LipMetab_II_TAG_FA_Oxidation_NOTES_2022_rev PDF

S.H. Ackerman, PhD Triacylglycerol metabolism & fatty acid oxidation 1 [email protected] 4213 Scott Hall LECTURE TITLE: TRIACYLGLYCEROL METABOLISM & FATTY ACID OXIDATION LEARNING OBJECTIVES 1. List the principal 3 sites i...

S.H. Ackerman, PhD Triacylglycerol metabolism & fatty acid oxidation 1 [email protected] 4213 Scott Hall LECTURE TITLE: TRIACYLGLYCEROL METABOLISM & FATTY ACID OXIDATION LEARNING OBJECTIVES 1. List the principal 3 sites in the body where triacylglycerol is synthesized and explain the similarities and differences in the pathways. 2. Explain the effects of glucagon, epinephrine, and insulin on the mobilization of TAG from adipose tissue. 3. Outline the mitochondrial pathway for fatty acid oxidation with attention to FA entry to the organelle, the 4 steps of the core β-oxidation pathway, and propionyl-CoA catabolism. 4. Recognize the names of the enzymes that are recruited to oxidize unsaturated fatty acids and explain why the oxidation of unsaturated FA’s yields less energy vs. saturated fatty acids of equal carbon number. 5. Compare/contrast the pathway of fatty acid oxidation in peroxisomes. 6. Define the characteristics of the following metabolic syndromes: Carnitine deficiency, MCAD deficiency, X-ALD, and Zellweger syndrome/Refsum disease. OUTLINE I. Triacylglycerol synthesis II. Mobilization of TAG stores for energy production A. Effects of epinephrine & glucagon versus insulin III. Mitochondrial pathway for fatty acid oxidation A. Fatty acid entry to mitochondrial matrix B. β-oxidation pathway C. Energetics D. Catabolism of propionyl-CoA (from odd chain FA’s) E. Auxiliary enzymes needed to oxidize unsaturated FA’s. VI. Peroxisomal pathway for fatty acid oxidation V. FA catabolism disorders associated with mitochondrial or peroxisomal proteins.  Triacylglycerol metabolism & fatty acid oxidation 2 Triacylglycerol (TAG) synthesis There are 3 principal sites in the human body for TAG: enterocytes that line the lumenal surface of the small intestine, hepatocytes, and adipocytes. In the intestine, TAG synthesis is part of the elaborate pathway that evolved to permit digestion of dietary lipid. Instead, the synthesis of TAG in liver and fat cells is attributed to anabolic metabolism, (de novo synthesis) which converts excess hydrocarbons from all nutritional sources to an energy form that can be storedindefinitely. Phosphatidate is an intermediate in TAG synthesis in both liver and adipose tissue. Whereas both cell types can obtain the starting material (glycerol-3P) by reducing dihydroxyacetone phosphate (DHAP), the liver uses the enzyme, glycerol kinase to synthesizes the precursor by direct phosphorylation of glycerol. Mechanism of TAG synthesis in intestinal cells: Digestive enzymes cleave dietary TAG to 2-monoacylglycerol (2-MAG) and two free FA’s, all of which are absorbed by enterocytes. Fatty acyl-CoA synthetases attach the FA’s to CoA. Acylglycerol acyltransferase transfers the acyl group from FA-CoA to the C1-OH of 2-MAG to make 1,2-diacylglycerol (DAG). DAG acyltransferase transfers the acyl group from FA-CoA to the C3- OH of DAG to make TAG. Rebuilt dietary TAG is complexed with other lipids and apolipoproteins to create amphiphlic particles known as chylomicrons, which eventually enter the bloodstream. Mechanism of TAG synthesis in hepatocytes: de novo FA synthesis from acetyl-CoA. Fatty acyl-CoA synthetases attach the FA’s to CoA. Glycerol-3-P. The liver phosphorylates the C3 position of glycerol directly using ATP and the enzyme glycerol kinase. Glycerol-3-P acyltransferase transfers acyl unit from FA-CoA to C1- OH of glycerol-3-P, generating lysophosphatidic acid (LPA). 1-acylglycerol-3-P acyltransferase transfers acyl unit from FA-CoA to C2-OH of LPA, generating phosphatidic acid (PA); aka DAG-3-P. Phosphatidate phosphatase hydrolyzes PA to yield Pi and DAG. DAG acyltransferase transfers acyl unit from FA-CoA to C3-OH, generating TAG. Liver-derived TAG is packaged in VLDL and released to blood for delivery to white adipose tissue.  