Lecture 13: Lipid Metabolism

Where in the body are ketone bodies used as fuel?

Acetoacetate and D-beta-hydroxybutyrate are fuel molecules that are normally used by the heart and renal cortex (kidney). Under starvation conditions, the brain also uses ketones

Where are all carbons in cholesterol derived from?

All carbons in cholesterol are derived from acetate (as acetyl-CoA)

How is cholesterol transported?

lipoproteins

Fatty acids can be used to build up larger lipids such as

triacylglycerols and glycerophospholipids

Diabetes

- A condition in which the body is unable to produce enough insulin, the hormone required for the metabolism of sugar - Body "thinks" you are starving b/c you can't uptake glucose into cells b/c of lack of insulin - As a result of insulin malfunction, cells can not absorb alot of glucose; therefore, cells are starved for glucose b/c glucose is "trapped" in blood. As a result, blood sugar is high

Fatty acid desaturation

- A fatty acid desaturase is an enzyme that removes two hydrogen atoms from a fatty acid, creating a carbon/carbon double bonds - The desaturation of a fatty acid is an oxidation reaction that requires molecular oxygen and two electrons; however, oxygen itself is not incorporated into the fatty acid chain but is released in the form of water - fatty acid desaturation increases membrane fluidity

Beta Oxidation

- A metabolic sequence that breaks fatty acids down to two-carbon fragments that enter the citric acid cycle as acetyl CoA - Beta oxidation forms Acetyl CoA - occurs in matrix of mitochandria - each time fatty acid undergoes beta oxidation, the chain is shortened by 2 carbons

What are the effects of a diet composed largely of fats on liver metabolism?

- Acetoacetate (ketones) concentration increases (b/c need to transport ketones around body to generate energy since have no carbs that can be broken down during glycolysis and TCA tp generate energy) - Oxaloacetate concentration decreases (b/c fats are 2C and all 2C are lost as CO2, which causes a depletion of TCA intermediates or OAA will be converted to gluconeogenesis intermediates). - Acetyl‑CoA is no longer able to enter the citric acid cycle (cant enter TCA if OAA is low) Oxaloacetate concentration decreases because it forms from pyruvate, a product of carbohydrate metabolism. If carbohydrates are not being ingested, the oxaloacetate concentration drops because it is shunted into gluconeogenesis to provide glucose. Since oxaloacetate is not available, acetyl‑CoA is unable to enter the citric acid cycle and it accumulates. Excess acetyl‑CoA is used in the formation of the ketone bodies acetate, acetoacetate, and D‑β‑hydroxybutyrate.

After fatty acids are broken down by beta oxidation, what are the 2 fates of acetyl CoA?

- Acetyl CoA can enter TCA cycle to generate energy of it be used to form ketones - Acetyl CoA generated from beta oxidation of fatty acids can only be used to generate ATP if there is enough oxaloacetate (OAA) around. When carb intake is low (ex: fasting or diabetes), the liver will use the majority of OAA to produce glucose via gluconeogenesis. OAA can be converted to malate and enter gluconeorgensis. (recall, pyruvate → OAA → PEP in gluconeorgenesis and OAA can be converted to pyruvate). Under these conditions, the liver diverts the acetyl CoA to form Ketone bodies.

Regulation of fatty acid synthesis

- Acetyl CoA carboxylase is regulated - ↑ Citrate activates Acetyl CoA carboxylase (indication of high energy; therefore, dont want to generate more by acetyl CoA going into TCA) - ↑ Palmitoyl CoA inhibits Acetyl CoA carboxylase b/c dont want to generate any more malonyl CoA if there is alot of fatty acids around - ↑ Glucagon means glucose and every levels are low. Therefore want to go into TCA (to generate energy) and glucoenoegenesis (to generate glucose) -

Coenzyme B12

- B12-containing enzymes catalyze molecular rearrangements and methyl transfer reactions - contain cobalt

