This damages nerve cells and usually leads to progressive neurodegeneration and death young iv. Screening for Hexosamidase A activity & gene mutations in at-risk parents & fetuses has reduced frequency of Tay Sach

10. Lipids as membrane anchors for proteins a. Attaching lipid to a protein pulls a cytoplasmically soluble protein to the membrane
b. c. d. e. f. Allows a cell to decide, under various signaling condition, whether to keep the protein in the cytoplasm, or attach it to the membrane (versus integral membrane proteins which will always be in the membrane) Palmitate (16:0) palmitoylation C14 myristate myrystoylation C15 isoprenoid farnesine farnesylation Modified phoshatidyl inositol GPI anchor

Lecture 27. STEROIDS AND FAT-SOLUBLE VITAMINS 1. Cholesterol

a. Important membrane lipid: reduces fluidity/permeability; cholesterol rafts b. Important derivatives: Bile salts, steroids hormones, Vitamin D, coenzyme Q c. Sources: i. Animal products in diet; Dietary cholesterol goes to liver as CEs in chylomicron remnants ii. Endogenous synthesis (primarily by liver, but also skin, intestines & kidney)

19

2. Cholesterol Synthesis uses Cytoplasmic Acetyl-CoA & NADPH

a. Similarly to FA Synthesis: i. Acetyl CoA from the Citrate Lyase pathway ii. NADPH from Pentose Phosphate pathway & NADPH-linked Malate Dehydrogenase

b. Cytoplasmic Acetyl-CoA is converted to HMG-CoA (Essentially the same as mitochondrial HMG-CoA formation to make ketone bodies) c. HMG-CoA converted to mevalonic acid i. HMG-CoA reductase is the major regulated step Cholesterol synthesis Cytoplasmic Acetyl-CoA (from citrate via citrate lyase) ii. Inhibited by: (signals that tell Thiolase Acetoacetyl-CoA the body to release energy) HMG-CoA synthase Major site of 1. Free cholesterol HMG-CoA regulation. HMG-CoA reductase (uses NADPH) Transcription & 2. Phosphorylation Mevalonate activity inhibited (3 ATPs, loss of CO2) (indirectly controlled by cholesterol. Isopentenyl-PP (5 carbon, isoprene) Farnesylated Inhibited by (Head to tail) by glucagon & proteins, dolichol glucagon via Gerenyl-PP (10 carbon) (sugar carrier), phosphorylation (Head to tail) epinephrine: cAMP Coenzyme Q by cAMPFarnesyl-PP (15 carbon) (electron carrier) dependent protein dep. PK activates a (Head to head, uses NADPH) kinase (indirectly). Squalene (30 carbon) second kinase) 3.5 hr half life. Squalene monooxygenase (uses O2 & NADPH) Squalene epoxide 3. Statin drugs (which
decrease the levels of circulating cholesterol by mimicking substrate/product and bind to the active site of the enzyme)
Cyclase Lanosterol (rst sterol) (other reactions, loss of 3 carbons) 7-dehydrocholesterol (nal reduction of double bond) Cholesterol (27 carbon)
UV Vit D.

d. Mevalonic acid (6C) is activated to form IPP (5C) e. Squalene is formed by condensation reactions of isoprene units i. Head (of DPP- an IPP isomer) to tail (of IPP) condensations, followed by
reductive head to head condensation of fanesyl pyrophosphate (15C)

f. Cyclization and maturation of cholesterol

i. Squalene Monooxygenase makes a Squalene epoxide ii. Cyclase holds the epoxide in the right orientation so it can form the four-ringed sterol structure (Lanosterol, 30C) iii. Demethylation followed by rearrangement forms cholesterol (27C)

3. IPP Condensations Yield Other Important Isoprenoids a. Ubiquinone uses isoprene units to make its hydrophobic tail units to stick into the membrane b. Dolichol (a sugar carrier) 4. Bile salts (bile acids) are synthesized in the liver from cholesterol to aid dietary fat absorption a. Emulsion: hydrophilic shell around the molecule in water allows pancreatic lipase to work b. 7- hydroxylase enzyme (7 position is where enzyme starts makes bile salts) 20

5. Enterohepatic Circulation: Preventing Excessive Cholesterol Loss

a. b. c. d. Bile acid & Cholesterol made in the liver Released by gall bladder to emulsify dietary fats Endogenous and exogenous cholesterol taken up and returned to liver as chylomicrons Active (energy requiring) uptake of bile salts, for return to liver

6. Control of Circulating Cholesterol and Heart Disease a. When cholesterol is too abundant, levels of circulating LDL increase, which is a root cause of atherosclerosis b. LDL levels can be controlled by: i. Ensuring proper LDL uptake by LDL-Receptors ii. Decreasing synthesis (HMG-CoA reductase inhibitors) iii. Decreasing dietary cholesterol iv. Promoting excretion of excess cholesterol (as bile salts)

