Fat Econ 2, Carb Econ 1-2, Prostaglandin

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Medium Chain Fatty Acyl-CoA Dehydrogenase Deficiency

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fatty acid oxidation disorder associated with inborn errors of metabolism. It is due to defects in the enzyme complex known as medium-chain acyl dehydrogenase (MCAD) and reduced activity of this complex. This complex oxidizes medium chain fatty acids (Fatty acids having 6-12 carbons) while reducing FAD to FADH2

The most important part of treatment is to ensure that patients never go without food for longer than 10–12 hours (a typical overnight fast).

Patients may also take daily doses of carnitine, which helps reduce toxic accumulation of fatty acids by forming acyl carnitines, which are excreted in the urine.

characterized by metabolic acidosis, elevation of MCFA, dicarboxylic acids and fatty acyl-carnitines in the blood.

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How to beta oxidize unsaturated fatty acids?

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Requires additional enzymes (e.g., isomerase to bypass blocks in pathway due to double bonds)

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Three main sources of regulation of fatty acid breakdown

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1) Source and availability of fatty acids (lipase activity); activated by glucagon (cAMP --> PKA phosphorylates lipase and perilipin), inactivated by insulin

2) Transport across the mitochondrial membrane (rate-limiting under many conditions). Allosteric inhibition by malonyl CoA of the fatty acid biosynthetic pathway.

3) Activity of enzymes in the pathway (regulation at the transcriptional level), on a longer time scale. PPAR.

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Perilipin

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protein that coats lipid droplets in adipocytes, the fat-storing cells in adipose tissue. Perilipin acts as a protective coating from the body’s natural lipases, such as hormone-sensitive lipase, which break triglycerides into glycerol and free fatty acids for use in metabolism, a process called lipolysis.

hyperphosphorylated by PKA,changes conformation, exposing the stored lipids to hormone-sensitive lipase-mediated lipolysis

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PPAR (Peroxisomal Proliferator Activator Receptor)

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Fatty acids bind to these transcription factors that bind to PPAR response elements to activate transcription of genes involved in fat breakdown (lipolysis, fatty acid transport, and mitochondrial (or peroxisomal) B-oxidation.)

Mechanism for increase of fatty acid oxidation when fatty acids are in excess.

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Where is the main site of ketogenesis?

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Liver

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What are the products of ketogenesis?

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Acetoacetate and B-hydroxybutyrate (from Acetyl CoA)

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What does excess AcetylCoA get converted into? Is it an intermediate in the TCA cycle?

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It is NOT an intermediate in the TCA cycle (Any intermediate can get converted to oxaloacetate --> gluconeogenesis). Excess Acetyl CoA is converted to acetoacetate (ketogenesis).

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What compound is excreted by the lungs (breath) in ketosis?

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Acetone

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Steps of ketogenesis

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Acetyl CoA --> 3OH3CHglutaryl CoA (HMG synthase)

---> acetoacetate (HMG CoA Lyase)

---> B-hydroxybutyrate dehydrogenase (NADH --- NAD+)

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How are the ketone bodies used in peripheral tissues?

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B-OH-Butyrate more highly reduced, taking reducing compound out of the liver

In muscles, B-OH-Butyrate dehydrogenase converts -> acetoacetate, then AcetylCoA
Succinyl-CoA-Transferase goes to work to make Acetyl CoA.

Now can be used for energy production in TCA cycle.

Peripheral tissues are good at clearing it and converting to AcetylcoA. Can put them thru the TCA cycle

Muscles preferentially use ketones for energy production

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3 reasons why ketogenesis is important

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1) Sink for excess Acetyl CoA

2) Easily utilized energy source for peripheral tissue

3) Primary energy source for brain during prolonged fasting (Enzymes for metabolism of 3-oxybutyrates are induced in the brain during prolonged fasting)

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Hormonal regulation of ketogenesis

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Glucagon stimulates (along with other activators of hormone sensitive lipase), by increasing the flow of fatty acids towards B-oxidation and the inhibition of the TCA cycle by gluconeogenesis activation.