Activation of Fatty Acids

- Before the fatty acid can make its way into the mitochondria, it has to be activated by an outer mitochondrial enzyme called acyl CoA synthase. Enzyme catalyzes the formation of a thioester bond between the carboxyl group of the fatty acid and coenzyme A - Fatty acyl CoA is activated form of fatty acid - uses fatty acyl‑CoA synthetase enzyme - uses 2 ATP

Beta oxidation vs fatty acid biosynthesis

- Beta Oxidation: Fatty acids are broken down to acetyl CoA by means of beta oxidation inside the mitochandria (fatty acids → acetyl coA) - Fatty Acid Biosynthesis: Fatty acids are synthesized from acetyl coA in the cytosol (acetyl CoA → fatty acids) Shuttle: - Beta oxidation: Carnitine gets fatty acids into matrix - Fatty acid biosynthesis: Citrate gets acetyl CoA into cytosol

What occurs if fatty acid is fully saturated and has an even number of carbon atoms?

- Beta oxidation process can occur over and over until we breakdown carbon atom until single acyl units

How is cholesterol synthesis regulated?

- By HMG-CoA reductase Have to balance biosynthesis of cholesterol with dietary uptake - ↑ Oxysterol inhibits HMG-CoA reductase: b/c do not to make more cholesterol if we already have alot in body - ↓ ATP inhibits HMG-CoA reductase: If ATP is low, dont have enough energy to build up molecules - ↑ Glucagon inhibits HMG-CoA reductase (high glucagon means there is low energy so you can not synthesize cholesterol) - ↑ Insulin activates HMG-CoA reductase

Beta Oxidation of polyunsaturated fatty acids (contain multiple double bonds)

- Enzymes reduces double bonds into a single double bond. The double bond is then moved to the right place so the intermediate can be placed in the beta-oxidation pathway - requires reductase and isomerase

Fatty acid Breakdown and Synthesis

- Fatty acid metabolism consists of a breakdown (beta oxidation) process and a synthesis process - Breakdown (beta oxidation): Oxidative process that releases an activated acetyl CoA unit, thereby shortening the hydrocarbon chain by 2 carbon atoms. The activated CoA units can enter the TCA, which can ultimately be used to form ATP molecules. Used to generate energy - Bioynthesis: This is a reductive process that builds up the hydrocarbon chain of fatty acids. Beginning with an activated acyl unit and mononyl unit. Want to synthesize fatty acids if we do not need more ATP or if we want to build a hormone, modified protein, or cell membrane

Beta oxidation of fatty acids with an odd-number of fatty acids

- Fatty acids with an odd number of carbon atoms are oxidized in the same way that even numbered fatty acids are, except, that one of the final products is a 3-carbon propionyl-CoA molecule. Propionyl-CoA molecule is utlimarly converted to Succinyl CoA which goes into TCA to help generate energy

acetyl group shuttle

- First step in fatty acid synthesis which moves Acetyl CoA from mitochondria to cytosol (by conversion to citrate from oxaloacetate). Citrate is moved across the membrane because Acetyl CoA cannot be moved across the membrane. - Citrate is the carrier molecule that can cross the membrane - process transfers acetyl CoA from matrix → cytosol - we need NADPH that is generated in the shuttle for the fatty acid synthesis process https://aklectures.com/lecture/fatty-acid-synthesis/initiation-of-fatty-acid-synthesis

What occurs during starvation?

- First, glycogen stores are depleted, then gluconeogenesis occurs. OAA is converted to gluconeogenesis intermediates so glucose can be produced - Gluconeogenesis depletes TCA intermediates (ex: OAA). This causes the TCA to slow, leading to acetyl CoA accumulation b/c acetyl coA cant combine with OAA to form citrate. Acetyl-CoA is diverted to ketogenesis instead

Breakdown of triglycerides

- Glycerol is broken down into glycerol and fatty acids - Glycerol ends up in liver and can be used to generate ATP (glycolysis) or glucose (gluconeogeneis)

Regulation of cholesterol metabolism

- HMG-CoA reductase is the target of many "cholesterol-lowering" drugs (statins). Statins act as competitive inhibitors. - Statins are competitive inhibitors that inhibit the biosynthesis of cholesterol - Statins bind to the active site of HMG-CoA reductase

What conditions lead to fatty acid biosynthesis?

