Cook Notes

Cook lectures- fatty acid metabolism
George Cook- pharmacology department
He covers fatty acid synthesis and metabolism. Following Stryer, which he says is a pretty horrible text for this..
2: Start with fat cell. Easy to isolate and use in solution. The cell in the photo is full of triacylglycerol. Very little cytoplasm.
3: This slide may be an exam question. 1 g glycogen stored- exists hydrated w 2 g water. Takes 6 g in cell to= 1 g fat. Fat is important energetically. Migratory birds could not fly without storing energy as fat.
Energy is from TCA –acetyl coA goes into TCA cycle.
Slide 4:Fatty acid oxidation- note similarity to TCA cycle:
1. the succinate dehydrogenase reaction to transform succinate to fumarate in TCA is like the oxidation(dehydrogenation)of the fatty acyl coA by acyl coA dehydrogenase(3 isozymes on slide 30) to form trans-delta2-Enoyl CoA. Both enzymes have a flavin, FAD, that forms FADH2 to donate electrons to the electron transport chain in the mitochondrion.
2. the conversion of fumarate to malate (hydration) in TCA is like the hydration of trans-delta2-Enoyl CoA to form L-3 - hydroxyacyl CoA by enoyl coA hydratase (see slide 31).
3. the conversion of malate to oxaloacetate (oxidation)is like the oxidation of L-3 hydroxyacyl CoA to form 3-ketoacyl coA (see slide 32) by beta-hydroxyacyl CoA dehydrogenase, which forms NADH. Then the ketoacyl-CoA is slplit to form an acylcoA shortened by 2 carbons and an acetyl-CoA that can go into the TCA cycle by thiolase (See slide 33). Why does it happen this way? When you find a way that works, you use it. See slides 29-33.
Look at the synthesis overview on the same slide(4) and refer to slides 63-71: Please note that all these reactions take place while the chain is attached to the Acyl Carrier Protein (ACP) in a massive complex of 7 proteins called Fatty Acid Synthase.
Activated acyl group +activated malonyl coA yields an acetoacyl group attached to the ACP. This condensation forms a diketoacyl ACP. Then there is reduction to form D-3 hydroxybutyryl-ACP, dehydration to form crotonyl ACP , and reduction using NADPH to form butyryl ACP, resulting in an activated acyl group to go through the cycle again until the cycle is finished with palmitate.
For synthesis and oxidation, the processes look like one is the reverse of the other, but the enzymes and locations are different. For these differences, see slide 62.
Slide 5: To do anything with fatty acids, you must get them into the cells first. Glycocholate is an example of a bile salt, which is important for the absorption of fat. Micelles form with the aid of bile salts in the intestinal lumen, which allow intestinal lipases access to digest the fats. Glucocholate is a Bile acid derived from cholesterol for lipase function.
Slide 6: Intestinal lipase clips triacylglycerol to hydrolyse off one fatty acid, to forms a diacylglycerol. Then further lipase action yields a monoacylglycerol in intestine, which is as far as metabolism goes there. No proteins were thought to transport fatty acids into the intestinal mucosal cells until recently. For short and medium-chain fatty acids, the monoacyl glycerol gets into membrane, flips, gets into cell that way. Long chain FAs appear to need help from fatty acid transporters (slide 7).. The fatty acids form triacylglycerols, which are packages in chylomicrons in the mucosal cells.
Chylomicrons go to lymph system. Why? Look at animal in fed state, see dilated blood vessels in intestine. The blood absorbs other nutrients, but chylomicrons to lymph. Do not want to deliver chylomicrons directly to liver, where they would overload the liver and be processed undesirably- want them to go to skeletal muscle and storage cells. Lymph empties in neck to general circulation. Lipids are not metabolized in liver first. Other Triacylglycerols get taken up and do go to liver.
In liver- some TAGs are put into VLDL. Good cholesterol- form HDL. Reverse cholesterol delivery- HDL brings cholesterol back to liver. Bad- LDL-leftovers when VLDLs get depleted a bit. LDLs form plaques.