Triacylglycerol metabolism & fatty acid oxidation 3 Mechanism of TAG synthesis in adipocytes: de novo FA synthesis from acetyl-CoA. Glycerol-3-P. The gene for glycerol kinase IS NOT expressed in adipose tissue. Glycerol-3-P is obtained exclusively by reduction of dihydroxyacetone phosphate that is formed by cleaving the glycolytic intermediate, fructose-1,6-bis-phosphate, when glucose is metabolized. Insulin effect on fatty acid synthesis in adipocytes. Insulin promotes reactions that load adipocytes with fatty acids and glycerol-3-P. 1. Insulin promotes the transfer of FA’s from lipoprotein carriers to adipocytes by stimulating capillary lipoprotein lipase to cleave FA’s from TAG carried in chylomicrons and VLDL’s. 2. Insulin stimulates GLUT-4 localization at plasma membrane, which increases glucose uptake for metabolism to DHAP. DHAP reduction provides glycerol-3-P TAG synthesized in fat cells remains stored in the adipose tissue. Mobilization of TAG stores for energy production White adipose tissue is a principle component of the body’s mechanisms for controlling energy homeostasis *due to the enormous capacity of adipocytes to store TAG in fat globules that are 4-10 X larger than other cell types. The utilization of stored fat begins with lipolysis inside the white adipose tissue. There are 3 different lipase enzymes in adipocytes: 1. ATGL: adipocyte TAG lipase (hydrolyzes TAG only) Rate limiting for TAG mobilization 2. HSL: hormone-sensitive lipase (hydrolyzes both TAG and diacylglycerol (DAG)) 3. MGL: monoglycerol lipase (hydrolyzes only monoacyl glycerol (MAG)) Hormone-linked function of perilipin in TAG hydrolysis Perilipin is a protein that coats lipid droplets in adipocytes. Perilipin exists in two forms: a non-phosphorylated form and a phosphorylated form. The non-phosphorylated protein monomers associate with each other at the lipid droplet periphery to create a continuous barrier that protects the contents from lipases.  Triacylglycerol metabolism & fatty acid oxidation 4 Phosphorylation causes perilipin to change conformation, which exposes gaps in the coating where lipases can bind. The initial signal for perilipin phosphorylation is hormone binding to a G-protein coupled receptor (GPCR) in the plasma membrane. The stimulant is either epinephrine, through a b-adrenergic receptor, or glucagon via the glucagon receptor. Either hormone leads to adenylyl cyclase activation and increased cAMP in cells. cAMP activates protein kinase A. PKA phosphorylates perilipin, which allows access to lipases. The hormone-sensitive lipase (HSL) is also phosphorylated by PKA, which hyperactivates the enzyme. The glycerol and fatty acid by-products enter the blood. Lipolysis is under strict hormonal control. Epinephrine & Glucagon: each promote lipolysis through phosphorylation of HSL and perilipin. Insulin: Among the most familiar downstream effects from insulin binding to its receptor in cell membranes is the inhibition of TAG lipolysis. Insulin causes down-regulates expression of the ATGL gene (slows rate-limiting step). Insulin reverses the effects of epinephrine/glucagon (causes dephosphorylation of HSL and perilipin) by promoting the activation of protein phosphatase 1 (PP1). Fate of fatty acids released by TAG lipolysis is related to acyl chain length SCFA – short chain fatty acid (fewer than 6 carbons) (no carrier required for transport in blood) MCFA – medium chain fatty acid (6-12 carbons) Bound to albumin for transport in blood LCFA – long chain fatty acid (13-21 carbons) “ VLCFA – very long chain fatty acid (22 or more carbons) “ SCFA’s and MCFA’s enter mitochondria by simple diffusion and are esterified to CoA through the action of acyl CoA synthetases in the matrix. LCFA’s are shuttled across the mitochondrial inner membrane esterified to a nitrogen-containing molecule called carnitine. LCFA transport is dependent on 3 proteins: 1. Carnitine acyltransferase I (CAT1,CPT1) (mito OM) catalyzes LCFA transfer from CoA to carnitine. The creation of fatty acylcarnitine esters by CAT1 is the rate-limiting step for b-oxidation of LCFA’s. The enzyme is inhibited by malonyl-CoA. 2. Carnitine acyltransferase II (CAT2,CPT2), attached to the matrix face of the IM, receives acyl-carnitine post transport and catalyzes FA transfer to CoA inside the organelle. 3. Acyl-carnitine/carnitine transporter of the IM transfers fatty acyl-carnitine into the matrix in exchange for free carnitine transferred out.  