Insulin has opposite effects, inhibiting fat breakdown and promoting fat synthesis.

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Type I diabetes -- insulin deficiency: what does it do?

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Results in mass utilization of stored fat, which results in lots of ketone body accumulation, more than the body can use. the acidic ketone bodies accumulate in blood (ketosis), upsetting the acid-base balance and leading to excretion in urine (ketonuria). Urinary excretion of potassium may lead to depletion.

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How are branched chain or odd chain fatty acids oxidized?

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Can work as long as Beta position is still free.
Instead of releasing acetyl CoA, we release propionyl CoA (not ketogenic like AcetylCoA), but there is sequence of reactions to go to succinyl CoA


(An aside: Possible survival mechanisms that are not ketogenic
Long voyage into space might want to carry along fats in form of branched fatty acid)

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Why can't glucose be made from fatty acids (or any other substrate that breaks down to Acetyl CoA)?

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Acetyl CoA is oxidized by the TCA cycle. It makes no net contribution to the intermediates in the TCA cycle, ie., it doesn't replenish intermediates diverted to other pathways (e.g., gluconeogenesis)

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How do Very Long Chain Fatty Acids (>20C) undergo β-Oxidation?

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It can't happen in mitochondria.

Eating these induces proliferation of peroxisomes with alternative oxidative pathway (mediated by PPARα).

Peroxisomes oxidize the first few carbons from a >20C fatty acid, converting it to a substrate for the mitochondrial pathway.

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How is β-Oxidation different in the peroxisome (vs mitochondria?)

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(1) in the first oxidative step electrons pass directly to O2, generating H2O2

(2) the NADH formed in the second oxidative step cannot be reoxidized in the peroxisome, so reducing equivalents are exported to the cytosol, eventually entering mitochondria.

The acetyl-CoA produced by peroxisomes is exported; Acetyl-CoA produced in mitochondria is further oxidized in the citric acid cycle.

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high insulin/glucagon ratio promotes:

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storage of chemical potential energy in fat.

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high glucagon/insulin ratio promotes:

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beta oxidation

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What is Acetyl CoA converted to when insulin is high? What enzyme? How is it regulated?

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Malonyl CoA by <b> Acetyl CoA Carboxylase </b>.

ATP is hydrolyzed, a CO2 is added to the alpha carbon of Acetyl CoA.

Major point of regulation. Anything related to glucagon (e.g., protein kinase) will inhibit, anything related to insulin/anabolic state will promote.

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Why does citrate activate Acetyl CoA Carboxylase (ACC?)

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Presence of citrate is a signal that there’s excess Acetyl CoA that needs to be dealt with

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Why does Palmitoyl CoA and Fatty Acids inhibit ACC?

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Product inhibition

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Sterol Response Element Binding Protein (SREBP)

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Involved in regulation of many lipid pathways
Involved in regulation of carbohydrate pathways (transcriptional level)
Induces AcetylCoA Carboxylase by binding to Steroid Response Element (SRE) on promoter (Activators of transcription)

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SREBP1-SCAP complex

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<b> Method by which insulin can upregulate Acetyl CoA Carboxylase (ACC) </b>

SREBP1 and SCAP are proteins in a complex in the ER membrane.

Normally a protein INSIG is associated with the SREBP1-SCAP complex to lock it in the ER. Binding of cholesterol and oxysterol promote this locking.

Insulin prevents binding of INSIG to the SREBP-SCAP. If not locked, the complex gets taken to the golgi where they're cleaved by proteases. The cleaved products associate with their target gene, ACC, to activate.

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Synthesis of Palmitic Acid

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Happens in cytosol by action of <b> Fatty Acid Synthase </b> a huge enzyme catalyzing 7 reactions.

1) Acyltransferase acylates 2 active sit sulfhydryl groups in Acetyl CoA and Malonyl CoA.

2) Condensation. Malonyl CoA loses it's CO2 group and alpha carbon attacks carbonyl group of Acetyl-SENZ.

3) Reduction of keto group. Like reverse beta oxidation. Reducing equivalents come from NADPH.

4) Dehydration of 3OH fatty acyl ACP.

