BICH exam 3

. (a) What accounts for the difference between the ATP yield of each of these fatty acids? (b) What would be the difference in oxidation if the unanticipated isomerization occurred?

(a) Double bonds in linoleic acid cost an FADH2 equivalent in two rounds when enoyl CoA isomerase is used in place of acyl-CoA dehydrogenase (an FADH2) for odd numbered double bond and use of 2,4-dienoyl CoA reductase followed by 3,2-enoyl CoA isomerase (a NADH) because of the presence of the even numbered double bond (b) About 20% of the time, 3,2-enoyl CoA isomerase will react with a beta oxidation substrate that is a Δ 2 and converts it to a Δ 3 double bond that is not. Notice in the blue box, this reaction is reversible by enoyl-CoA isomerase. Thus, the action of 3,5-2,4-dienoyl CoA isomerase is required to shift the double bond, producing a substrate for 2,4-dienoyl CoA reductase followed by 3,2-enoyl CoA isomerase. Thus, this added step costs an additional NADPH 20% of the time.

β-Oxidation of FAs

(begin with even number carbon, saturated FAs) • Fatty acyl-CoA (activated form of fatty acids), proceeds through oxidation (loss of electrons) in which 2- carbon units are removed, producing acetyl-CoA and the remainder of the fatty acyl-CoA molecule until all possible 2-carbon units have been converted to acetyl Co-A. 1. Formation of the trans-double bond - catalyzed by acyl-CoA dehydrogenase - fatty acyl-CoA is converted to trans-Δ 2 -enoyl-CoA - 2 e from the flavin end up as part of H2O at the end of the electron transport chain * double bond between alpha and beta carbon - transfer to Q cycle/CoQ-> ETC through complex 3 - hydride transfer forms FADH2 *This is a flavin containing enzyme that participates in redox reactions, thus the forming of the double bond is an oxidation step and the flavin is reduced. The electrons from the flavin are transferred through a series of electron transfers involving CoQ, ultimately ending up as part of H2O at the end of the electron transport chain. Thus, electrons from β-oxidation produce about 1.5 ATP per electron pair generation in the formation of the trans-double bond (remember FADH2 equates to 6H+ and 4H+ is needed to generate an ATP ready for use in the cytoplasm).

Thiolase - Mechanism

** β-ketoacyl-CoA thiolase (thiolase) catalyzes a Claisen ester cleavage reaction. Before this mechanism is understood, a few points about Claisen esters formed through condensation reactions and the significance of chemistry available with a β-keto ester. 1. As a reminder, a Claisen condensation reaction produces a carbon-carbon bond between two esters and results in in a β-keto ester or a β-diketone. 2. Thus, the product is the original ester with an acyl group added ( i.e. an acylation reaction has occurred) - which is what β-ketoacyl-CoA is - an acylated β-keto ester. In β-oxidation, thiolase breaks the carbon-carbon bond in β-ketoacyl-CoA. The active site of thiolase contains a cysteinyl residue that functions as a nucleophile (step 1) to generate an enzymethioester carbanion intermediate, stabilized by the thioester. The carbanion intermediate (step 2) is short-lived as the collapse of the tetrahedral intermediate results in the cleavage of carboncarbon bond, producing fatty acyl-CoA bound to enzyme and the first product acetyl CoA, which is formed by a proton donated from an acidic group on the enzyme thiolase (step 3). The enzyme thioester intermediate (enzyme utilizes covalent catalysis) reacts with CoA, as the sulfhydral serves as a nucleophile forming a second tetrahedral oxyanion intermediate (step 4). The collapse of the oxyanion intermediate releases the second product, acyl-CoA from thiolase

The Ketone Zone: Blood Ketones

** graph; Under starvation conditions, gluconeogenesis will deplete the TCA cycle intermediates trying to make glucose to keep up with the body's demands, thus acetyl-CoA only has a single path for use in energy production - ketogenesis. Further, under starvation, fat levels are depleted and levels of malonyl-CoA are low. As high levels of malonyl-CoA level inhibit the carnitine shuttle and thus inhibit fatty acid degradation, in the absence of malonyl-CoA, carnitine shuttle is highly active, shuttling acetyl-CoA to the matrix for the purpose of producing ketone bodies. In an individual suffering from starvation, there is no longer fat available for producing acetyl-CoA. So what is the source of acetyl-CoA? Degradation of amino acids, the body begins breaking down muscles under starvation in order to supply the body with acetyl-CoA via the production of ketone bodies. KB- brain doesnt have enough energy GNG- liver glucose-> fats-> proteins (muscle)-> ketoacidosis and less CAC intermediates

Ketone body degradation

**Brought into cells by the monocarboxylate transporters (MCTs) - recognize KB - acetoacetate is the most beneficial for cells to metabolize - any B-hydroxybutyrate is converted to acetoacetate ( red to ox) and NAD+-> NADH - 3-ketoacyl-CoA transferase-> acetoacetyl-CoA (HS-CoA + GTP Succinyl-CoA Synthetase (TCA cycle) ) - source of CoA from succinyl-COA - happens in TCA cycle producing a GTP - product of GTP favorable because of coupling of loss of thioester bond. CoA degradation- bypass succinyl CoA TCA step and use succinyl CoA for make acetoacyl-CoA and succinate thiolase- last sep of B-ox to create acetyl-CoA -> fates-> energy * dec energy ( fasting) 1. adipocytes allow for FA release to cells 2. liver utilized B-hydroxybutyrate as an energy source ( fat burning mode) to make ATP - ATP important to keep up with GNG to supply glucose to brain and KB biosyn so other cells can use KB as energy source * pathway happen in other ells -not in liver becasue then other cell wont have access to KB --Liver lacks 3-ketoacyl CoA Transferase -Liver mitochondria lack this enzyme; starvation causes the brain and some other tissues to increase the synthesis of βketoacyl-CoA transferase, and therefore to increase their ability to use these compounds for energy - B-hydroybutyrate -> acetoacetate are released for transport by liver to blood stream

Chylomicrons

- Composed of dietary TAGs, cholesterol and cholesteryl esters ( thats why its exogenous) - Chylomicrons are formed in intestinal mucosal cells after absorption and are highly soluble for release into the intestinal lymph for transport through lymphatic vessels to large veins for delivery throughout the body as part of the exogenous pathway * chylomicrons and VLDL deliver energy rich TAGs to cells in the body largest SA, decprotein

• VLDL

- Composed of dietary TAGs, cholesterol and cholesteryl esters - The remnants of chylomicrons are related to VLDL • VLDL is biosynthesized in the liver using cholesterol-rich chylomicron remnants and triacylglycerols - Transport TAGs and FAs from liver to tissues - VLDLs are released to the blood stream as part of the endogenous pathway.as part of the endogenous pathway. low density lipoproteins 1. TAGS for energy 2. FA for membrane integrity maintenance

• Emulsification

- Digestion is greatly aided by emulsification, the breaking up of fat globules into much smaller emulsion droplets. - Bile salts and phospholipids are amphipathic molecules that are present in the bile. ( OH group is polar in biles salts) - Bile salts help solubilize fat droplet into micelles * fat globules are target for liapses which are a class of esterases that hydrolyze glycerol backbones of TAGS usually at C1 or C3 polar interacts with phillic solvent and phobic with fat globule smaller drops= micelles

• IDL

- IDL is formed from the remnant of VLDL after TAGs are removed. - The "transition particle" between triacylglycerol transport (VLDL) and cholesterol transport (LDL) • Some IDL is reabsorbed by the liver, other IDL will pick up cholesteryl esters from the HDL to form more LDL. - IDLs operate as part of the endogenous pathway. ** VLDL-> IDL-> LDL-> HDL

ApoE

- Multiple isoforms that associate with chylomicrons and chylomicron remnants - Found in VLDL and IDL because of conversion of lipoproteins in the endogenous pathway - Enhance binding to LDLreceptors primarily in liver, but also in central nervous system • Recently defects in Apo4 have been correlated to increased chances of developing Alzheimer's Disease **as VDL is converted to LDL via IDL it remains bound to IDL, multiple copies of ApoE allow IDL to bind to the LDL receptor with a high affinity.When IDL is converted to LDL, the ApoE leaves the particle and only the ApoB-100 remains. Thereafter, the affinity for the LDL receptor is much reduced. ApoE receptors, though typically found in the liver, are also present in the central nervous system and facilitate the tranfer of cholesterol to neuron good apoE is broken down and solution with peptides is cleared out of brain - defects causes ApoE to interact with amyloid precursors and phobic precursors-> plaques which kill off healthy neurons leading to alzheimers

What about other FAs?

- Unsaturated FAs • Monounsaturated - ie: Oleic acid • Polyunsaturated - ie: Linoleic acid - Odd-numbered FAs • Producing propionyl-CoA 22 **Need additional enzymes!!

Diabetic Ketoacidosis

- absence of insulin ( type 1; autoimmune disease where pancreatic cells destroyed) - dec insulin and no response to high BS ( type 2) - inc blood glucose because cells cant respond to glucose without insulin - inc glucagon, in GNG and glycogen breakdown, fats-> KB in liver , in KB - fat cells release FA-> glycerol taken up by cells -> muscle cells - Amino acids not needed by proteins are broken down and utilized as carbon skeletons for glycolysis intermediates plan A= glucose ( fastetes) plan B= fats ( KB ( fast in liver), FA chain ( energy rich and longer) plan C= amino acids ***Diabetics (Type I) are individuals who suffer from an autoimmune disorder whereby pancreatic β-cells are destroyed and the body has no way of producing insulin, although the insulin receptors and the body's ability to respond to insulin if it were produce are possible - they lack the response insulin offers signaling high blood glucose, so cells can take up glucose for use or in the case of fat cells, halt fatty acid degradation and the carbohydrate source of energy is present. Diabetics (Type II) suffer from lack of response for insulin binding its receptor, often attributed to genetic factors or environment such as poor diet/exercise combination, and experience DKA in final stages of uncontrolled diabetes. These individuals lose hormonal regulation for the body to respond to high blood glucose levels, whereby in the presence of insulin binding to receptors, glucose would be taken up by the liver or muscles cells for the purpose of energy production and insulin-binding inhibits fatty acid degradation in fat cells. Instead, the binding of insulin to receptors is over-loaded so to speak, the response by the binding event does not produce the intended response even though insulin is being produced. As a consequence, these individuals suffering from diabetes are marked by unique metabolic levels of ketone bodies, which are produced in significantly higher amounts to compensate for energy starvation of the cell. As the cells are not getting adequate supply of glucose, glucose is constantly being made in the liver using gluconeogenesis, causing a complete depletion of the TCA cycle intermediates. Levels of malonyl-CoA (which we will see is starting material for fatty acid biosynthesis) is diminished This is the body's way of compensating, switching to fatty acid metabolism and use of ketone bodies when the levels of carbohydrates for cellular catabolism are low inc fat breakdown, inc KB, inc [H+], dec pH

examples

1. C6 sat FA -2 FADH2, 2NADH2, 3 ACetyl Coa 2. cis double bond - 2, NADH, 1 FADH2, 3acetyl coA * trans double bond cost of 1 FADH2 3. cis double bond one carbon over - 2 FADH2, use NADPh, 1 NADPH for payback and 1 net NADH, 3 acetyl CoA