Triacylglycerol metabolism & fatty acid oxidation 5 CAT1 and 2 are also known as CPT1 and 2; CPT for carnitine palmitoyltransferase. Silly b/c the enzyme accepts all long chain fatty acids as a substrate. VLCFA’s are degraded inside peroxisomes until reaching the size of a long chain fatty acid, which then gets released to the cytoplasm and converted to a carnitine ester for transport into mitochondria. Switching pathways is critical b/c fatty acid catabolism in peroxisomes is not a major source of energy. Fatty acid b-oxidation Saturated fatty acyl-CoA’s are degraded to acetyl-CoA in four steps that comprise the mitochondrial b-oxidation pathway. Opposite to the anabolic pathway, fatty acids are oxidized in the direction moving from the acyl end to the methyl end of the molecule. Step 1: Dehydrogenation of Alkane to Alkene Catalyzed by isoforms of acyl-CoA dehydrogenase (AD) located in the inner mitochondrial membrane LCAD (long-chain fatty acyl-CoA dehydrogenase) MCAD (medium-chain fatty acyl-CoA dehydrogenase) SCAD (short-chain fatty acyl-CoA dehydrogenase) Introduces a trans double bond between carbons 2 and 3. ETF-FAD CoQ ETF-FADH2 CoQH2 Step 1 is coincident with oxidative phosphorylation; fatty acyl-CoA’s are bona fide respiratory substrates that transfer electrons to Coenzyme Q. Anything that interferes with oxidative phosphorylation blocks b-oxidation of FA’s.  Triacylglycerol metabolism & fatty acid oxidation 6 Step 2: Hydration of Alkene Catalyzed by enoyl-CoA hydratase: Water adds across the double bond yielding the L stereoisomer of a hydroxyl intermediate. Step 3: Dehydrogenation of Alcohol Catalyzed by b-hydroxyacyl-CoA dehydrogenase The enzyme uses NAD cofactor as the hydride acceptor and yields NADH, which feeds electrons into the respiratory chain at complex I (NADH DH). Only L-isomers of hydroxyacyl CoA act as substrates Step 4: Transfer of Fatty Acid Chain Catalyzed by acyl-CoA acetyltransferase (aka b-ketothiolase) Thiolysis of carbon-carbon bond that releases one acetyl-CoA and transfers the shortened fatty acyl chain to an incoming CoA molecule. The b-oxidation pathway is recursive; each round of 4 reactions releases acetyl-CoA and shortens the FA by 2C The b-oxidation pathway is sufficient to completely catabolize fully saturated FA’s that are composed of an even number of carbons.  Triacylglycerol metabolism & fatty acid oxidation 7 Energy yield from b-oxidation of an even-numbered saturated fatty acid. Each round of b-oxidation yields: 1 FADH2 in STEP 1 1 NADH in STEP 3 * 1 Acetyl CoA in STEP 4 * In the final round of b-oxidation, the substrate for b-ketothiolase is a 4-C ketoacyl group and STEP 4 yields 2 moles of acetyl CoA. Each round of b-oxidation consumes 1 H2O and 1 CoA The stoichiometry of palmitoyl-CoA oxidation to acetyl-CoA is: Palmitoyl-CoA + 7 FAD + 7 NAD+ + 7 CoA + 7 H2O ® 8 acetyl CoA + 7 FADH2 + 7 NADH + 7 H+ 7 FADH2 x 1.5 ATP/FADH2 = 10.5 ATP and 7 NADH x 2.5 ATP/NADH = 17.5 ATP 28 ATP are yielded from palmitoyl-CoA oxidation to 8 acetyl-CoA. The number drops to 26 ATP for palmitate oxidation b/c energy equivalent to 2 ATPs is spent in converting the free fatty acid to palmitoyl-CoA. Every acetyl-CoA burned to CO2 and H2O yields 3 NADH,H+ 1 FADH2, and 1 NTP by the combined reactions of the TCA cycle and oxidative phosphorylation. 10 ATP per acetyl-CoA. Palmitate catabolism yields 8 acetyl-CoA x 10 ATP/acetyl-CoA, for another 80 ATP Palmitate catabolism to CO2 and H2O yields 106 ATP. Palmitate (256 g/mol) weighs 1.4 x more than glucose (180 g/mol) but yields 3.3 X more ATP (106 ATP vs. 32 ATP) because it is much more reduced.  Triacylglycerol metabolism & fatty acid oxidation 8 Odd-chain and unsaturated fatty acid catabolism The complete oxidation of odd-chain and unsaturated fatty acids requires additional enzymes. (a) Odd-chain fatty acids The substrate for the final cleavage reaction in STEP 4 is a 5-carbon acyl CoA and the products of the reaction are acetyl CoA and propionyl CoA. Propionyl CoA is also a product of the metabolic breakdown of some amino acids. Propionyl CoA is converted in 3 steps to succinyl CoA, which feeds directly into the TCA cycle 1. propionyl CoA is carboxylated to form D-methylmalonyl CoA, catalyzed by propionyl CoA carboxylase. Propionyl carboxylase contains a biotin cofactor and the reaction consumes 1 ATP. 2. D-methylmalonyl CoA is converted to L-methylmalonyl CoA. This reaction is catalyzed by methylmalonyl CoA epimerase. 