- High ATP (therefore dont want to produce more by acetyl CoA going into the citric acid cycle). Recall anabolism requires high energy - High levels of citrate (indicative of high ATP) - When ATP in the matrix is high, ATP inhbits isocitrate dehydrogenase. This causes the buildup of isocitrate. If isocitarte builds up, citrate, will also buildp

What domains of fatty synthase have thioester- forming SH groups?

- KS and ACP domains - SH group allows the binding of certain molecules; malonyl groups are esterfied to the -SH group

How are ketone bodies used as fuel?

- Ketones are water soluble and move through the blood to target cells. Once in the target cells, ketones are converted back to acetyl CoA. In the target cell, acetyl CoA can be combined with OAA to enter TCA to generate energy (ATP)

What happens if an individual is Vitamin B12 deficient?

- L-methylmanoyl CoA → Succinyl CoA reaction will not occur so there will be a buildup of L-methylmanoyl CoA - deficiency interferes with breakdown of odd chain fatty acid - deficiency can occur if a person has a plant-based diet

What molecule is responsible for coordinating regulation between fatty acid synthesis and breakdown?

- Malonyl Co-A, the dedicated enzyme for biosynthesis - ↑ malonyl coA inhibits carnitine acyl-transferase 1 (this enzyme commits fatty acid to beta oxidation to form acetyl coA). If we break down fatty acids and that's not regulated, fatty acids will be imported into the mitochondria via carnitine and broken down to acetyl coA

Methylmalonyl-CoA mutase

- Methylmalonyl-CoA mutase catalyzes a carbon skeleton rearrangement in the conversion L-methylmanoyl CoA → Succinyl CoA - Succinyl CoA can go into TCA to help generate ATP - B12

How many H+, FADH2, and NADH molecule are produced for each round of beta oxidation?

- NADH: 1 - FADH: 1 - H+: 1

Head group attachment in Sphingolipids

- Open hydroxy is used for head group attachments

What is the precursor for triacylglycerols and glycerophospholipids?

- Phosphatidic Acid - Phosphatidic Acid consists of 2 fatty acids to L-glycerol 3-phosphate

Beta Oxidation of Palmitoyl-CoA

- Recall, each cycle shortens the carbon chain by 2; therefore, since Palmitoyl-CoA is a 16 carbon molecules (16./2 = 8), 8 Acetyl CoA's are formed - 7 rounds - NADH and FADH2 molecules move on the ETC after formation https://aklectures.com/lecture/fatty-acid-breakdown/atp-yield-in-fatty-acid-oxidation

How much ATP does the Beta Oxidation of Palmitoyl-CoA generate?

- Recall, in TCA, each acetyl CoA generates 3 NADH, 1 FADH2, and 1 GTP/ATP

Beta Oxidation of unsaturated fatty acids (contain a single double bonds)

- Requires additional enzymes that will ultimately convert the fatty acid into an intermediate that can be placed in the beta-oxidation pathway - The trans enoyl-CoA can be can enter the beta oxidation pathway and be acted upon by enoyl CoA hydratase

Why is OAA depleted during starvation and diabetes?

- Starvation and diabetes are indicators of low sugar absorption in cells (i.e. liver cells) which promotes gluconeogenesis (creation of glucose). Gluconeogenesis uses OAA b/c OAA can be converted gluconeogenesis intermediates that are used in the first bypass reaction. Since there is low OAA, acetyl CoA cant combine with OAA to form citrate, so acetyl CoA starts to accumulate.