Biosynthesis of Other Lipids

1. TAG energy reservoir 2. membrane 3. signaling - sphingmyelin- choline or ethanolamine; cerebroside v ganglioside - monosacchride v oligosacchride ** disease states occur in lipids - glycerophospholipid- 2 FA, glycerol and 1 phos tay sachs- improper breakdown og gangliosides ( CNS) - early symptoms and death

FA synthesis is largely the reverse of βoxidation

1. compartmentalization 2. tagged 3. e flow ( red -> FA, NADPH e carrier-> NADP+) 4. stereospecificity (L-> D) 5. C 2 unit -acetyl CoA v fatty ACP AMP v ACP diff between carrier proteins

β-Oxidation of FA pt.2

2. Hydration of the double bond - catalyzed by enoyl-CoA hydratase - trans-Δ 2 -enoyl-CoA is converted to 3-Lhydoxyacyl-CoA - H2O adds across a double bond, similar to fumarase of TCA cycle OH ends up with B carbon 3. NAD+ -dependent dehydrogenation - catalyzed by 3-Lhydroxyacyl-CoA dehydrogenase - 3-L-hydroxyacyl-CoA is converted to βketoacyl-CoA and NADH is formed - B picks up H+ and hydride transfer to NADH-> complex 1-> ETC-> 10H+-> 2.5 ATP - create B-carbonyl group which is the goal of B-oxidation 4. β-ketoacyl-CoA is cleaved by a reaction with the thiol group of coenzyme A - catalyzed by β-ketoacylCoA thiolase (sometimes just called thiolase) - β-ketoacyl-CoA is converted to acetyl CoA (2-carbon unit for the TCA cycle) and the remaining fatty acyl-CoA that is 2-carbons shorter - nuc attack by CoA - deposit S group at B carbon - tetradduct -> oxyanion - drive product release to gain acetyl-CpA - resonance stabilized structure to pick up H+-> acetyl coA substrate for CAC - facilitate thioester bond formation - CopA nuc attack-> break covalent bond and regenerate enzyme for next round of AT - END 2C shorter than FA at beginning *The ketone function on the β carbon (C-3) makes it a good point for nucleophilic attack by the thiol group (-SH) of CoA, and the acidity of the α carbon makes the terminal -CH2-CO-S-CoA (acetyl CoA portion) a good leaving group, facilitating breakage of the α-β bond.

Ketone Bodies

A special source of fuel and energy for certain tissues • Some of the acetyl-CoA produced by fatty acid oxidation in liver mitochondria is converted to - Acetone - acetoacetate - B-hydroxybutyrate • These are called "ketone bodies" • Source of fuel for brain, heart and muscle • Major energy source for brain during starvation • They are transportable forms of acetyl-CoA! (- acetoacetate - B-hydroxybutyrate) inc in liver because of B-ox of FA - ketone bodies are these three structures - typical brain uses glucose; if cant be met because of GNG= starvation ( atypical) and uses ketone bodies The brain is about 2% of the body's mass yet it consumes on average about 20% of the body's energy. The brain consumes glucose at much faster rates, though only under times of starvation, ketone bodies can serve as the brains alternate fuel source in the absence of glucose as they can cross the blood-brain barrier

Acetyl- CoA and the Tricarboxylate Transport System

Acetyl-CoA serves as the building blocks for malonyl-CoA. - Acetyl-CoA is generated in the mitochondrion: 1. pyruvate dehydrogenase complex or ( sugar and AA)-> pyruv 2. beta oxidation • Acetyl-CoA must therefore be transferred out of the matrix to the cytosol and is transported using the tricarboxylate transport system, where acetyl-CoA is moved in the form of citrate. • Citrate is formed by the addition of acetyl-CoA to oxaloacetate in the mitochondria; citrate is transported out via the tricarboxylate transport system, whereby ATP-citrate lyase catalyzes the conversion of citrate to oxaloacetate and acetyl-CoA in the cytosol in a reaction that resembles the reverse of citrate synthase. -Oxaloacetate can then be converted to malate by malate dehydrogenase, then malate is decarboxylated by the malic enzyme to form pyruvate, which can then reenter the mitochondrial matrix. The acetyl-CoA liberated from the ATP-citrate lyase reactions becomes the source for biosynthesis of malonyl-CoA Citrate + CoA + ATP → Acetyl-CoA + OAA + ADP + Pi inc acetyl CoA inc matrix inc E - citrate synthase - inc citrate in matrix dec TCS inc E-> moved to cyto inc citrate inc citrate lyase - invest ATP - acetyl-CoA 1. red biosyn- makes NADPH -pyruv H+ symporter inc OXA-> acytl CoA-> citrate 2. carbon skeletons To synthesize lipid, must export acetyl-coA to cytoplasm (via citrate) Only "new" enzyme IS ATP-citrate lyase

b-Oxidation in Peroxisomes

Animals • Shortens very long FA (>22C) • Carnitine acyltransferase and a transferase independent of carnitine that recognizes longer chain acyl groups are present in the peroxisome for transporting fatty acids into the peroxisome • Sent to mitochondria for further degradation Plants: glyoxysomes (peroxisome) method of FA β-oxidation - used by seedling to produce sugars synthesized from fats until it is mature enough to produce them by photosynthesis using light - glyoxylate cycle ( acetyl coa lost as CPO2 so must bypass CO2 evolving steps) First reaction is different • Acyl-CoA oxidase: FA-CoA + O2 enoyl-CoA + H2O2 • Does not feed into e- transport chain - Peroxisomes don't have an e- transport chain 2 (1.5 ATP) less ATP because of loss of 1 FADH2 - Catalase is present H2O2 → H2O + O2 Second enzyme and third enzymes are the same • Peroxisomal enoyl Co-A hydratase • Peroxisomal thiolase - does not recognize substrates with eight-carbons or less, thus the incomplete oxidation of FAs. - use ULFA transports v carnitine transporters to mito why does this happen? flux> efficiency *very long FA use perioxizomes-> break down into smaller FA to be fed into mito so most efficient FA in mito that will allow for max flux - long FA from diet ( fed state) FA oxidation in peroxisomes: • Produce 1.5 fewer ATP per 2-C units than does the mitochondria • Further, the enzyme catalase is present to address the generation of H2O2 , utilizing it as a substrate for conversion to molecular oxygen and water Subfamily of the ATP-binding cassette (ABC) transporters - not dep on carnitine • Shorten very long chain fatty acids • Transport is via an ALD protein which is not dependent on carnitine • 1 st enzyme is acyl-CoA oxidase • Catalase breaks down the hydrogen peroxide to H20 and O2 • Peroxisomal thiolase is inactive with acyl-CoAs of C8 or less *deficiency of the ABCD1 gene is linked to adrenoleukodystrophy or ALD

Which enzyme is being described by the following characteristic? (4 - 7 each) 4) Transfers acyl portion of fatty acid to coenzyme A in the mitochondrial matrix. I 5) Catalyzes fatty acid activation, condenses fatty acids with Coenzyme A with simultaneous hydrolysis of ATP to AMP and PPi. 6) Cleaves beta-ketoacyl-CoA with addition of Coenzyme A 7) Enzyme in peroxisomes which converts fatty acyl-CoA to trans-Δ2 -Enoyl-CoA

Answer: Carnitine palmitoyl transferase II Answer: Acyl-CoA synthetase . Answer: β-ketoacyl-CoA thiolase (KT) Answer: Acyl-CoA oxidase (ACOX)

A fatty acid with 24 carbon units can start its beta-oxidation in mitochondria.

Answer: False / Fatty acids composed of over 22 carbons should be first cleaved in peroxisome in animals or glyoxysome in plant

HSL is activated by hormones which in turn activates receptors in liver and adipose tissue

Answer: True / Release of hormones, such as glucagon or epinephrine, triggers HSL which will produce free fatty acids from stored fats.

Workouts and Nutritional Ketosis

Carbohydrates • Anaerobic exercise is characterized by shorter bursts of energy, - weight training or - high-intensity interval training. - **Carbohydrates are the primary fuel for anaerobic exercise - gluc-> pyruv-> lactate (2 ATP) Fats • Aerobic exercise, also known as cardio exercise, is anything that lasts over three minutes. - Lower intensity, steady-state cardio is fat burning, - **Making it very friendly for the keto dieter *TARGETED KETOSIS: A good rule of thumb is to eat 15-30 grams of fast-acting carbs, such as fruit, within 30 minutes before your workout and within 30 minutes after. This will ensure you provide your muscles with the proper amount of glycogen to perform during the training and also recover. It allows the carbs to be used exactly for this purpose and prevent any risk of leaving ketosis. --fast acting carbs= simple sugars (Apple)

COX-2 Inhibitors: Specific

Celebrex and Vioxx COX-2 produced in high levels in response to pain, specific inhibitors called coxibs became important for treatment of inflammatory diseases like arthritis.