3. L-methylmalonyl CoA is converted to succinyl CoA by the enzyme, methylmalonyl CoA mutase, which uses cobalamin (adoB12) as a cofactor. The consumption of raw eggs can cause propionyl-CoA to accumulate in serum at levels that are above normal. An abnormally high amount of methylmalonyl-CoA in serum is a marker for potential vit. B12 deficiency. b-oxidation of unsaturated fatty acids When unsaturated fatty acids are catabolized, metabolic intermediates with cis double bonds are produced that are NOT substrates for the enzymes of the b-oxidation pathway. Depending on whether the double bond is on an odd-numbered carbon or an even-numbered carbon (typical of PUFA’s), one or two additional enzymes are required for the catabolic pathway (an isomerase, by itself, or in combination with an NADPH-linked reductase) D3D2 Enoyl-CoA Isomerase, by itself, is required to degrade a fatty acyl-CoA with a double bond on an odd-numbered carbon. D3D2 Enoyl-CoA Isomerase, in combination with 2,4-dienoyl-CoA reductase + NADPH, is required to degrade a fatty acyl-CoA with a double bond on an even-numbered carbon.  Triacylglycerol metabolism & fatty acid oxidation 9 IMPORTANT b-oxidation of unsaturated fatty acids yields less energy relative to a fully saturated FA of equal carbon number. This makes sense b/c the energy produced during catabolism is correlated with oxidation reactions. Unsaturated fatty acids are partially oxidized compared to their saturated counterpart and require fewer oxidation reactions to be degraded. The actions of the D3D2 Enoyl-CoA Isomerase and 2,4-dienoyl-CoA reductase/NADPH are not described here in detail b/c this information is beyond the scope of the course. If you are curious about their activities, see the uploaded document entitled beta-oxidation of unsaturated fatty acids. Peroxisomes in human FA metabolism Mammalian peroxisomes specialize in the oxidation of very long fatty acids (VLCFA (22-30 carbons) and branched chain FA (phytanic acid). The peroxisomal pathway is essentially the same as in mitochondria with the noted difference that there is no connection between the FAD-linked dehydrogenation of fatty acyl-CoA and ATP synthesis in peroxisomes. Re-oxidation of the FADH2 cofactor bound to the fatty acyl- CoA DH is through direct reduction of O2 to H2O2. NADH released from the b-hydroxyacyl-CoA DH transfers to the cytoplasm and is re-oxidized by either the glycerol-3-P or Malate/Aspartate redox shuttle. Acetyl-CoA is exported to the cytosol. It is not oxidized further, but used as a substrate for new lipid synthesis. Once a VLCFA has been reduced to a LCFA, the fatty acyl intermediate enters the mitochondria by way of the carnitine shuttle.  Triacylglycerol metabolism & fatty acid oxidation 10 Disorders associated with defective FA catabolism: In mitochondria Primary and secondary carnitine deficiencies CATI is the rate-limiting enzyme of fatty acid catabolism and is inhibited by malonyl-CoA (avoids futile cycle of degrading newly synthesized FA’s). Even though carnitine can be synthesized from lysine in liver and kidney, the diet is the major source of carnitine in skeletal muscle. Primary carnitine deficiency is marked by reduced intracellular carnitine, which can result from nutritional deficit (e.g. strict vegan diets), defective hepatic synthesis, or defects in uptake from blood. Secondary carnitine deficiency is caused by defects leading to the accumulation of acylcarnitines, which deplete the cell of free carnitine. Such defects may map to the transporter or CATII, or to a metabolic defect that depletes the mitochondrial matrix pool of free Coenzyme A (e.g. MCAD deficiency). Symptoms range from mild muscle cramping to severe muscle weakness and death. Genetic deficiencies in CATII expression can lead to cardiomyopathy; rarely observed for the other 2 proteins b/c lethal. MCAD deficiency is the “most common” FA catabolic defect (1 in 12,000 births). causes secondary carnitine deficiency 𝝎-oxidation of MCFA’s lead to accumulation dicarboxylic acids in serum and metabolic acidosis. accumulation of octanoic acid linked to hyperammonemia and mitochondrial damage. Inefficient use of FA’s for energy impairs liver ability to meet demands when blood glucose decreases, leading to hypoglycemia. In peroxisomes: X-Linked Adrenoleukodystrophy (X-ALD) affects the ABCD1 gene, coding for the peroxisomal VLCFA transporter. Abnormal accumulation of lignoceric acid (C24:0) and cerotic acid (C26:0) has greatest effect on CNS and adrenal glands. Causes deficit in adrenocorticosteroids and demyelination of nerves in the brain. Zellweger syndrome/Refsum disease is caused by an autosomal recessive mutation in one of the proteins required for peroxisome biogenesis. Accumulation of phytanic acid (branched FA) in blood causes a severe neurological phenotype, including deafness and blindness. Zellweger syndrome extends also to X-ALD.