5) Reduction of double bond.

6) Fatty acyl group transferred back to sulfhydryl group, keep going 7x to release palmitic acid.

<img src = "http://dolly.biochem.arizona.edu/Bioc462b_Honors_Spring_2009/ighare/images/figure_12a.jpg">

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Differences between Beta oxidation and fatty acid synthesis

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Location: B oxidation is in the mitochondria; Fatty acid synthesis is in the cytosol

Acyl Group Carrier: Uses an acyl carrier protein (ACP) was the carrier.

Reducing equivalents: Synthesis (like anabolic pathways) uses NADPH. Oxidation (like catabolic paths) using NADH.

4) Carbon source: Synthesis starts with malonyl coA, whereas B oxidation ends with Acetyl CoA

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What is the rate limiting step in fatty acid synthesis? What regulates it?

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Formation of malonyl coA by Acetyl CoA Carboxylase (ACC).

Inhibits: Palmitoyl CoA, oleoyl CoA, CAMP, AMP protein kinases (from glucagon)

Activates: citrate

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Biotin: what does it do?

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It's the carboxyl binding cofactor in 4 carboxylase reactions.

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Citrate shuttle

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Transports citrate from mitochondria to cytosol, where <b> citrate lyase </b> regenerates acetyl-CoA to become substrate for fatty acid synthesis.

Signifies excess acetyl coA is available, and activates ACC.

Promotes synthesis of fatty acids.

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Elongase

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Enzyme in the ER membrane/microsome that act like fatty acid synthase.

Purpose is to synthesize long chain, unsaturated fatty acids for incorporation into membrane lipids and synthesis of biologically active metabolites.

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Desaturase

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Located nearby elongases in the ER, these enzymes introduce cis double bonds into specific locations in fatty acids (e.g., stearic acid --> oleic acid)

They use oxygen as a receiver of electrons in removing the electrons and forming the unsaturated C=C bond!

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What would the accumulation of 20:3o9 in blood and tissues signify?

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Essential fatty acid deficiency.

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Regulation of <b> unsaturated </b> fatty acid synthesis.

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1) Transcriptional regulation (long term): fatty acids, sterols mediated by nuclear transcription factors, including PPARa, SREBP1 and LXR.

2) Competition for the same enzymes among the substrates.

3) As long as Omega6 and 3 FAs are available, they are preferentially elongated, with the preference going to the one in the highest concentration, so it's important to have the balance in the diet.

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Why are fish oils containing EPA/DHA preferable to other omega 3 sources, like flax seed?

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Though linolenic (in flax) acid can be converted to EPA and DHA, it has marginal effects on tissue levels of EPA or DHA during prolonged feeding.

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Where are triglycerides made? (3)

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Adipose tissue, intestine, and liver

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Source of fatty acyls used in triglyceride synthesis

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Usually they're selected from newly made OR dietary sources by the long chain fatty acyl CoA synthetases (that make fatty acyl CoA from long chain fatty acids).

The substrate specificity of these enzymes assures that short and medium chain fatty acids are not usually found in triacylglycerols of animals with high dietary intakes.

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Are short, medium, and long chain fatty acyls all made into triglycerides?

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NO

Mainly long chain fatty acyls become triglycerides.

Short and medium chain are preferentially degraded by Beta oxidation or resynthesized into long chain fatty acids.

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How does the oxygen concentration affect desaturation reactions?

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Oxygen is used as the electron acceptor in desaturation reactions to synthesis oleic acid.

Oxygen is more soluble in colder water.

Therefore, cold water fish have more unsaturated fatty acids because more oxygen is available

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Difference in how plants vs. animals desaturate fatty acids.

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Both are starting from steric --> oleic acid (18:1o9).

Plants preferentially desaturate toward the methyl end, changing the omega classification.

Animals preferentially desaturate toward the carboxyl end, never changing the omega classification. (Animals can only make omega 9 fatty acids without plant linoleic and linolenic)

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What is the first/second step of triglyceride synthesis?

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Can start with <b> either </b> alpha glycerol phosphate OR dihydroxyacetone.