Carnitine carries FA into mitochondria for oxidation (degradation)

Do we want this to happen all the time? - no; regulated to respond to high/low energy states - B- oxidation v. FA synthesis - dec E= acyl CoA brought in by carnitine to fuel ETC an PMF-> ATP synthesis - high E= extra carbons are regulated by malonyl-CoA - carbon skeletons exported from CAC from cycle inte4rmediates to matrix-> CAC-> malonyl-CoA -> FA synthesis inc malonyl-CoA; inhibits carnitine and inc E and inc FA synthesis Carnitine acyl-transferase I is inhibited by malonyl-CoA malonyl-CoA is used for FA synthesis

elongation

Elongation beyond 16 C on ER and in mitochondria Substrate is acyl-CoA rather than acyl-ACP FA elongation in mito is unusual in using acetyl-CoA rather than malonyl-CoA *Elongases operate in the endoplasmic reticulum where by condensation reactions can occur using malonyl-CoA with acyl-CoA, this process utilizes NADPH and CoA derivatives as compared to ACP derivatives used in the formation of fatty acids catalyzed by fatty acid synthase red-> ox what happened to e? lost as water Animal cells have Δ4 , Δ5 , Δ6 and Δ9 FA desaturases Recall: all natural double bonds in FA are "cis" - must get FA with double bond past C9 from diet Animals can't desaturate FA beyond Δ9 ** Must get linoleic acid etc. from diet -The Δ12 and Δ15 double bonds are formed from plants that contain the specific desaturases and therefore are essential fatty acids obtained from diet. omega 3 v omega 6 ( count from last carbon to double bond)

Energetic Potential of FAs

FATS → ENERGY RESERVOIRS • Fats - offer energy storage sufficient for 2-3 mos • Glycogen - offers energy storage sufficient for 1 day - Complete oxidation of glucose yields ~30 ATP -32 (depending on shuttle) • How does oxidation of fatty acids compare? ** fats can be stored for 2-3 months

When comparing the net yield of ATP within one cycle of beta

False / Peroxisome doesn't have electron transport chain which means that FADH2 cannot be oxidized to produce ATP. Hence, mitochondria produce more ATP than peroxisome does within a cycle of beta-oxidation.

syn of cholesterol

First step is same as for ketone body synth except done in cytoplasm rather than mito. (reverse of last step of β-ox) Second step also same as for ketone bodies (HMG-CoA Synthase) join to form cholestrol - ring, isoprene units from acetyl CoA and C3. OH group

Descriptions of the enzymes of β-oxidation

For β-oxidation DO NOT USE ABBREVIATIONS FOR ENZYMES 1. Acyl-Co A dehydrogenase: creates a trans double bond between C2 and C3 of the fatty acyl-CoA. FAD → FADH2 because oxidizing a C-C bond 2. Enoyl- CoA Hydratase: Adds water across the newly created trans C2=C3 bond. The OH is transferred to C3 3. 3-L Hydroxylacyl- CoA dehydrogenase: oxidizes the newly created OH to a ketone NAD → NADH because oxidizing a C-O bond 4. β -ketoacyl-CoA thiolase (thiolase): Cleaves the bond between C2 and C3 to create acetyl-CoA and a new acyl-CoA with 2 fewer C atoms . 5. Enoyl- Co A isomerase: Isomerizes a cis double bond between C3 and C4 (cis C3=C4) to a trans double bond between C2 and C3 (trans C2=C3) 6. 2, 4 dienoyl-CoA reductase: Reduces two double bonds (2,4 dienoyl) to a single trans double bond between C3 and C4. The 2,4 dienoyl consists of a trans double bond between C2 and C3 (trans C2=C3) AND a cis double bond between C4 and C5 (cis C4=C5). Utilizes NADPH → NADP Reconversion of NADP → NADPH requires NADH → NAD 7. 3,2 enoyl-CoA isomerase: Isomerizes a trans double bond between C3 and C4 (trans C3=C4) to a trans double bond between C2 and C3 ( trans C2=C3) 8. 3,5-2,4-dienoyl-CoA isomerase: Isomerizes the Δ3 trans double bond (trans C3=C4) to a Δ2 trans double bond (trans C2=C3)so it becomes a substrate for 2,4-dienoyl-CoA reductase

hormonal regulation

Glucagon and epinephrine activate a cascade of reactions that stimulate glycogen breakdown and inhibit glycogen synthesis in liver and muscles, respectively glucagon- dec blood glucose ( fed<-> fast) - NK ( dec glucose but fed) - inc glucagon-> inc cAMP-> PKA-> inc glycogen breakdown and dec glycogen synthesis - dec pKA, dec F2,6 BP-> dec glycogen and inc GNG-> inc blood glucose

Uptake of Free Fatty Acids cont

Glucogon or epinephrine trigger hormone sensitive-lipase-> (HSL) HSL produces free fatty acids ->Glycerol, monoglycerides, and fatty acids, are absorbed through diffusion directly across the cell membrane->Albumin aids in transport of the predominantly hydrophobic fatty acid-> CD36 -> Activation of FAs and use of carnitine transport trigger is based on energy- glucagon is released signaling dec blood sugar and directs fat cells to release fat - fat is used as energy reserve -epinephrine- fight or flight; take action involving release of fat from fat cells 1. stored tags mobilized in phobic environment 2. hydrolysis of tags by HSL produces free FA 3. FA released in blood - albumin aids in transport of FA with inc phobicity because dont want aggregation 4. CD36 oxidizes fat cells allowing for b- ox of fats to provide acetyl coA to fuel ETC 5. activation of FA to move FA/ acetyl CoA from cytosol to matrix using carnitine transport how fats from adipocytes are mobilized. This is a hormonally regulated process where the activity of the enzyme hormonesensitive triacylglycerol lipase (sometimes called hormone-sensitive lipase or HSL) is regulated by covalent modification in response to low blood gluose levels (high glucogon) or in response to energy mobilization (think epinephrine and fight-or-flight response). Thus, the rate of hydrolysis of triacylglycerols is what dictates the release of free fatty acids released in the bloodstream. Once in the bloodstream, free fatty acids bind to serum albumin (usually referred to as albumin), which aids in transport of the predominantly hydrophobic fatty acid. Albumin actually accounts for about half of the protein in the blood

Lipid Metabolism

I. Digestion • Mainly triacylglycerols (~90%) - Remainder is cholesterol, cholesteryl esters, phospholipids, and free fatty acids * oxidation of fatty acids occur in a repetitive 4 step process called B-oxidation • Important enzymes: - Pancreatic lipase - Colipase - Cholesterol esterase • Micelle Formation II. Absorption • Formation of chylomicrons III. Transport • Lipoproteins * each with unique uses - chylomicrons, VLDL, LDL, IDL, HDL • Apolipoprotein- protein portion of lipoproteins

Transport

Introduction to Lipoprotein Structure - Major structural components: 1. Triacylglycerol- glycerol and 3 FA 2. Phospholipid - phillic head and phobic tail to pack closely with TAGS and chol 3. Cholesterol- cholesterol esters ( phobic_ and chol with OH ( phillic) 4. Apoprotein * TAGS form nonpolar core and proteins which interact with soluble lipids to form the outer layer and aid in solubility; solubility is important for transport • Lipoproteins are named according to their density ( density of outer core v inner core) - Increases density in direct proportion to their protein content 1. Chylomicrons- exogenous ( move from lymph to small intestine); largest in SA, dec protein 2. Very Low Density Lipoprotein (VLDL) - TAGS 3. Low Density Lipoprotein (LDL)-fats 4. Intermediate Density Lipoprotein (IDL)- fats 5. High Density Lipoprotein (HDL)- bile salts * VLDL,LDL,HDL are syn by the liver yo transport internally produced TAGs and chol form the liver to the tissues; lipoproteins aren't the same and dont have same levels in body and thats why routine health check require lipoprotein panels for cholesterol and to determine health status ** inc protein content, smaller, inc density; density inc, size dec, inc % total protein

What is the only other source of energy the brain can rely on aside from glucose? Why is that?

Ketone bodies can be used by the brain under starvation conditions, aside from glucose, these are the only other energy molecules that pass the blood brain barrier.

Acetyl-CoA carboxylase (ACC)

Key regulatory step ↑ by citrate ↓by FA-CoA Regulated by a kinase and Phosphatase •Insulin -> activation •Epinephrine or glucagon -> inactivation cov mod ( hormones) - AE ( cell sense internally) citrate 1. malonyl CoA -> biosyn FA and reg with carnitine --AE (+) FA CoA - AE(-) - feedback inhibiton - inc FA in liver-> inc VLDL insulin CAMP ind ( dephos Ser) -- FA biosyn inc E - diet gives mal -cAMP dep ( need E) - produces energy mole; halt FA biosyn -phos of ACC-> inactive recall that malonyl-CoA inhibits carnitine acyl transferase-1

Structure data has been used to design drugs that inhibit only COX-2

Marketed as • Celebrex • Vioxx Reduce inflammation without gastrointestinal side effects However, have cardiovascular side effects Lead Attorneys In Vioxx Settlement Get $315.3M Oct 20, 2010 • The federal judge overseeing the thousands of lawsuits filed over withdrawn painkiller Vioxx has ruled that the lead lawyers in the case will get $315.3 million, while other lawyers who sued Merck & Co. will share $1.2 billion. • U.S. District Judge Eldon J. Fallon, in a ruling Tuesday, wrote that the lead lawyers deserve 6.5 percent of the $4.85 billion settlement. That's less than the 8 percent they initially sought. • Merck withdrew Vioxx from the market on Sept. 30, 2004, after its own research showed the onceblockbuster arthritis pill doubled the risk of heart attack and stroke Medication must weight cost and benefit

Phosphatidic acid is key intermediate for synth of TAG & other complex lipids

Note: starts with DHAP or Gly-3P not glycerol *The biosynthesis of triacylglycerol will require the glycerol backbone. This comes from the biosynthesis of glycoyerol using carbohydrate metabolism. As part of the biosynthetic pathway by which dihydroxyacetone is used to produce glycerol backbones of TAGs, phosphatidic acid is generated. This molecule along with 1,2-diacyl glycerol is also used in the biosynthesis of phospholipids. glycerol 3- DH glycerol-4 acyltransferase -LPA with 1 FA and phosphate head ( singaling mol) - OH still open for FA - phosphaditic acid-> lipid biosyn, phospholipids (membrane) and TAGS ( energy storage) *Triacylglycerols are an essential storage form of energy, but can also be used for the synthesis of other compounds, namely membrane lipids and cholesterol Lysophosphatidic acid is also an important signaling molecule

Eicosanoid Biosynthesis

PLA2 releases arachidonic acid - a precursor of eicosanoids • Eicosanoids are local hormones • The cyclooxogenase (COX) oxidizes and cyclizes. • Tissue injury and inflammation triggers arachidonate release and eicosanoid synthesis • Eicosanoids - named based on the fact that all are twenty carbon containing compounds (Greek eikosi - twenty). • These compounds act in very low concentrations in a local environment and are not generally transported in the bloodstream to have their impact. Thus, these molecules are products of the precursor arachidonic acid - Stored in the cell membrane as a C2-ester of phosphatidylinositol or other phospholipids that when are hydrolyzed by phospholipase are utilized in metabolism that is cells specific. - For example, arachidonate (20 C) metabolism in platelets produces thromboxanes, which facilitates the aggregation of platelets. In endothelial cells, the process of arachidonate metabolism produces prostacyclins, which prevents aggregation of platelets. Local Hormones • Eicosanoids exert their effects at very low concentrations (~10-14 M) • Eicosanoids have very short half lifes (between 20 - 200 seconds) • Eicosanoids act at sites near their biosynthesis, • Eicosanoids are synthesized from arachidonic acid in the ER. • The first step of eicosanoid biosynthesis is the epoxidation and cyclization by COX. • Release of eicosanoids is stimulated by histamines, hormones & proteases. • Eicosanoids are also produced when tissues are injured producing inflammation and pain. • Thromboxan A2 is produced by platelets to stimulated platelet aggregation. • Aspirin used to block pain and reduce fever • Aspirin mechanism of inhibition prevents biosynthesis of prostogladins via acetylation of a serinyl residue that blocks arachidonate from reaching active site of COX