How can Succinyl CoA be used to used in gluconeogenesis?

- Succinyl CoA can be converted to gluceoneogenesis intermediates and used in the first bypass reaction where pyruvate → PEP in first step of gluconeogenesis

What are Sphingolipids derived from?

- They are derived from serine and palmitoyl-CoA (fatty acid). These form the backbone

How can we get to triacylglycerols and glycerophospholipids from Phosphatidic Acid?

- Triacylglycerols: Once we have phosphatic acid, we get to triacylglycerol by releasing the phosphate group forming a diacylglycerol compound. We then esterify a third fatty acid to compound to form a triacylglycerols - Glycerophospholipid: Add polar head group to the phosphodiester bond.

What are the fates of fatty acids after fatty acid synthesis?

- Used directly - Chain lengthening (longer fatty acids) - Desaturations (Note: some desaturations humans can not do—essential fatty acids)

Can long chain desaturated fatty acids be synthesized from palmitate?

- Yes - Some desaturations cannot be performed by mammals. The products must be supplied in the diet as "essential fatty acids. For example alpha-linolenic acid (an omega-3 fatty acid) and linoleic acid (an omega-6 fatty acid) need to be supplied in the diet because humans can't make them. Only plants can - essential means humans cant make it, so we have to get it through our diet

ketoacidosis

- a high level of ketones in the blood - caused by prolonged alcohol use, starvation, and type 1 diabetes

Ketone bodies

- compounds produced during the incomplete breakdown of fat when glucose is not available in the cells - includes: acetoacetate, acetone, and D-beta-hydroxybutyrate. - Derived from acetyl-CoA - Important fuels - water soluble so can move through blood

Acetyl CoA carboxylase

- enzyme catalyzes the rate-limiting step in fatty acid synthesis in which acetyl co-A → malonyl CoA Enzyme contains 3 different things: 1) Biotin 2) ATP (energy source) 3) Carbon source (HCO3-): Carboxylase attaches CO2 onto acetyl CoA, elongating the molecule by 1 carbon. Thats why we have HCO3- - Enzyme has 2 active sites connected by a biotin carrier molecule. The left active site charges the biotin so it can carry CO2, and the second binding site carries out the actual catalysis of making malonyl CoA

Ketogenesis

- formation of ketone bodies - occurs during carb (i.e., glucose) deficiency - occurs in the liver

Committed step in fatty acid synthesis

- formation of malonyl CoA Malonyl-CoA (C3) is the major carbon source for FA biosynthesis

palmitic acid

- most common fatty acid in humans - 16 carbons atoms long - activated by palmitoyl CoA

Stages of fatty acid oxidation (fatty acid catabolism)

1) Beta oxidation of acyl chain → acetyl-CoA: Process of clipping fatty acid into smaller components. Releases energy in the form of reducing equivalents (NADH and FADH2) and also releases acetyl CoA 2) Oxidation of acetyl-CoA → CO2: Acetyl CoA enters the TCA and is converted into CO2 3) Electron transfer → ATP: In stage 3, reduced cofactors go to ETC and are oxidized. This process releases ATP https://www.youtube.com/watch?v=ZufZvbhPpws

How do triglycerides make their way to other cells in the body? (ex: of fatty acid breakdown)

1) Breakdown and mobilization of triglycerides into fatty acid and glycerol 2) Activation of fatty acids and their transport into the mitochondria 3) Degradation of fatty acids into acetyl coA and ATP generation

How are head groups attached to Glycerophospholipids?

1) CDP is an activating molecule. It activates the OH group on the head (shown on right) or on the diaclyglycerol 2) Nucleophilic attack occurs 3) CDP is a good leaving group and leaves as a CMP 4) Head group is attached to Glycerophospholipid - note: one of phosphates comes from CDP

How is acetyl CoA converted to malonyl CoA?