Convert between these with glycerol-3-phosphate dehydrogenase.

The second (really, first) step of triglyceride synthesis involves adding a fatty acyl group to the 1st carbon of glycerol (the one at the other end of the molecule from the phosphate group)

Acyltransferase is the name of the enzyme.

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Acyltransferases

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They add the Acyl CoA groups to the phosphoglyceride, forming triglycerides.

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Third/Fourth Step in triglyceride synthesis

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Third step can be dehydrogenase conversion between the two products of the second reaction.

Fourth step is the same for both -- Another Acyl CoA group gets added to the middle carbon.

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Fifth Step in triglyceride synthesis

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Catalyzed by phosphatidate phosphatase

Phosphate group gets removed from the 3rd carbon, leaving a hydroxyl group.

This step is probably most subject to short-term regulation.

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phosphatidate phosphatase

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Removes phosphate group from the 3rd carbon in 5th step of triglyceride synthesis.

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Sixth Step in triglyceride synthesis

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Addition of the final acyl coA group, resulting in a triglyceride!

(acyltransferase is the enzyme, of course)

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Where does triglyceride synthesis take place?

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Most de novo TAG synthesis takes place in the smooth endoplasmic reticulum of liver, which exports TAG in VLDL, or adipose tissue, which stores TAG as fat droplets

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Lipid rafts

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Fatty acyl CoA synthetases that provide substrate for triacylglycerol synthesis are located here. (ER membrane)

fatty acid transport protein is colocated with fatty acyl-CoA synthetase and triacylglycerol synthesis

The plasma membrane of cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains

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Phosphoglyceride synthesis

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Branches off from triglyceride synthesis at either of two points, depending on which precursor is activated.

1) Salvage pathway: called "salvage" because we're salvaging choline from diet or other. Branches off after the synthesis of diacylglycerol. Choline/ethanolamine precursor is activated as a CDP derivative which donates its phosphorylcholine moiety to diacylglycerol.

2) De novo pathway: most important is de novo synthesis of phosphatidylcholine by methylation of phosphatidylethanolamine. Only in liver

Overall, the body doesn't make enough phosphatidylcholine to not get some from dietary sources.

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Does the body make enough phosphatidylcholine to not get some from dietary sources?

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No, especially in times of growth or stress.

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How is phosphoglyceride synthesis regulated with fatty acid and sterol pathways?

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Probably through fatty acid availability via transcriptional activation of early acyltransferases by PPAR and activation by SREBP1 and SREBP2.

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Salvage pathway of phosphotidylcholine synthesis

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Starts with choline that has been "salvaged" from food, etc.

<b> Choline kinase </b> adds phosphate group, resulting in <b> P- Choline. Cytidylyl transferase </b> adds a CDP to choline, resulting in <b> CDP choline </b>. Then <b>phosphocholine transferase </b> adds the CDP choline to the diacylglycerol (from the triglyceride synthesis pathway).

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How is glycerolipid synthesis inhibited?

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By the phosphorylation (cAMP dependent kinase) of phosphatidate phosphatase and cytidylyl transferase, the rate limiting steps.

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The rate limiting step for both triglyceride synthesis AND phosphoglyceride synthesis is what?

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The conversion of the diacylglycerol to the product (whether triglyceride or phosphoglyceride).

Phosphatidate phosphatase in triglyceride synthesis is inhibited, and cytidylyl transferase in phosphoglyceride synthesis is inhibited.

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Transcriptional up regulation of lipid biosynthesis in absence of insulin

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In a high fat, low carb diet, the liver senses the buildup of fatty acids. PPARalpha upregulated triglyceride synthesis and other things that insulin would normally do.

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Prostaglandins: broad overview of structure

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20 carbon structure with a cyclopentane ring formed by a carbon-carbon bond between carbons 8 and 9.

Based on the structure of "prostanoic acid"

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Prostaglandins: broad overview of function

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Minor structural differences dictate wide range of functions, such as inflammatory AND antiinflammatory actions.