Metabolic Water

Palmitoyl-CoA + 15 FAD + 31 NAD + 8 GDP + 8 Pi + 15 H20 → Co A + 16 CO2 + 8 GTP + 31 NADH + 15 FADH2 Converting reduced cofactors to ATP and H2O 15 x (1 FADH2 + 1.5 ADP + 1.5 Pi + ½ O2 → 1 FAD + 1.5 ATP + 2.5 H2O) 31 x (1 NADH+ 2.5 ADP + 2.5 Pi + ½ O2 → 1 NAD + 2.5 ATP + 3.5 H2O) Palmitoyl-CoA +23 O2 + 108 GDP + 108 Pi → 108 ATP + 16 CO2 + CoA + 131 H20 - complex 4 1/2O2-> H2O - reduced cofactors to ATP and H2O * B-ox allows for inc in energy and water

Synthesis of Ketone Bodies: KETOGENESIS

Produced in the liver mitochondria • First step in ketone body biosynthesis is reverse of last step in β-oxidation • HMG-CoA is key intermediate in both ketone body and cholesterol biosynthesis • HMG-CoA lyase is the first committed step in ketone body biosynthesis **Know structure 1. thiolase- reverse of last step of B-ox - combine 2 acetyl CoA for release of CoA and energy to create acetoacetyl-CoA 2. HMG-CoA Synthase- 2 carbon moiety as acetyl-CoA to join and release CoA to get HMG-CoA ( enz for cholesterol biosyn) ** intermediate for chol syn and ketone body syn 3. HMG-CoA Lyase- break into 2 products; acetyl CoA ( TCA cycle energy) or KB biosyn or acetoacetate-> KB generation to create acetone and -B-hydroxybutyrate 1. Two molecules of acetyl CoA condense to form acetoacetyl-CoA in a reaction catalyzed by the enzyme thiolase, also called acetyl-CoA acetyltransferase. • This is thiolase in β-oxidation working in the reverse direction 2. A Claisen ester condensation catalyzed by HMG-CoA synthase facilitates the reaction of acetoacetyl-CoA and a third acetyl CoA to produce β-hydroxy-β-methylglutaryl-CoA (MHMG-CoA). 3. HMG-CoA is degraded to acetoacetate (β-ketoacid) and acetylCoA by HMG-CoA lyase. • HMG-CoA is a precursor in cholesterol biosynthesis; compartmentation prevents interference with cholesterol biosynthesis as HMG-CoA lyase is mitochondrial and cholesterol biosynthesis is cytosolic

What is the final product of -oxidation of an odd-chain fatty acid? Show structures and list enzymes involved in the conversion. What does the muscle cell do with this molecule - explain with words and think of how to diagram the process?

Propionyl CoA, it is converted to succinyl-CoA to feed into the TCA cycle - (1) it can stay as an TCA cycle intermediate to help drive flux through the TCA cycle or (2) it can be utilized to form malate, which is then exported out of mitochondria for conversion to pyruvate. Pyruvate is transported back to matrix for conversion to acetyl-CoA. Then energy is gained by the acetyl-CoA being oxidized as part of the TCA cycle. **In order for succinyl CoA to be consumed for oxidation and energy gain, the C4 skeleton must exit the TCA cycle (not serving as an intermediate) for conversion to pyruvate for reentry to mito to produce acetyl-CoA.

b-oxidation of palmitic acid (16:0)

Repetition of the cycle yields a succession of acetate units • Each round of b-Oxidation yields 1 NADH, 1 FADH2 and 1 acetyl-CoA • Each acetyl-CoA enters TCA generating 1 FADH2 and 3 NADH, 1 GTP b-Oxidation of Palmitoyl-CoA (C16) • yields 8 acetyl-CoAs, 7 FADH2 , 7 NADH • Oxidation of the 8 acetyl-CoAs yields 8 GTP, 24 NADH, 8 FADH2 Totals : 31 NADH 77.5 ATP 15 FADH2 22.5 ATP 8 GTP 8 ATP 108 ATP Payback: FA FA-CoA -2 (ATP AMP, PPi→2Pi) 106 ATP

Acetyl-CoA Carboxylase

The "ACC enzyme" commits acetate to fatty acid synthesis • Carboxylation of acetyl-CoA to form malonyl-CoA is the irreversible, committed step in fatty acid biosynthesi s • ACC uses bicarbonate and ATP (AND biotin!) • E.coli enzyme has three subunits • Animal enzyme is one polypeptide with all three functions - biotin carboxyl carrier, biotin carboxylase and transcarboxylase **in a mechanism previously seen with pyruvate carboxylase and propionyl-CoA carboxylase. 1. pyruvate carboxylase ( GNG pyruv-> OXA) 2. propinyl CoA ( odd # FA in B-ox) - > succinyl COA - propinoyl carboxylase • The acetate units are activated by the formation of Malonyl-CoA at the expense of ATP. • Overall reaction: some fold independently and have unique functions

. Why does the oxidation of fat provide water?

The final electron acceptor of molecular oxygen will form water FADH2 + ½ O2 H20 NADH + H+ + ½ O2 H20

Lipid Synthesis Overview

The tricarboxylate transport system transfers acetyl-CoA into the cytosol for fatty acid synthesis. • Fatty acid synthesis begins with the carboxylation of acetyl-CoA to generate malonyl-CoA. • Fatty acid synthase carries out seven reactions and lengthens a fatty acid two carbons at a time. • Elongases and desaturases may modify fatty acids . • Triacylglycerols are synthesized from glycerol and fatty acids

What is the correlation between fatty acid uptake and glucagon or epinephrine?

These hormones signal low blood glucose or fight-or-flight response, thus cAMP and phosphorylation cascades are produced to activate protein kinase which phosphorylates hormone-sensitive lipase to enhance TAG hydrolysis and release of fatty acids in liver and fat cells

What type of dietary issues would be expected for individuals with blocked bile ducts?

Those individuals would lack proper digestion and absorption of lipids, thus their body would eliminate lipid products and their body would lack necessary absorption of not only fats from their diet, but lipid soluble vitamins like A, D, E, and K.

Fates of Ketone Bodies

Ultimately, AcAc and β-HB are converted to acetyl-CoA 1. B-hydroxybutyrate (NADH/NAD+) to the blood stream or tissue-> form acetyl COA within target cell and undergo oxidative phosphorylation to create ATP 2. transported as acetyl-CoA * if not needed then excreted by urinary clearance 3l. acetone- (20%) - lipids, proteins, exhalation - urinary clearance

The complete oxidation of palmitoyl-CoA to carbon dioxide and water is represented by the overall equation: Palmitoyl-CoA + 23O2 + 108Pi + 108ADP -> CoA + 16CO2 + 108ATP + 23H2O Water is also produced in the reaction ADP + Pi -> ATP + H2O, but is not included as a product in the overall equation. Why?

Water produced in the reaction is utilized in the hydration of double bond in trans-∆ 2 - Enoyl-CoA, which is the second step of beta-oxidation in each cycle. Thus, it is not included as a product in the overall equation

FA biosynthesis

We will first look at the top part (on next slide) How acetyl-CoA ->distal 2 C priming reactions (1a and1b) - malonyl ACP- E from decaroxylation at active site MAT - acetyl COA and malonyl COA release CoA - KS is the condensing enzyme *Each of these seven different reactions have a specific role in the formation of fatty acids and are described below as part of either the mobilization or the elongation portion of fatty acid biosynthesis. Mobilization- A. Acetyl Transacylase activity whereby the acyl portion of acetyl CoA is transferred to acyl carrier protein (ACP), releasing CoA. B. Malonyl Transacylase activity whereby the malonyl portion of malonyl CoA is transferred to ACP, release CoA. 2a. transacytal portion to enz ( cov catalysis) 2b- malonyl decarboxylation release CO2 - nuc attack on carbonyl - 4C acetoacetyl-ACP formed *Distal two C come from acetyl-CoA Subsequent C come from malonyl-CoA 2 3. B-keto red to OH - NADPH-> NADP+ 4. dehydration and form of double bond ( D v. L) 5. red with NADPH to saturated double bond to 2 C added *Elongation- 1. β-ketoacyl-ACP synthase "condensing enzyme" activity serves to condense malonyl-ACP and acetyl-ACP, decarboxylation occurs in the process allowing for the formation of the 4-carbon unit acetoacetyl bound to ACP. 2. β-Ketoacyl ACP reductase uses NADPH to reduce a ketone producing a hydroxyl group in formation of β-hydroxybutyrylACP. 3. 3-Hydroxyacyl-ACP dehydratase removes water in a dehydration reaction, forming a double bond in the product α,β-trans-butenoylACP. 4. Enoyl ACP-reductase reduces the double bond using NADPH to form butyryl-ACP. 5. This process repeats using the acyl-malonyl ACP condensing enzyme until 7 cycles have been completed and form palmitoylACP. See figure to right.6. Thioesterase catalyzes the final step, hydrolyzing palmitate from ACP endpoint- palmitate ( C16;0) - longest product formed MAT with AT or MT - ACP nuc attack to SH - condensing enz - ACP use SH for nuc attack when malonyl condenses to MAT; beak ester - decarboxylation and energy to break thioester -ACP with 4 C attached in ox form but need r4ed form - reduction rxns with NADPH ( e donot) - dehydration - red reaction with NADPH reactions with domains domain 2 1. B-ketoacyl reductase 2. dehydratase 3. enoyl-ACP reductase - 16 C unit saturated *Structurally, fatty acid synthase contains six active sties for seven reactions as steps 1 and 2 are carried out in the same active site. Fatty acid synthase is a dimer so two fatty acids can be biosynthesized simultaneously. These subunits associate forming an 'X' that are loosely connected and allow for a 180° rotation. domain 3- thioester release ACP and liberation of palmitate - 2 halves work in concert

. (a) Draw the structure of linoleic acid (C18:2Δ9,12). (b) How many rounds of beta oxidation can oxidation will occur to fully oxidize this molecule? Calculate values and negate the occurrence of unanticipated isomerization reaction. (c) What is the total yield of ATP?