FA transporters in fat tissue and muscle cells:
CD36 or Fatty Acid Transporter- most important. As originally described, it allows transport of FAs, docking to system, access so that lipases can hydrolyze triacylglycerol. 6 isoforms found in mouse and human cells. Rat different. Still questions about them . FA transport in yeast in bacteria-they form acyl coA. Free FAs have detergent function and are dangerous. FA transporters from mice can be explored in yeast. Isoforms 1 and 2 are transporters. 5 and 6 may be synthetases. 4 may be enzyme involved in cholesterol transport. Some transport not clear.
In adipose tissue cells resynthesize TAG. Stored in TAG form. To release- form free fatty acids. Lipases called hormone sensitive lipase and adipose tissue lipase are involved in forming free fatty acids for release into blood(slide 9). The glycerol liberated in adipose cells released by hormone sensitive lipase and adipose tissue lipase can be used for gluconeogenesis or glycolysis (slide 11). Hormone sensitive lipase clips first 2 fatty acids and monoacylglycerol lipase cleaves off the last fatty acid. Monoacylglycerol is active all the time.
Slide 10: Hormone sensitive lipase is highly regulated. Activated by epinephrine, norepinephrine, adrenocorticotropic hormone (ACTH). ACTH is secreted from the pituitary in response to corticotropin releasing hormone (CRH) from hypothalamus. ACTH acts on adrenal gland to produce norepinephrine and epinephrine from medulla. Hormone sensitive lipase has to be phosphorylated to be activated. Fatty acid binding protein is important in helping triacylglycerol lipase to associate with the lipid droplet after the enzyme has been phosphorylated.
11:Glycerol can be made into a primary substrate for gluconeogenesis.
12: Fatty acids released from adipose tissue are transported by albumin during fasting. Albumin has 3 binding sites. 6 disulfide bonds create hydrophobic lipophilic pockets.3 Fatty acids bound per molecule. Binds steroid hormones as well, but main function is fatty acid transport.
13: To oxidize fatty acids, must have acyl coA form. This form is necessary for oxidation or for esterification to form TAGs. ATP is hydrolyzed to inorganic pyrophosphate and AMP. The AMP is attached to the fatty acid, then HS-CoA joins to form acyl coA.
14: Steps to get to fatty acid oxidation (FRAGMENT- this comes BEFORE the steps in the summary in the beginning of the lecture, and allows the FAs to get into the mitochondrion.):
1. Acyl coA synthetase in mammals on external mitochondrial membrane catalyzes the reaction in slide 13, creating a high-energy coA bond.
2. carnitine palmitoyltransferase (CPT, his research chemical)- There isn’t an acyl-coA transporter to get through the impermeable inner mitochondrial membrane, so another form of the fatty acid has to be transported- mitochondrion has its own synthesis for coA, so we get it back.
15:Acyl carnitine is transported into mitochondrion, carnitine out. Carnitine can go in or out on transporter, but not acylcarnitine out.
Carnitine acyltransferase actually does the transfer across the membrane.CPT does the reaction to get the carnitine-acyl bond.
16:Malonyl coA inhibits fatty acid oxidation. It Is a product in synthesis used for making Fatty Acids. Oxidation and synthesis go on in same cell. Oxidation occurs in the mitochondrion, synthesis in cytosol. Malonyl coA is important to keep things in balance. Pathways are regulated in opposite directions.
There is more than one acetyl coA carboxylase. Acetyl-coA carboxylase I is free in cytosol for FA synthesis. Acetyl CoA carboxylase II has one transmembrane region and is bound to mitochondrion on outside to make malonyl coA. Heart and fatty tissue have both. There are 2 carnitine palmitoyl transferases,too. One on outside inhibited by malonyl coA and one on inside is not.
CPT activated by cardiolipin is found in inner membrane and contact sites. How FA crosses outer membrane has yet to be solved.Lipids will not go through porins- porin is too charged.
20: 3 isoforms of CPT-1. Outer membrane may contain CD36.