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Thromboxanes

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Similar to prostaglandin in structure and function except they have an oxane ring instead of a cyclopentane ring. Nomenclatures are similar. (TXXn)

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TXA2

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A thromboxane that mediates inflammation and is a potent platelet activator.

Precursor is arachidonic acid.

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Leukotrienes

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Similar structure to prostaglandins, but they have no ring. (LTXn)

They're secreted by leukocytes and mediate inflammatory responses, tumorigenesis, plaque destabilization, etc.

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Prostaglandin synthesis

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Enzymes are cyclooxygenase/prostaglandin synthase.

Start with arachidonic acid.

1) endoperoxidase rxn
2) hydroperoxidase

Yields PGH2, the precursor of all prostaglandins

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CLASSES OF CYCLOOXYGENASES (prostaglandin synthases): COX-1

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COX-1: Constitutively expressed. Produces prostaglandins that maintain stomach mucosa against acidic environment

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CLASSES OF CYCLOOXYGENASES (prostaglandin synthases): COX-2

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Induced response to growth factors and cytokines at inflammation and tumor sites

Pathway produces inflammatory/pain mediating prostaglandins.

Targeted by COX-2 inhibitors (celecoxib)

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Resolvins

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Active metabolites of 22-carbon polyunsaturated fatty acids.


Extensive class of lipids thought to mediate some of the antiinflammatory properties of DHA and EPA

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Nonsteroidal antiinflammatory drugs (NSAIDS) : how do they work

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They inhibit cyclooxygenases

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Steroid antiinflammatory drugs: How do they work?

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inhibit phospholipase A2 and release of the fatty acid precursors to all biologically active eicosenoids (leukotrienes, prostaglandins, thromboxanes).

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What is the concern of using COX-2 inhibitors?

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elevate the risk of myocardial infarction and stroke, apparently by knocking down production of antithrombogenic PGI2.

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What do platelets release?

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Active platelets secrete prostaglandins and cytokines that increase permeability of arterial endothelium

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What activates platelets? Inhibits platelets?

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TXA2 activates
PGI2 and PGI3 inhibits

TXA3 doesn't inhibit, but it doesn't activate either (TXA3 is the thromboxane made from EPA as precursor. TXA2 is the thromboxane made from arachidonic acid as precursor.)

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Why is aspirin better to take for heart attack prevention than tylenol or other NSAIDS?

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It <b> irreversibly </b> inhibits all classes of COX

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How can you tell how many double bonds will be in a prostaglandin?

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Depends on the fatty acid that you started from. It'll be the number of double bonds in the fatty acid - 2.

(review: PGE1 has one double bond, but the fatty acid precursor had three. PGE2 has two, but the precursor had 4.)

Different prostaglandins produced from different fatty acid precursors.

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Phospholipase A2

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In the short term, it regulates synthesis of eicosenoids by releasing precursor fatty acids (eg., eicosatrienoic, arachidonic) from membrane phosphoglycerides.

PLA2 activity is substantially regulated by inhibitory proteins which are induced by steroids (mechanism for steroidal antiinflammatory agents like cortisone)

Since steroids block synthesis of ALL eicosenoids, they're more effective antiinflammatory agents than NSAIDS which do not block lipoxygenase or monoxygenase.

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How does aspirin prevent myocardial infarction?

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Based on the idea that platelets preferentially produce inflammatory <b> thromboxanes </b>, while endothelial cells produce antiinflammatory <b> prostacyclins</b>, which inhibit platelet activity.

Aspirin irreverisibly inhibits the COS in both cell types, but the platelets can't make more bc they have no nucleus.

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How does eating eicosapentanoids (EPA) reduce risk of atherosclerosis?

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Because the fatty acid precursor dictates which class of prostaglandin gets produced.

TXA3 produced by platelets from EPA does not activate platelets.

PGI3 produced from EPA DOES inhibit platelets.

Net effect is to inhibit platelets.

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What is the difference between TXA2 and TXA3?

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Despite being quite similar, one of them (TXA3) is synthesized from EPA, and does not activate platelets. The other (TXA2) is synthesized from arachidonic acid and it DOES activate platelets.