7. Steroid Hormones

21

8. Fat Soluble Vitamins a. Isoprenoids required as cofactors or signaling molecules that cannot be synthesized de novo or for which de novo synthesis may not be sufficient (e.g. Vitamin D) i. A (light reception & hormone) ii. D (hormone controlling calcium absorption) iii. E (antioxidant) iv. K (important cofactor for blood clotting) b. "The FAT cat is in the ADEK (attic)": Fat soluble vitamins are A,D,E,K
c. Are hydrophobic enough that they need to be transported in the blood using lipoproteins; Not soluble in water- cannot be easily excreted

d. Great excess (>10x RDA) can be toxic, especially for D & A

i. Excess of fat soluble vitamins are stored in adipose and can cause health issues

9. Vitamin A a. The three faces: similar structure, differing only at one (terminal) carbon b. Retinal: aldehyde i. Bound to opsin, forms rhodospsin ii. Light induces a cistrans isomerization, causing dissociation and the conformational change in opsin is the first signal in vision 1. Vitamin A deficiency results in night blindness c. Retinol: alcohol d. Retinoic: carboxylic acid i. Hormone important in growth and differentiation; Activates transcription factors of the steroid receptor class 10. Vitamin D
a. Penultimate intermediate in cholesterol biosynthesis has a single double bond at the 7 position

Only occurs in the skin; can synthesize with sufficient UV exposure Activates Vitamin D hormone receptor transcription factors Promotes intestinal Ca++ absorption and bone formation Deficiency and Toxicity both promote demineralization of bones a. Deficiency: poor calcium absorption, bone demineralization and rickets (in children) or osteomalacia (in adults) b. Toxicity: excess serum calcium: renal stones & metastatic calcifications; calcium taken from bonesdemineralization 11. Vitamin E a. Important antioxidant; terminates free radical oxidation of unsaturated FAs by taking the free radical onto itself 12. Vitamin K a. Essential co-factor for blood clotting; allows the -carboxy-glutamate to be formed, forming a vitamin K epoxide in the process which can be recycled back to vitamin K hydroquinone b. Blocking this process results in internal bleeding i. Vit K Epoxide reductase inhibitors (dicourmarins like warfarin & coumarol) cause uncontrolled bleeding ii. Poisons at high doses, lower doses can treat thrombosis (too much blood clotting) c. Vit. K deficiency can cause hemorrhagic disease in premature infants 22

b. c. d. e.

13. Integration of Lipid Metabolism a. Hormonal Controls: i. Glucagon or Epinepherine (blood glucose is low, cells need energy); inactivates all the new biosynthesis; Signaling cascade causes protein phosphorylation by cAMP-dependent protein kinase (PKA) 1. Inactivates Acetyl-CoA carboxylase 2. Inactivates HMG-CoA reductase (inhibit cholesterol synthesis) 3. Inactivates glycogen synthase 4. Activates hormone sensitive lipase (release FA) 5. Activates glycogen phosphorylase (release glucose from glycogen stores) ii. Insulin bound to a receptor tyrosine kinase has multiple opposing effects (not as simple as removal of phosphates added by PKA) b. Intracellular regulation by small molecules: i. Cholesterol: 1. Inhibits HMG-CoA reductase ( increases LDL uptake) 2. Decreases LDL Receptor level 3. Increases ACAT activity (form CE deposits and lipoproteins) ii. Fatty acyl CoA and Malonyl CoA inhibit Acetyl CoA carboxylase iii. Citrate activates Acetyl CoA carboxylase iv. Malonyl CoA inhibits Carnitine acyl transferase I (CATI) (decrease B oxidation) c. Other limitations: i. CHOs needed for G3P in adipose to make TAGs ii. CHOs needed for OAA to make citrate for FA & cholesterol synthesis iii. Liver lacks 3-ketoacylCoA transferase (makes but does not use KBs)
1. RBCs also cannot use ketone bodies, only can use glucose

iv. Cant get glucose from FAs (no enzyme to make pyruvate from Acetyl-CoA); Can only use the glycerol backbone from fat to make a small amount of
Pancreas viaLipase Mouth glucose gluconeogenesis Intestinal Wall Phospholipids Cholesterol Phospholipids Gall Bladder Bile Salts

Use of Ketone Bodies

Bile Acids Cholesterol Phospholipids AcCoA Protein FA TAG KB Citrate Liver Glucose ATP Glucose Blood

Summary & DAG Fat MAG Catabolism TAG of 2-MAG TAG FA LCFA Transport Protein
Glycerol Chylomicron Remnants Chylomicrons VLDL IDL FA-Albumin LDL LPL KB ATP Glucose FA ATP FA Glucose LPL HSL

Intestinal Lumen

Use of Fatty Acids MCFA

Glycerol

HDL

TAG 3-GP Adipose Tissue

Brain

KB

Extra-Hepatic Tissue

FAs & KBs cant do everything: Erythrocytes (RBCs) use anaerobic glycolysis for energy and NADPH formation.

23

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