1) Energy generated by the hydrolysis of ATP helps us attach a CO2 onto the biotin 2) CO2 is transferred from biotin onto the acetyl CoA molecule to generate malonyl CoA - rate limiting step - requires energy - enzyme: Acetyl CoA carboxylase

What are fatty acids 4 important roles:

1) Fatty acids are used for fuel 2) Fatty acids are used to build cell membranes 3) Fatty acids are used to diversify protein function 4) Fatty acids are used to build hormones and other messenger molecules

What occurs during starvation?

1) Gluconeogenesis depletes TCA intermediates (b/c intermediates are being pulled out of TCA and used for gluconeogenesis) 2) TCA slows and acetyl-CoA builds 3) acetyl-CoA → acetoacetate (ketones) Ketone bodies sent to brain, muscle, heart, and kidneys 4) Regenerate acetyl-CoA from ketone bodies to use as fuel in target organs

What happens in type 1 diabetes?

1) In type 1 diabetes, lack of insulin will decrease absorption/uptake of glucose into the liver cells 2) Lack of glucose absorption in cells will make cells think they are "starving." This will decrease the production of OAA in the liver cells b/c OAA will be used for gluconeogensis to produce glucose. 3) As fatty acids are absorbed into the liver, they are converted into acetyl CoA 4) Since OAA levels are low, acetyl CoA can not combine with OAA to enter the TCA. The only pathway left for acetyl CoA is ketogenesis 5) Ketone bodies are released into the blood causing ketoacidosis https://aklectures.com/lecture/ketogenesis/diabetic-ketoacidosis

ketogenesis steps

1) Two molecules of acetyl CoA are combined to generate acetoacetyl CoA. This is reversible and catalyzed by thiolase 2) Mitrochandrial HMG CoA synthase catalyzes the condensation reaction in which it adds another acetyl-CoA molecule onto acetoacetyl CoA. The hydrolysis of the thiosteser bond drives this reaction forward. This is the rate limiting step of ketone body formation 3) The enzyme HMG CoA lyase then cleaves the HMG CoA, thereby releasing CoA and forming acetoacetate. This is a metabolizable ketone body. It can be broken down to form other ketone bodies such as acetone, and D-beta-hydroxybutyrate - occurs in liver

Cholesterol biosynthesis

1) synthesis of mevalonate (C6) from acetate (3 C2): committed step in cholesterol biosynthesis 2) activated isoprene (C5) production: activate compound by phosphorylating it 3) condensation of activated isoprene units: Isopropene units are polymerized into a precursor, a linear precursor called squalene 4) cyclization of squalene

What are the 4 steps of beta oxidation?

1. oxidation: Double bond created 2. hydration: Double bond hydrated and an OH group is placed 3. oxidation: OH group is oxidized to form a keto group 4. thiolysis: Releases shortened fatty acid component attached to CoA (acyl-CoA) and acetyl CoA https://www.youtube.com/watch?v=ZufZvbhPpws

Cholesterol

A lipid that forms an essential component of animal cell membranes and acts as a precursor molecule for the synthesis of other biologically important steroids.

Where does the beta oxidation chemistry occur?

Beta carbon of the fatty acid

Why would fatty acids undergo beta oxidation?

Beta oxidation is an oxidative process that releases an activated acetyl CoA unit, thereby shortening the hydrocarbon chain by 2 carbon atoms. The activated acetyl CoA units can enter the TCA, which can ultimately be used to form ATP molecules. Used to generate energy

Fatty Acids

Contain: 1) A long hydrocarbon chain (hydrophobic) 2) A Carboxylic group (hydrophilic)

Is more energy generated from the oxidation of glucose or fatty acids?