18: Peroxisomal fatty acid oxidation- very long chain polyunsaturated Fatty acids from fish oil are oxidized here. May have same transport- difficult to isolate. Yeast does not oxidize FA in mitochondrion- oxidize FA in peroxisome. CPT is in ER- found in microsomes. Transport systems may get FAs in to make VLDL and chylomicrons.
19:CPT on outer membrane- 2 transmembrane segments.
CPTII in matrix- has matrix targeting sequence. Cleaves and is active, but in vitro is active even without cleaving the targeting sequence.
Peroxisomal form has same specificity. COT metabolizes octanoic acid- same chain specificity as CPT.
carnitine acyl transferase (CAT)-short chain specificity. CPT I is only one with multiple isoforms.
20: alpha found in liver and kidney. Sensitive to inhibition by malonyl coA.
beta sensitive to malonyl coA.
Gamma- binds malonyl coA,important function, may be exclusive to brain. Not active with any tried substrate so far. Not known if it works on substrate. Alpha and beta are on outside of mitochondrion.
Malonly coA concentration does not vary a lot.
Mitochondrial CPT in fed animals- potently inhibited by malonyl coA. No inhibition in fasting.
Mostly level of malonyl CoA never gets over 5 micromolar.
22-23: Fasting and diabetes on hepatic CPT-Ialpha. Ki increases in diabetic animal. In liver- FA synthesized, do not want FA synthesis to be inhibited. Change prevents FA oxidation after synthesis in hepatocytes.
27:For CPT1-alpha, in liver levels change. Heart and brain- much less. Gamma really is not specific for brain.
26 and 28: More regulation- look at thyroid hormone T3 receptor. It has lots of accessory factors that bind and stimulate mRNA synthesis.PGC-1 added to cells stimulates transcription of CPT-1alpha.
29-32:Through CPT- fatty acid gets into mitochondrion- oxidized in matrix.Get acetyl coA.
First oxidation step uses FAD to get FADH2. Get 2 ATP out.
Long chain, med chain, short chain FA have their own dehydrogenases.
If you have unsaturated FAs, they come mostly from plants. All natural ones are cis to double bond. All same direction. Makes more flexibility in membrane.
See pathway on slide 38 for odd-chain fatty acid oxidation. Slides 35-45 show how propionyl coA is broken down. Most fatty acids have an even number of carbons, reflecting their synthesis. A few have odd numbers of carbons. Succinyl coA only found inside mitochondria.
Malonic acid inhibits the TCA cycle. No malonyl coA in mitochondrion.
Lecture 2:
email: gcook@utmem.edu
Will review as he goes today.
Yeast fatty acid oxidation is different from that of mammals.
Fat: The real high energy food slide could be exam question. Comparison slide of degradation and synthesis – list differences was question last year. Not on there this year, but good to know. May ask about comparison to TCA cycle.
Can also ask differences in peroxisomal and mitochondrial fatty acid oxidation See Slide 48 and Stryer 22.3.4, or if you have Lehninger 4th edition, a really nice diagram in figure 17-13..
Picture of peroxisome in slide 46- has single membrane packed with stuff. Crystalline structure may be urate oxidase, artifact of fixing cells. Shows how tightly packed they are.
Peroxisomal FA oxidation- important for long chain fatty acids. Phytol and phytanic acid degradation pathways are important. Toxic byproducts of degradation of chlorophyll. Can get a lttle energy from them. Literature of peroxisomal FA oxidation- he says FA are oxidized completely in peroxisome.
Zellweger syndrome- few peroxisomes. Retarded brain development.Another disease- deficiency acechoic (??wrong spelling, but that’s how he pronounced it) oxidase in peroxisomes- get same symptoms of retarded brain development and decreased myelin sheath and buildup of long chain FAs.
First step in peroxisome is acyl coA oxidase- oxidase, not dehydrogenase.
48: Early interest in peroxisome proliferators was in drugs for lipid lowering by increasing oxidation in peroxisomes. Stimulates synthesis carnitine palmitoyl transferase 1 amountt peroxisome formation in animals is more than in humans. Peroxisome proliferation in animals is associated with liver cancer - not necessarily in humans.