a. Linoleic acid: b. Rounds of beta oxidation? 8 rounds c. Yield of ATP: 7 FADH2, 7 NADH, 9 acetyl- CoA 1 acetyl CoA = 1 FADH2, 3 NADH, 1 GTP (x 9) FADH2 total = 7 + 9 = 16 FADH2 x 1.5 ATP/ FADH2 = 24 ATP NADH total = 7 + 27 = 34 NADH x 2.5 ATP/NADH = 85 ATP GTP total = 9 = 9 ATP TOTAL ATP = 118 ATP - 2 ATP (transport cost) = 116 ATP YIELD

Fates of HMG-CoA

also in KB biosyn - isoprene units are 5 C - exists in 2 forms with C=C at diff locations - easily interconverted - dec CoQ ( ETC - lipid soluble carrier)

• LDL

bad cholesterol - The function of LDL is to: 1. deliver cholesterol to cells, where it is used in membranes 2. for biosynthesis of bile salts in the liver 3. for the synthesis of steroid hormones. • LDL is the major cholesterol carrier in the blood stream. - As VLDL is stripped of triacylglycerols, they are remodeled in the liver to become IDL and ultimately LDL as part of the endogenous pathway inc LDL inc fat inc LDL inc chol ->> plaque lead to phobic contact points which is bad

Lipid synthesis

dec E- catabolism ; oxidation - 1. sugars-> pyruv-> acetyl coA-> CAC-> ETC-> ATP 2. fats (FA)-> carnitine-> B-ox-> acetyl oA inc E; anabolism ; reduction 1. citrate ( acetyl CoA + OXA)-> move out 2. ATP citrate lyase-> OXA-> pyruv-> CAC or -> acetyl CoA -> ACC-> malonyl CoA 3. FA syn-> 16:0-> 18;0-> 18;1 - FA synthase - malonyl CoA inhibit carnitine and B -oxidation *reciprocal regulation inc FA syn, inc malonyl CoA< dec B-ox, dec FA in matrix dec malonyl COA, dec FA syn, imc carnitine and inc B-ox and FA in matrix

Dietary Lipids

degradation => absorption transport-> exogenous and endogenous • The processing of dietary lipids (which is primarily fats) occurs in 8 steps: 1. Bile salts mix with fats in the small intestines to form mixed micelles 2. Intestinal lipases degrade triacylglycerols to form the products of fatty acids and glycerol 3. Fatty acids are taken up by the intestinal mucosa and converted into triacylglycerols 4. Reconverted triacylglycerols are packaged with dietary cholesterol to from lipoprotein aggregates called chylomicrons. 5. Chylomicrons are shuttled through the lymph system and the blood stream to various tissues. Apolipoproteins are lipid binding proteins that aid in the transport of triacylglycerols. Their protein moieties are recognized by receptors on cells surfaces of muscle and fat cells. 6. In the capillaries, extracellular lipoprotein lipase is activated by apoC -II to hydrolyze the fatty acids and glycerol. 7. The fatty acids and glycerol are then taken up by cells. 8. In muscle cells the fatty acids can be used, in adipocytes they are reesterfied for storage as triacylglycerols .

What about 'Keto' diets??

diabetic ketoacidosis - dec glucose, not enough entering the blood stream to cells - no insulin so dont have response to mobilize glucose - cells starving for sugar so live inc glucose production by GNG, fats breakdown, kidneys are overwhelmed ( KB from liver too) and start to have issues, sweet smell because kidney is trying to excrete so much glucose because cells ant * perpetual cycle - dangerous - B-OHB; 15-25 mM nutritional ketosis- B-OHB; 0.5-3 mM - dec dietary glucose, body relies on fat by liver producing KB because it can be quickly converted to aceytl CoA - glucose generated by GNG to inc gluc and supply brain cells-> KB as major source of acetyl-CoA so wont compete with brain -acetone fates

• HDL

good cholesterol * non-liver cells called extrahepatic cells are the major contributor to HDL as the cells cannot metabolize cholesterol - Biosynthesized in the liver and intestines for release in the blood stream as a protein rich particle ; liver is the only place where cholesterol can be consumes for production of bile acids • HDL is used for cholesterol recovery - Clean up excess cholesterol from blood for excretion ( only way to get out of body) excreted as bile or bile salts * fats and chol trapped in different lipoproteins but HDL can excrete it and carry to liver and intestines-> blood stream • HDL delivers cholesterol to steroidogenic tissues - Adrenal glands, ovaries, testes - HDLs operate as part of the endogenous pathway * precursor is formed by the liver and small intestine and gather chol from cell surface membrane extraction to form mature HDL HDL picked up by liver-> bile salts-> role in digestion

Nutritional Ketosis

hormones- ↑ glucagon ↓insulin Activate receptors in liver and adipose ↑ cAMP -> activate protein kinases (enhanced glycogen breakdown and GNG activation; PKA) ↓ lipid synth. Activate hormone sensitive lipase allow glucose to BS - fats within liver itself to serve as energy source (ATP) -*utilized in different organs-> heart, kidney - B-HB released by liver - muscle tissue takes up B-HB - interaction with CAC fueld by fats ( CD36) acyl-CoA actiavted-> carnitine transporter-> acyl-CoA -> B-ox-> acetyl-CoA-> CAC - skeletal muscle (MCT) bring in KB and fats with CD36 - glucose comes in only if brain isnt starved through GLUT transporter; inert response and muscles switch to fat burning mode - adipose tissue released FA to muscles and transports using serum albumin ( carry into phillic environment and transport into cell) - liver produces limiting amount of glucose-? brain -- transports to brain cells with a high affinity so it doesnt compete with other cells - is the brain meant to function on KB? NO becasue starvation *healthy individuals GNG supply glucose to brain and if KB used then liver isnt keeping up and go into starvation

ACC Allosterically Regulated

inactive protamers Active Polymers • Palmitoyl CoA inc - inhibit FA biosyn and feedback inhibition - T state • Citrate inc - act ACC - R state *Citrate is an allosteric activator that stimulates levels of fatty acid biosynthesis, whereas high levels of fatty acylCoAs serve as feedback inhibitors cat committed step in FA biosyn - protamers= T stae polymers= R state

diagnosing diabetes

inc fat breakdown, inc fat in plasma, inc ketogenesis, inc ketouria, dec alkali response, inc acidosis hyperglycemia, dec glucose uptake, inc protein breakdown inc AA in liver, inc GNG, hyperglycemia renal failure, dec electrolytes, dehydration coma***Blood acidosis can lead to a number of conditions including hypotension (low blood pressure), hyperkalemia (high potassium levels - recall in 'membrane and membrane transport' sections from BICH410 - the body expends a lot of energy regulating potassium and sodium levels necessary for cellular function), hyperventilation (exhaling more than inhaling, leading to loss of CO2 which can impact hemoglobin function and oxygen delivery as well as impact the blood buffering system - recall details from BICH410), and respiratory fatigue (often requires ventilation machinery and aid in maintaining CO2 levels). Left untreated for too long, a coma, and in some cases death can occur.

Pancreatic lipase

is enhanced by a process known as interfacial activation - a phenomenon that describes rate enhancement of lipase when it contacts the lipid-water interface ** requires bile salts, mixed micelles of phosphatidylcholine and colipase - breaks down micelles; produced in pancreases; portal vein receives blood and then blood leaves through the hepatic vein - pac man; ester hydrolysis reaction -phobic portion of FA esterfied to phillic region - phospolipase A2 attack in FA--> micelle destroyed *unique-polar -- interaction at surface in which enzyme can attack micelle • Lipase functions in a 1:1 ratio with colipase (yellow) - (a) absence of micelle - (b) presence of micelle - **Notice structural rearrangements of the magenta residues called the lid and the β5 loop in orange * more definitive a helical structure - residues= lid structure and interact more favorably with lipase; help form active site so phobic ( aliphatic side chains) can be broken down - allows access to a hydrophobic active site of lipase - Thus colipase facilitates a hydrophobic platform that aids in binding lipid surfaces

Stoichiometry of Fatty Acid Synthase

malonyl COA is 3 C bur supply 2 C to FA chain because 1 C lost as CO2 - 14C + 2C-> 16 C palmitic acid FA syn stops at palmitic acid so use elongases and desaturases to modify FA

NNT and ROS

nicotinamide nucleotide transhydrogenase - ratios of NADH to keep up NADPH - inc NADH and use e to drop e at complex 1 and balance NADH with NADPH - inc NADPH is important( ROS) - glutathione reductase and peroxidase dec H2O2 by conversion to water - superoxide is most common ROS and prevents form of hydroxide radical ( dangerous)-> fenton chem to hydroxide radical - keeps H2O2 low - needs GTR and GTP - sensitivity to cell to ROS when NADPh and NADH arent balanced H, proposed model for the role of NNT in regulating the TCA cycle activity. Left diagram, NNT catalysis contributes to reductive carboxylation (RC) in the mitochondrion via production of NADPH. Under NNT activated conditions, cells exhibit an adequate balance between the contribution of glucose (blue)- and glutamine (red)- derived carbons to the oxidative TCA cycle. Kg, -ketoglutarate. Right diagram, NNT loss of function decreases the NADPH/NADP ratio, which inhibits the formation of RC-derived citrate and increases reactive oxygen species (ROS) levels and mitochondrial uncoupling (Mit. uncoupling). This decreases the NADH/NAD ratio thereby stimulating the contribution of glucose catabolism into the TCA cycle, depicted as an increased percentage of the blue color in the oxidative TCA cycle

Polyunsaturated FAs

• 3 issues that could require four enzymes • Problem 1: The presence of the β, double bond (need α,β for enoyl-CoA hydratase). • Answer: Enoyl-CoA isomerase moves the location of the double bond - Specifically moves the location of the cis-Δ 3 double bond to trans-Δ 2 , between α,β carbons • The round of β-oxidation can continue. • Problem 2: The presence of the Δ4 double bond inhibits enoyl-CoA hydratase . • Answer: The NADPHdependent enzyme 2,4- dienoyl-CoA reductase reduces the Δ4 double bond. -NADPH e donor - moves double bond from C3.4 to C2,3 for B-ox with regular enxymes - **In mammals, the enzyme 3,2-enoylCoA isomerase • A Δ 2 double bond product that is a substrate for 3,2-enoylCoA isomerase, where 20% of the time the Δ2 double bond product is converted to a Δ3 double bond product • Problem 3: the Δ3 double bond product is not a substrate for enzymes in β-oxidation. • Answer: 3,5-2,4-dienoylCoA isomerase functions to convert the trans Δ 3 double bond to a trans Δ2 double bond so it becomes a substrate for 2,4-dienoyl-CoA reductase