Fatty acids - Per carbon, Palmitoyl-CoA yields 106 ATP/16 carbon = 6.625 ATP/C - Per carbon, glucose yields 30/6 or 32/6 = 5 to 5.333 ATP/C - Per C atom oxidized, fatty acids yield 6.625/(5 to 5.333) = 1.24 to 1.325 times more ATP Fats provide more energy from oxidation and are easier to store (hydrophobic so don't require water to be stored)

Fatty acid biosynthesis

Fatty acids are synthesized from acetyl CoA in the cytosol - requires high levels of acetyl coA in the cytosol and high levels of NADPH https://www.youtube.com/watch?v=WuQS_LpNMzo

Transport of Fatty Acids into the Mitochondria

Fatty acids need to be transported from the cytosol → mitochondria matrix Steps: 1) Once activated, the fatty acid must be transported into the matrix via a transport protein called translocase. To do this, the acyl CoA must first react with carnitine to form acyl carnitine 2) The acyl carnitine then moves into the matrix via translocase. In the matrix, the acyl group is transferred back onto CoA to reform acyl CoA in matrix, while the carnitine is shuttled into the cytoplasm - Formation of acyl carnitine is the rate-limiting step in fatty acid oxidation; it is inhibited by the fatty acid precursor malonyl-CoA

Cholesterol biosynthesis: Condensation

Form 30 carbon molecule

Entry of glycerol into the glycolytic pathway

Glycerol → L-glycerol 3-phosphate → DHAP → D-glycerol 3-phosphate → glycolysis

What are determination of blood groups?

Glycosphingolipids - Sphingolipids are the antigens on the surface of erythrocytes that determine blood group. - sugar determines specific blood group

What common precursor are triacylglycerols and glycerophospholipids synthesized from?

L-glycerol 3-phosphate

What are the fates of cholesterol?

Need cholesterol as not only a membrane component, but also as a precursor to give us other molecules, hormones, etc.

Are fatty acids synthesized directly from acetyl CoA?

No - needs help of Malonyl-CoA

Formation of Palmitate (fatty acid)

Palmitate is a 16 C fatty acid

What problem would arise if the excess acetyl‑CoA were not converted to ketone bodies?

Problem: Fatty acid beta oxidation would stop when all of the CoA is bound as acetyl‑CoA. Recall, activation of fatty acids for beta oxidation requires CoA Solution: When ketones are formed the enzymes converting acetyl‑CoA to acetoacetate liberate the CoA to be used in further fatty acid oxidation

How is Succinyl CoA formed from Propionyl-CoA?

Steps: 1) Propionyl-CoA → D-methylmanoyl CoA: The Propionyl-CoA is converted into D-methylmanoyl CoA by the activity of the enzyme prnopinyl CoA carboxylase. This enzyme depends on the B-vitamin biotin. 2) D-methylmanoyl CoA → L-methylmanoyl CoA 3) L-methylmanoyl CoA → Succinyl CoA: Intramolecular rearrangement reaction. This rearrangement occurs in the presence of vitamin B12 - anaplerotic reaction b/c Succinate can then be fed into TCA

Steps of fatty acid synthesis

To start: KS subunit loaded with acetyl-CoA, ACP subunit loaded with malonyl-CoA Condensation: - Add molecule in KS unit onto malonyl-CoA in ACP unit - lose CO2 Reduction: - Carbonyl on beta reduced to hydroxyl - NADPH used Dehydration: - Remove water to form double bond between alpha and beta Reduction - Double bond removed; two hydrogens added - NADPH used Afterwards: growing chain moved to KS, ACP reloaded with malonyl-CoA; cycle repeats until cleavage by thioesterase - 2 carbons are gained each cycle - 2 NADPH are used each cycle https://www.youtube.com/watch?v=WuQS_LpNMzo

In vertebrates, what does desaturation to a monounsaturated fatty acyl-CoA involve?

an electron transport chain

What enzyme carriers out fatty acid biosynthesis?

fatty acid synthase - Fatty acid synthase contains 7 different active sites - ACP moves molecules across all the catalytic active sites of protein

General nomenclature of fatty acids

fatty acyl-CoA

Where are fatty acids stored?

in adipocytes (adipose cells) as triglycerides