49:Looking at rat, mouse, and humans CPT1-alpha genes- why do FAs stimulate formation of CPT1 protein? All have long second intron. Lots of elements for thyroid hormone receptor binding there. Other regulatory properties there, including PPAR regulation.
50: Relation fatty acid oxidation and glucose oxidation- In heart and skeletal muscle- switch from FA to glucose oxidation. heart can live and pump in glucose solution happily for a few days. Add FAs- FAs oxidized, and glucose oxidation stops. End up w lactic acidosis-
Pyruvate dehydrogenase kinase is PDK.FA oxidation does not block glycolysis, so lactate builds up. Cant be transferred. To get lactate to pyruvate, must get rid of reducing equivalents. Heart has 4 isoforms of PDK, PDK4 most important. Pyruvate carboxylase is constitutively produced, not regulated.
51: Ketone body synthesis- fatty acids in liver and kidney are converted to ketone bodies.Muscle uses fatty acids for energy, converting them to CO2.
53: Ketone bodies are used by brain for energy during fasting.HMGcoA synthase is highly regulated, by cortisol (increased during fasting, increased gluconeogenesis thru PEP carboxykinase and stimulation of HMG coA synthase) and by glucocorticoids.
54: Lyase pushed to make more ketone bodies.
56: Measuring 3-hydroxybutyrate-acetoacetate ratio tells information about ketone body formation in liver.
57: Tissues that don’t make ketone bodies have enzymes to use them. Ketone body utilization happens all the time- Pathway not induced or capacity induced. Utilization is increased.
58: Ketone bodies go too high due to increased fatty acid oxidation- urine acidic.
59: Insulin regulates lipolysis and inhibits FA oxidation in liver.
60: Acetyl coA carboxylaseI in cytosol and fatty acid synthase reactions shown.Fatty acid synthase stops at palmitate. Acetyl coA carboxylase is Regulated by phosphorylation (see slide 61).
62 was a question last year.
63: ACP is a large carrier molecule with a phosphopantetheine group. These Enzymes are free in bacteria.
FA synthesis: see beginning of lecture and slides 64-71.
FA synthase genes are linked in mammals. Continuous AA sequence from one enzyme to another.
72: Some citrate from mitochondrion is transferred out by tricarboxylate carrier.Reacts using ATP-citrate lyase releases acetyl coA.
oxaloacetate to malate by malate dehydrogenase is another shuttle important to this pathway.
74: Peroxisomes- 2 fatty acid oxidation enzymes are combined.
75-6: Regulation of synthesis:
Acetyl CoA carboxylase- regulated by phosphorylation and by citrate. Citrate regulation slide he says is wrong. w 10mM citrate with pure enzyme, get same activation w phosphorylated and dephosphorylated form. No citrate- no activation in phosphorylated form, activation in dephosphorylated. Binding constant changes. Binds better or not as well.
77: Chicken ac coA carboxylase forms filaments in solution with citrate.
78: Enzyme important in obesity: data from gene chips-Stearoyl CoA desaturase induced in obesity. Stearoyl coA converts to monounsaturated oleoylcoA. Desaturated. Plants make unsaturated bonds by elongation of the chain and desaturation, alternating. Animals can insert an unsaturated bond after the chain is formed.
79: Linoleate and linolenate cannot be synthesized in animals.Essential for mammals.Omega-3 designation means 1st point of unsaturation (double bond) is 3 carbons from omega end.
80: Prostaglandin synthesis-
comes from FAs.Arachidonates come from phospholipids. Phospholipase A2 cuts off middle FA of TAG. to make arachidonic acid, then prostaglandins using cyclooxygenase. NSAIDS inhibit synthesis by inhibiting cyclooxygenase. COX 2 is present in inflammation- inducible enzyme. aspirin prevents inflammation by blocking COX in many tissues. Some prostaglandins protect stomach. Others protect heart and kidneys, so inhibiting them is bad. Glucocorticoids are also antinflammatory. They bind to nuclear receptors and regulate phospholipase A2 expression- knock it down.
For test:
First question usually about comparing one thing and another. What are possibliities for differences...? He has 2 questions on the test.