ACC Isoforms

• ACC can exist is in different isoforms based on tissue specificity. - Adipose tissue contains cytosolic ACC1 where fatty acid biosynthesis occurs, including lipogenic tissue like adipose tissue. - Other tissues that lack fatty acid biosynthesis have much higher ratios of ACC2. - Why? (hint think roles for ACC product) • As ACC is necessary in the formation of malonyl-CoA for fatty acid biosynthesis, it is believed that tissues producing ACC2 uses malonyl-CoA for a regulatory role to inhibit import of acyl-CoA via carnitine palmitoyl transferase I. • Liver has both - why? FA biosyn with ACC1 and entry to matrix with ACC2 - GNG-> liver use4s fats for energy -or uses KB 1. malonyl COA sub for FA biosyn 2. malonyl COA cant reg FA matrix B=ox **Adipose tissue contains ACC1 where fatty acid biosynthesis occurs, other tissues that lack fatty acid biosynthesis have ACC2. As ACC is necessary in the formation of malonyl-CoA for fatty acid biosynthesis, it is believed that tissues producing ACC2 uses malonyl-CoA for a regulatory role to inhibit import of acyl-CoA via carnitine palmitoyl transferase I liver (ACC1) muscle (ACC2)- reg malonyl CoA, inhibit carnitine transporter and block FA -> B-ox

ALD

• Abnormal adrenoleukodystrophy protein (ALDP protein) is located in a part of the cell called the peroxisome, and is responsible for breaking down very long chain fatty acids. - The accumulation of very long fatty acids damages the protective covering of nerves (myelin sheath), resulting in neurological problems. - Encoded by the ABCD1 gene mutation. in ABDC1 gene - build up of LCFA leads to heath issues

Release of Ketone Bodies from Liver

• During pro-longed periods of fasting, where glucose is limiting, the energy source of the body becomes the breakdown products of fat and thus ketone bodies • Gluconeogenesis will kick in to meet the demands of the brain for glucose 1. glucose is limiting -> used in brain 2. switch to breakdown fat ( adipocytes and liver) 3. KB (liver) 4. GNG to inc glucose for brain **fasting not starvation • Ketone bodies are used for energy. • Ketone bodies are transported from the liver to other tissues in the bloodstream, where acetoacetate and β-hydroxybutyrate can be reconverted to acetyl-CoA to produce energy . • The heart gets much of its energy from ketone bodies, although it also uses a lot of fatty acids acetyl CoA-> TCA or acetoacetate - exported to blood stream to allow for transport -succinyl CoA generates acetoacetyl-CoA and then acetyl CoS * fast acting because quicker conversion to acetyl CoA compared to FA 1. brain 2. heart for constant pumping ( fats-> FA and KB)

Transport of Activated Fatty Acids into the Mitochondrion: Carnitine

• Activated acyl-CoA occurs in the cytosol, but the process of fatty acid oxidation occurs within the mitochondria. • The carrier carnitine functions to accept the acyl portion (fatty acid) of the activated fatty acid through a transesterification reaction catalyzed by carnitine palmitoyl transferase I in the outer-mitochondrial membrane . - Releases CoA in cytosol for use in more activation reactions The product, acyl-carnitine, has similar free energy of hydrolysis as acyl-CoA, preserving the energy investing in the activation of the fatty acid catalyzed by the acyl-CoA synthetases while producing a molecule capable of being transported across the inner mitochondrial membrane. carnitine 1 and 2 because passes from cyto-> matrix through the OMM and IMM - system on cyto and matrix side - carrier protein is carnitine - carnitine 1 is on OMM - carnitine react with FA CoA-> hydrolysis of CoA ( free cytosolic) and energy maintained in acyl- carnitine as carrier protein - same energy because of acyl ground and carnitine bond - acyl CoA is substrate for B-oxidation in the matrix The carrier carnitine functions to accept the acyl portion (fatty acid) of the activated fatty acid through a transesterification reaction catalyzed by carnitine palmitoyl transferase I. This reaction releases free CoA for future use in fatty acid activation in the cytoplasm. -mediated by carnitine carrier protein. Upon entry into the matix, the acyl portion is transferred to CoA using as transesterification reaction catalyzed by carnitine palmitoyl transferase II, regenerating carnitine for the transport process and acyl-CoA in the matrix for use in fatty acid β-oxidation

Alcoholic Ketoacidosis

• Alcohol: 1. diminishes hepatic gluconeogenesis 2. leads to decreased insulin secretion, 3. increased lipolysis, 4. impaired fatty acid oxidation, and 5. subsequent ketogenesis how body processes ethanol ethanol-> acetate(hangover) exogenous uptake of ethanol overwhelms system - messes with NADh/NAD+ levels ( pools dec) 1. poor food intake - dec glycogen, ketogenesis, nausea and vomiting, metabolic acidosis 2. NADH/NAD+ pools - NADH>>NAD - dec fat breakdown - inhibit GNG, dec blood sugar, dec glucagon and insulin - fats used but NADH/NAD+ pools inhibit GNG so cant fix it= stress and dehydration***Water is then collected in higher amounts to form urine to handle the influx of sugar and ketone bodies, leaving the blood stream, which exacerbates acidosis while causing dehydration

ApoA-1

• Apolipoprotein A-1 (ApoAI) binding occurs in chylomicrons and HDL and add a high degree of hydrophilicity. • ApoAI is a homotetramer producing a structure that can ideally wrap around an HDL particle. ( figure 8/ infinity sign) • ApoAI is to help clear fats and cholesterol from white blood cells in arterial walls, helping ensure health of white blood cells. faces that are nonpolar and polar because of solvent and clean fats and chol from WBCs * LCAT or lecithin-cholestrol acyltransferase is an enzyme activated by ApoA-1 that converts cholesterol to cholesterol esters • Apolipoprotein A-1 - Facilitates interactions with SR-B1 receptor within HDL at the liver - sometimes called reverse cholesterol transport - Allows HDL to bind to SR-BI and transfers its component lipids to the cell - Depleted HDL (remHDL) dissociates from the receptor and reenters circulation for scavenging cholesterol**ABCA1 - ATP-binding cassette transporter ABCA1 (member 1 of human transporter sub-family ABCA), also known as the cholesterol efflux regulatory protein (CERP) is a major regulator of cellular cholesterol and phospholipid homeosta sis. ABCA1 mediates the efflux of cholesterol and phospholipids to lipidpoor apolipoproteins (like apo-A1) to form nacent HDL (nHDL). Lecithin-cholesterol acyltransferase (LCAT, also called phosphatidylcholine-sterol Oacyltransferase) is an enzyme activated by ApoA-I that converts free cholesterol into cholesteryl ester , which is then sequestered into the core of the newly synthesized HDL sphere and forcing the reaction to become unidirectional since the particles are removed from the surface. The ABC transporter ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. - The ABC transporters: • ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. • ABCA1 mediates the efflux of cholesterol and phospholipeds to lipid-poor apoliproteins like ApoA-I to form nacent HDL (nHDL). * HDL pick up of free chol-> bile salts - recycle certain pieces of HDL and chol for ability to make bile salts and transports into epithelial of intestine ** defects lead to progressive atheroma and premature coronary artery disease

Digestion cont

• Bile salts or bile acids - amphiphilic molecules - synthesized from cholesterol in liver - stored in the gall bladder - released into the small intestines for digestion purposes - the action of bile salts produces solubilized fat globules - known as micelles in a process known as emulsification * taurocholic acid is a bile salt involved in fat digestion and also available for commercial use from the byproduct of the meat-processing industry ( groups in blue are hydrophobic and red are hydrophillic. in an aqueous environment the sidedness of this molecule allows it to interact well with hydrophobic fats while in solution) HDL important for this process of syn of cholesterol in liver * structure phobic> phillic - phillic interactions -> sidedness - phobic interactions with fats

fatty acid oxidation or β-oxidation

• Both used to describe the process of fatty acids degradation in 2C-units through an oxidative process involving the β-carboxyl group ( carbon that would be ox) - 2C-unit β-oxidation products proposed by Franz Koop in 1904 using dogs and phenyllabeled fatty acids where excretion products were monitored - Confirmed in the 1950's • A Repeated Sequence of 4 Reactions w 4 unique enzymes • Strategy: create a carbonyl group on the beta-C -oxidation ( FAD e acceptor, hydride transfer- FADH2), hydration( h2o split across double bond), oxidation (NADH-> ETC) and cleavage( acetyl coA + acyl CoA products; rest goes through B-ox until 2 C units remain) # rounds of ox = #C in FA/2 -1 ** # acetyl coA produceed is = #FA C/2

Acetone

• Conversion to lactate and then pyruvate (all fates of pyruvate) - EPA Toxicology Review of Acetone, 2001 • Radiolabeled carbon atoms from acetone were found inconsistently in: - proteins - lipids - exhaled CO2 acetone-> lactate-> pyruvate-> many fates acetone on the breathe

Absorption

• Digestion produces a mixture of fatty acids and mono - and diacylglycerols that must be absorbed by mucosal cells in the small intestines. - Only freely dissolved monoglycerides and fatty acids can be absorbed, not micelles • Bile salts also aid in absorption by better facilitating diffusion of lipids into mucosal cells and also for absorption of lipid soluble vitamins A,E,D,K 1. bile salts-> micelles ( emulsification) and facilitate diffusion of lipids to inc absorption to intestinal cells 2. lipase- cleave bonds ( C1, C3 or both) to greate the monoacylglycerols; only freely dissolved monoacylglycerols can be absorbed after conc in mucosal cells; role of I-FABP • Intestinal fatty acid-binding protein (I-FABP) interacts with fatty acids forming a complex inside the intestinal cells, increasing solubility and dec unfavorable phobic interactions - rats by Dr.Jim Sacchettini Once inside the mucosal cell, FAs are reesterfied into TAGs • TAGs, cholesterol esters, apoproteins, vitamins are packaged to form chylomicrons • Chylomicrons are transported via the lymph system as part of the exogenous pathway for lipid metabolism - exogenous= directly from diet ( digestion) micelles can be brought into cell but not big globules because too big

Fates of Acetoacetate

• Fates of acetoacetate : - Conversion to βhydroxybutyrate; redox reaction catalyzed by βhydroxybutyrate dehydrogenase ( reverisble reaction) - B carbon oxidation to make red product and ox NAD+ - Acetoacetate is a β-keto acid that can undergo nonenzymatic decarboxylation to produce acetone and energy • What dictates fates of acetoacetate? - inc NADH, dec NAD+ = Bhydroxybutyrate - dec NADH, inc NAD+- acetoacetate • Ratio of AcAc to β-HB is usually maintained around 1:1, though it is based on NADH to NAD+ concentrations

FA Transport in Cells from Bloodstream

• Fatty acids are catabolized in the mitochondria, thus they must be transported across multiple membranes - CD36: accessibility of CD36 is directly related to the influx of fatty acids into the cell inc FA in mito inc CD36 vesicles with CD36 The accessibility of CD36 is directly related to the influx of fatty acids into the cell. The following figure shows both the short term and long term regulation involving CD36 (ref: Biochimie, Volume 136, May 2017, Pages 21-26). CD36 can be sequestered intracellularly for short-term regulation, in which CD36 is subcellularly recycled through the actions of vesicle fusions involving the plasma membrane and endosomes. Or CD36 can be used for long-term regulation in which increased cytosolic concentrations of long-chain fatty acids (LC-FA) allow for fatty acid oxidation (β-oxidatoin) and use in 2 other cellular functions, also triggers a signaling event that allows for the upregulation of transcription and thus enhanced biosynthesis of the protein product CD36.

Strategy for Fatty Acid Biosynthesis

• Fatty acids are constructed by the addition of 2-carbon units derived from acetyl-CoA. • The acetate units are activated by the formation of Malonyl-CoA at the expense of ATP. • Driving force of the condensation reaction is the the exergonic decarboxylation of MalonylCoA. • Chain elongation stops at palmitoyl-CoA. ATP ( biosyn, NADPH, ATP) inc energy - decarboxylation release E - FA synthase elonate malonyl COA (product of ACC)-> palmitoyl COA (16;0)-> elongases and saturases

ACC Also Under Hormonal Control

• Hormonal control involves phosphorylation of Ser79 - Catalyzed by AMP-dependent protein kinase (AMPK) that is part of a cAMP-independent pathway leading to ACC inactivation. - ACC levels are also regulation by glucagon and epinephrine, which activate protein kinase A, phosphorylation inhibits enzyme. - Insulin has the opposite impact promoting the dephosphorylation of ACC and leading to enzyme activation Glucagon and epinephrine dec glucose - catabolic producing E, reduce inactive carboxylase inhibiting FA biosyn - citrate (+ AE)- forms partially active carboxylases insulin - inc E - cAMP dep ( glucagon and epinephrine) and ind ( insulin)

Ketoacidosis

• Increased concentration of acetoacetate and D-βhydroxybutyrate, a carboxylic acid that ionizes readily in the bloodstream, ionization causes a decrease in blood pH • Possible to smell acetone on a diabetic's breath • Marked by unique metabolic levels of ketone bodies, which are produced in significantly higher amounts to compensate for energy starvation of the cell • Glucose is constantly being made in the liver using gluconeogenesis - Causes a complete depletion of the TCA cycle intermediates - ate= deprotonated state -- inc conc of acetoacetic acid and B-HB which readily ionize and give up H+ thus inc [H+} and dec pH =acidosis GNG inc so will use CAC intermediates to inc glucose

ApoB-100

• LDL contains apolipoprotein B100 (ApoB-100) in a ratio of 1:1 • Over 4000 residues, one of the largest monomers knowns, and covers over half of the LDL surface • Plays are role in LDL-receptormediated endocytosis. • LDL is the major cholesterol carrier in the blood stream wraps around LDL -> specificity for receptor • LDL -receptors target ApoB -100 - Receptors are transmembrane glycoproteins. - These LDL -receptors specifically target apoB - 100 in clathrin -coated pits that produce a clathrin - coated vesicle for endocytosis ;. Once triskelions are produced from depolymerized clathrin, the uncoated vesicles fuse with acidic endosomes that facilitate the dissociation of LDL from the LDLreceptor. The LDL-receptors are likewise recycled to the plasma membrane. - ApoB -100 and cholesteryl - esters are hydrolyzed producing amino acids, fatty acids, and cholesterol *but the impact of cholesterol production from LDL activates acylCoA:cholesterol acyltransferase (ACAT) which catalyzes formation of cholesterol esters, while inhibiting HMGCoAReductase (catalyzes ratelimiting step in cholesterol biosynthesis) and LDL receptor biosynthesis.** Defects in LDL receptor regulation lead to high level of circulating cholesterol, leaving the individual at high risk of heart disease. LDL is target - breakdown by lysosomes inc chol body shuts down chol biosynthesis ( regulation for number of receptors and amount of chol made_ inc LDL brought in 1. chol biosyn dec 2. LDL receptors dec on the surface of the ell ** how the body deals with recycling of fats and chol inc LDL into cell inc LDL conc in the blood and in the cell so uptake of LDL dec and stays in blood which leads to plaques, arteriosclerosis, and strokes

Apoproteins

• Lipoproteins function largely based on the apoproteins associated with their structures ( ex interior of LDL is phobic) - Apoproteins are the protein portion of lipoproteins - Dictates interactions B-100- LDL ApoA-I-> chylomicrons and HDL ApoE-> VLDL-> IDL-> LDL

Lysophosphatidic acid signaling in vertebrate reproduction

• Lysophosphatidic acid (LPA) has important roles for receptor-mediated LPA signaling in multiple aspects of vertebrate reproduction. • These include ovarian function, spermatogenesis, fertilization, early embryo development, embryo implantation, embryo spacing, pregnancy maintenance and parturition. • LPA signaling can also have pathological consequences, influencing aspects of endometriosis and ovarian cancer. pain Pain-associated signals, acidosis and lysophosphatidic acid, modulate the neuronal K(2P)2.1 channel *Pain is a physiological state promoting protective responses to harmful episodes. However, pain can become pathophysiological and become a chronic disruptive condition, damaging quality of life. The mammalian K(2P)2.1 (KCNK2, TREK-1) channel, expressed in sensory neurons of the dorsal root ganglia, is a polymodal molecular sensor involved in pain perception. Here, we report that two pain-associated signals, external acidosis and lysophosphatidic acid (LPA), known to rise during injury, inflammation and cancer, profoundly down-modulate human K(2P)2.1 activity. pain-> inhibitor

Cholesterol Maintains the Fluidity of our Membranes.

• Maintains the fluidity of membranes. - Helps maintain order at high temperatures • Offers rigidity for increased movement of FA tails at high temperatures - Helps offer a better barrier at low temperatures • Offers increased interactions when movement ceases at lower temperature • Phase change from fluid to gellike solid - increases breadth of membrane fluidity? membranes- fluid mosaic - phospholipid- phillic head and 2 aliphatic side chains ( saturated) nonpolar tail - rotational movement at C to C bonds allow for fluidity - unsaturated- kink C=C creates rotation and takes up more volume; not too tight of packing - inc temp, inc E< inc entropy, dec aggregation and membrane is destroyed -- inc rigidity and dec entropy - dec temp dec movement leads to gel like solid -- chol combats phase change and adds rigidity chol stabilizes membrane structure at both high and low temps

Acyl-CoA dehydrogenase

• Mitochondria have four different acyl-CoA DH - As the acyl-CoA becomes shorter, it requires enzymes with different substrate specificity • Used for FA of different lengths (4C - 18C) • Deficiency of medium chain acyl-CoA DH is associated with sudden infant death syndrome ≈ 10% of cases - medium-chain acyl CoA dehydrogenase (MCAD) deficiency, first reported in 1983, is considered the most common β-oxidation deficiency - the majority of SIDS cases were suggestive of long-chain fatty-acid-oxidation defects (J Pediatr. 1998;132:924-933) • Most fatty-acid-oxidation disorders are amenable to therapy by diet modification, and early recognition may prevent catastrophic illness. Oxidation of the Ca-Cb bond • A family of three soluble matrix enzymes short (C4-C8), medium (C4-C14), and long (C12 - C20) • Mechanism involves proton abstraction, followed by double bond formation and hydride removal by FAD • Electrons are passed to an electron transfer flavoprotein, and then to the electron transport chain Electron Transferring Flavoprotein • Electron input to electron transport bypasses complex I and II • Net result : 1.5 ATP synthesized per electron pair transferred; 6H+ per FAHD@-> 4H+ = 1 ATP = 1.5 ATP`

Cholesterol

• Modulates membrane fluidity • Precursor of - Bile - Steroid hormones - Atherosclerotic plaques • Heart attack • Stroke *precursor of biosynthesis of biological molecules having numerous functions including formation of steroid hormones, bile salts/acids, and ubiquinone to name a few. Cholesterol is characterized as having rigidity due to a fused four-ring structure, an aliphatic tail, and a hydrophilic -OH group that can further be esterified to form a cholesterol-ester. • Compose of 4 fused Rings which make it rigid. • Weakly Amphiphilic due to the lone hydroxyl group. Cholesterol is Synthesized from Acetyl CoA -Acetyl-CoA ->HydroxyMethylGlutaryl-CoA ->Isoprene units ->Cholesterol or Vit A, D, E or K Carotenoids Ubiquinone Hormones Rubber etc. 4 rings inc rigidity -rotational movement with aliphatic side chains - inc phobicity; prone to aggregation - a ring has OH- only source of polarity 1. structural role- modify membrane fluidity 2. metabolic precurosr - bile salts, lipid digestion ( emulsificaton) - testosterone and estradiol - aggregation leads to plaques and disease states - block movement and issues with pressure inc phobicity - OH= polar head group - amphipathic (NP and polar)

Advantages to Nutritional Ketosis

• New studies show that the ketogenic diet as part of dietary therapy, a high-fat, calorierestricted diet used to treat epileptic seizures in children, alters genes involved in energy metabolism in the brain, which in turn helps stabilize the function of neurons exposed to the challenges of epileptic seizures. - 2005, Johns Hopkins useful for weight loss -autism, epilepsy, cancer (ROS), dementia (ROS), migrane

ACC as a Drug Target

• Nonalcoholic fatty liver disease (NAFLD) - characterized by hepatic accumulation of excess triglycerides - Secretion into plasma as verylow-density lipoprotein (VLDL) triglycerides • Cardiovascular disease is the leading cause of morbidity and mortality in patients with NAFLD • ACC catalyzes committed step in FA biosynthesis - Success of ACC inhibitors for fatty liver disease and hepatocellular carcinoma inhibit ACC- loose acetyl COA and loose ability to block fats to B-ox via carnitine

Odd-numbered FAs

• Odd-numbered FAs proceed through β-oxidation as all FAs would based on the degree of saturation - Details have been discussed • So what is the issue? 3 C unit left over • What is the fate of 3C unit propionyl Co-A? - Converts to succinyl CoA - 3 enzymes involved 1. Propionyl-CoA Carboxylase 2. Methylmalonyl-CoA Racemase 3. Methylmalonyl-CoA Mutase - Where have we seen succinyl CoA? TCA intermediate 1. Propionyl-CoA Carboxylase - an ATP-dependent enzyme that catalyzes the carboxylation of propionyl-CoA to methylmalonyl-CoA using the prosthetic group biotin. This mechanism is the same as that of pyruvate carboxylase, formation of high-energy carboxyphophate using ATP to activate CO2 essentially and drive the reaction forming the biotin-bound carboxyl group for transfer to propionyl-CoA in a three step process. **Can you draw this mechanism?? You should be able to do so! 2. The stereospecific (S)-methylmalonyl-CoA product is converted to the R-form by the enzyme methylmalonyl-CoA racemase. 3. The (R)-methylmalonyl-CoA product is now used in an unusual rearrangement of the carbon skeleton using free radical chemistry to produce succinyl-CoA by the enzyme methylmalonylCoA mutase. This enzymes uses 5'-deoxyadenosylcobalamin (coenzyme B12) as a cofactor. *Methylmalonyl-CoA mutase utilizes a free radical mechanism to produce the carbon skeleton rearrangement depicted. The first step of the mechanism involves (1) homolytic cleavage, where one electron is transferred to Co and the other to 5'-deoxyadenosyl. Second, (2) the 5'- deoxyadenosyl-CoA radical abstracts a hydrogen atom from methylmalonyl-CoA, generating a methylmanolyl-CoA radical. Third, (3) a carbon skeleton rearrangement using what is a proposed cyclopropyloxy radical intermediate. Fourth, (4) the succinyl-CoA radical abstracts a a proton from 5'- deoxyadenosyl to produce succinyl CoA and 5'- deoxyadenosyl-CoA radical. Finally, (5) the product succinyl-CoA is released from the enzyme allowing for the reformation of the C-Co atoms to regenerate 5'-deoxyadenosyl coenzyme. **xethylmalonyl-CoA involves the unique B12 cofactor 5'-deoxyadensylcobalamin, whose structure was discovered by Dorothy Crowfoot Hodgkin in 1956 using x-ray crystollagraphy • Succinyl-CoA does not enter the TCA directly, rather it is converted to malate, which is then converted to pyruvate. • Why? - TCA cycle is a cycle - it would not be degraded for energy, rather just converted as a pool of TCA intermediates. propinyl CoA-> succinyl CoA to inc flux of CAC intermeidates or converted to malate in cyto-> pyruv-> PDHC-> CAC - as as sub to inc e and energy * malate aspartate shuttle • Malate produced using TCA cycle enzymes: - succinyl-CoA synthetase, - succinate dehydrogenase, - Fumarase • Malate is transported from the matric to the cytosol via the malate aspartate shuttle • Malate is then utilized by malic enzyme for oxidative decarboxylation to produce pyruvate and CO2 • Pyruvate enters matrix through pyruvate dehydrogenase complex and is converted to acetyl-CoA • Acetyl-CoA fuels the TCA cycle

What is Ketosis

• Oftentimes referred to as nutritional ketosis (NK), this is a diet that is high in fat and protein and low in carbohydrates - NK is usually consistent diet with daily consumption below 20 g of carbs(glucose, fructose) - Comparison: • Low carbohydrate is usually in the range of 50-100 g of carbs • Recommendation for weight loss is 50-150 g • Normal carb consumption is between 225-325 g (low estimate/healthy est)• For most people, NK simply relies on the liver to keep up with glucose for the brain and thus fats and ketone bodies are supplied for the peripheral tissues DEC carbs, inc fats and protien - less than 20 g carbs - hard to do - dec carbs so GNG and KB ( fast) produced in liver - supplement FA B-ox and fats release from adipocytes ( longer acting) fats-> liver KB -- dec adipose tissue - inc HSL, serum albumin, Box * good supply fo acetyl CoA for ATP production

Activation of Fatty Acids: Overview

• Once in the cytosol of muscle cells, FAs must be activated - modification FA + CoA + ATP ↔ acyl-Co + AMP + Ppi PPi → 2Pi **Requires 2 ATP investment for FA activation; costly Catalyzed by acyl-CoA synthetases, utilize same mechanism but differ on their specificities for the FA recognized for acylation with CoA 34 * specific to length of FA chain ATP nuc attack by FA at a phosphate - release beta and gamma phosphate as pyrophosphate and hydrolyzed to inorganic phosphate to be used as an energy source to drive reaction - acyladenylate has CoA nuc attack to force acyl CoA and AMP - acyl CoA moved from cytosol to matrix because that is the site for b-ox and entry into CAC R- CH chain that varies in length Acyl-CoA synthetase condenses fatty acids with CoA, with simultaneous hydrolysis of ATP to AMP and PPi • Formation of a CoA ester is expensive energetically • Reaction just barely breaks even with ATP hydrolysis • But subsequent hydrolysis of PPi drives the reaction strongly forward • Note the acyl-adenylate intermediate in the mechanism! ATP is utilized to activate the fatty acid through the formation of aceyladenylate mixed anhydride intermediate for nucleophilic attack of CoA. Following completion of the reaction, acyl-CoA, AMP, and PPi are produced. Pyrophosphate (PPi) is then hydrolyzed to inorganic phosphates to complete overall reaction, thus the equivalent of 2 ATP is required for activating each fatty acid.

Lipid Energy Metabolism

• Products from Beta oxidation feed into - Citric acid cycle - Electron transport / oxidation phosphorylation each 2 C unit= formation of acetyl CoA - substrate for oxidation in matrix of mitochondria 1 CAC= 3 NADH2 and 1 FADH2-> PMF_> ATP syn = 2 acetyl CoA drive CAC-> ETC-> Ox phsophoryaltion

Monounsaturated FAs

• Reminder: most FAs have cisdouble bonds - β-oxidation make use of transdouble bonds • β-oxidation proceeds until the double bond is reached. - Problem: trans-double bond fatty acid is not a substrate for the four enzymes involved in βoxidation - Answer: enoyl-CoA isomerase will catalyze the isomerization reaction converted the cisdouble bond to the transdouble bond. • β, cis-double bond becomes an α,β trans-double bond

Long distance bird migration

• Some humming birds cross Gulf of Mexico (>450 miles nonstop) during migration from South America to Canada. • Initial weight = 4.7 gram 2 grams fat range > 600 miles @ 25 mph* • Hummingbirds consume a high sugar diet and have fasting glucose levels that would be severely hyperglycemic in humans, yet these nectar-fed birds recover most glucose that is filtered into the urine . • Hummingbirds accumulate over 40% body fat shortly before migrations in the spring and autumn • Among the highest mass-specific metabolic rates known • Quickly enter torpor (state of lethargy or inactivity) and reduce resting metabolic rates by 10-fold *evening allow for glucose to be main E source Oxidation of fat energy + water FADH2 + ½ O2 H20 NADH + H+ + ½ O2 H20 1 kg of fat ≈ 1.4 liters H20 camels, bear (hibernation), and orca whales

Digestion

• TAGs are insoluble ( glycerol back bone and FA tails) • Enzymes operate in a soluble environment with an insoluble molecule • Lipid digestion is very minimal in the mouth (unlike carbohydrate digestion) and stomach (like protein digestion) • Stomach has gastric lipase, where fat droplets are reduced in size • Lipids are transported to the small intestines essentially intact - then digestion truly begins (lipid digestion) 1. carbohydrate digestion in the mouth by chewing and amylase 2. protein in stomach ( folds) with churning and protease ( pepsin) 3. lipid digestion in small intestines ( folds and villi) dudodenum, jejunum, ileum, amylase, protease, lipase, nuclease, nutrient and water absorption 4. liver- bile 5. gall bladder- stores and concentrates bile 6. pancreas- makes enzymes for small intestine

Transport of Activated Fatty Acids into the Mitochondrion: Carnitine cont

• Translocation → carnitine carrier protein. • Matrix → carnitine palmitoyl transferase II - Acyl portion is transferred to CoA using as transesterification reaction - Regenerates carnitine for the transport process and acyl-CoA in the matrix for use in fatty acid β-oxidation

Uptake of Free Fatty Acids

• We have already covered digestion, absorption, and transport of dietary lipids - Fed state, part of dietary metabolism ( high energy • What about fatty acids that come from adipocytes - fat storage cells in adipose tissue? - Low energy, fasting state - when the body deals with energetic demands because the diet is not sufficient * stored fats must be mobilized

Ketosis vs Ketoacidosis

• When excess ketone bodies accumulate, this abnormal (but not necessarily harmful) state is called ketosis. • When even larger amounts of ketone bodies accumulate such that the body's pH is lowered to dangerously acidic levels, this state is called ketoacidosis. - Associated with diabetics who cannot utilize glucose in high enough amounts to keep up with energy demands, hence diabetic ketoacidosis both inc KB but by vastly different levels

Beta Oxidation

• fatty acids are degraded in the mitochondria by removal of 2 -C units • the 2 -C unit released is acetyl -CoA, not free acetate • The process begins with oxidation of the carbon that is "beta" to the carboxyl carbon, so the process is called "beta - oxidation"

Activation of hormone sensitive lipase

• ↓blood glucose -or- fear/excitement ↑ glucagon ( liver and adipose) ↑ epinephrine • Activate receptors in liver and adipose ↑ cAMP -> activate protein kinases ( release/activate adenyl cyclase (AMP) ↓ lipid synth. & Activate hormone sensitive lipase (breakdown TAG for release from cells) Epinephrine or glucagon ↑ cAMP active cAMP dependent protein kinase (PKA) lipid droplets -> more accessible to hormone-sensitive lipase The rate of HSL hydrolysis dictates rate of diffusion of fatty acids passing across the adipocyte membrane to bloodstream 1. hormone ( glucagon or epinephrine) bind to receptor and structural changes to inc cAMP 2. cAMP bind to regulatory subunit to be released from catalytic subunit - activate kinases through a phosphorylation cascade - target-> activate hormone sensitive lipase 3. HSL lipid droplets that are stored to be released as TAGS and hydrolyzed and be released to blood stream as free FA - inc HSL inc diffusion and fats released to BS free FA have long phobic tails -FA transported in blood via serum albumin; binds to FA and enhance rate that they are pumped into the blood stream and enter to muscles cells easier - enters muscle cells (myocyte) et al. via FA transporter from cytosol to matrix( b-ox happens and start TCA/ETC)