Lipid Structure and Metabolism I & II

Wayne V. Vedeckis, Ph.D. Department of Biochemistry and Molecular Biology And Stanley S. Scott Cancer Center Room 4B6, 4th Floor Clinical Sciences Research Building 533 Bolivar Street 504-599-0572 (Office) wvedec@lsuhsc.edu

LIPID STRUCTURE AND METABOLISM I 09/26/07 • LEARNING OBJECTIVES • 1) To identify the basic structure and properties of lipids • 2) To list the major pancreatic enzymes involved in lipid metabolism • 3) To describe the overall digestion and absorption of lipids • 4) To illustrate the role of bile in lipid metabolism • 5) To distinguish the forms (chylomicrons, lipoproteins) in which lipids are transported in the blood • 6) To discuss the importance of lipoprotein lipase in the uptake of fatty acids from the blood

II. INTRODUCTION TO LIPIDS (Fig. 15.1) A) Water Insoluble, heterogeneous B) Functions 1) compartmentalize cellular components and biochemical pathways 2) provide energy 3) coenzymes, signaling molecules C) Hydrophobic, lipophilic D) Major factor in obesity, diabetes, atherosclerosis

III. DIGESTION (Fig. 15.2) A) Adults – lingual lipase (acid stable; from tongue) swallowed. Different acid-stable lipase, gastric lipase, also made in stomach. Small amount of digestion begins in stomach (triacylglycerols containing short- and medium-chain fatty acids) B) Small intestine 1) emulsification 2) bile salts and acids(Fig. 15.3) 3) peristalsis

C) pancreatic enzymes 1) pancreatic lipase (Fig. 15.2); colipase (anchors pancreatic lipase) Triacylglycerols  2-monoacylglycerol + 2 FAs2) cholesterylesterase (Fig. 15.2) 3) phospholipase (Fig. 15.2) phospholipase A2 + lysophospholipase

IV. LIPID ABSORPTION A) Mixed micelles (Fig. 15.5) – disc- shaped structures with charged, hydrophilic portions on surface (water-soluble) and hydrophobic portions facing interior. Approach unstirred water layer at brush border of intestinal mucosa. Aid in transport of lipids through cell membrane. B) Short and medium chain fatty acids – soluble without forming micelles. Directly absorbed into intestinal cells.

V. LIPID RESYNTHESIS A) Long chain fatty acids - activated by fatty acyl CoA synthase (thiokinase) (Fig. 15.6). Veryimportant - 2 high energy bonds are used to activate a fatty acid. In typical reactions, ATP (2~) is converted to ADP (1~) and Pi (0~). In the thiokinase reaction, ATP (2~) is converted to AMP (0~) and PPi (1~). However, there is a ubiquitous pyrophosphatase present is all cells that converts PPi (1~) to 2 Pi (0~). Thus FA activation requires 2~ (or 2 ATP “equivalents”; ATP  ADP + Pi). 2 fatty acids are esterified to 2-monoacylglycerol to re-form triacylglycerol. B) Short/medium chain fatty acids – pass through cell, bind to albumin C) Lysophospholipids – reacylated to form phospholipids D) Cholesterol – reacylated to form cholesteryl esters 2Pi

2Pi VI. LIPID SECRETION (Fig. 15.6) A) Chyle (contains chylomicrons; not chyme) secreted into the lymphatics and then the bloodstream B) Chylomicrons – Large, TAG-rich, lipoprotein particle

VIII. USE OF DIETARY LIPIDS BY TISSUES A) Skeletal muscle and adipocytes B) Minor tissues - heart, lung, kidney, liver C) Lipoprotein lipase - from muscle and adipocytes; digests triacylglycerols; anchored to the endothelium on the interior of blood vessels D) Fate of free fatty acids – energy and resynthesis of triacylglycerols E) Fate of glycerol – converted to glycerol 3-phosphate in the liver F) Chylomicron remnants - cholesteryl esters, phospholipids, protein, some triacylglycerol. Bind to liver and are endocytosed and metabolized.

LIPID STRUCTURE AND METABOLISM II 09/26/07 I. LEARNING OBJECTIVES 1) To identify the basic structure of a fatty acid, and its state of saturation 2) To illustrate common fatty acids using the three methods (common, carbon 1 numbering, w carbon numbering) 3) To identify the citrate shuttle 4) To describe the rate limiting enzyme in fatty acid biosynthesis,acetyl CoA carboxylase, and how it is positively and negatively regulated in various ways 5) To distinguish the steps in fatty acid synthesis, and the way in which it is catalyzed by fattyacid synthase 6) To explain how fatty acids are used for the synthesis of triacylglycerol

II. INTRODUCTION A) Fatty acids - free (FFA) and fatty acid esters B) Plasma levels - varied with fasting and starvation C) Utilization – mostly liver and muscle D) Important source of energy E) Structural components of phospholipids and glycolipids F) Precursors of signaling molecules G) Synthesis versus degradation – pathways(Fig. 16.1) H) Central role for Acetyl CoA

III. STRUCTURE AND NOMENCLATURE A) General structure – amphipathic (Fig. 16.2) B) Features (Fig. 16.3) - saturated; unsaturated; cis double bonds; rancidification; melting temperature. The melting temperature of a FA is lower with shorter chain length and with a higher degree on unsaturation (more double bonds – adds “kink” in the chain).

C) Naming - three methods (Fig. 16.5) 1) common (Table 16.4) 2) carbon 1 numbering - # of carbons: # of double bonds (carbons at which double bonds occur); delta () naming (or N- or n- naming); carbon #1 is carboxyl, #2 is called a carbon, # 3 is b carbon, etc.

3) w carbon numbering – terminal methyl group is always the omega carbon. Begin counting at omega carbon (#1); most common are w-6 or N-6 or n-6 (linoleic) and w-3 (N-3; n-3) (linolenic) fatty acids. These two are “essential” fatty acids. Arachidonic acid becomes essential in linoleic acid deficiency. *To determine the type of omega fatty acid, subtract the first carbon in the highest numbered double bond from the total number of carbons in the FA. Must number carbons starting at carboxy carbon!!

IV. FATTY ACID BIOSYNTHESIS A) Sites - major (liver, lactating mammary gland) and minor (adipose tissue, kidney) B) Precursors and cofactors – acetyl CoA, ATP, NADPH, CO2 C) Cytoplasm - cellular synthesis site D) Cytoplasmic acetyl CoA – from mitochondrial acetyl CoA equivalents E) Citrate = form in which acetyl CoA is transported (Fig. 16.6) - mitochondrial citrate synthase (OAA + Acetyl CoA)  citrate  cytoplasm  cytoplasmic citrate lyase (OAA + Acetyl CoA) F) acetyl CoA is precursor for fatty acid synthesis; energy charge (ATP levels) must be high in the cell

(1~) (1~) (2~) (1~) G) Carboxylation/decarboxylation – provides energy and mechanism for synthesis (Fig. 16.7) – acetyl CoA carboxylase (covalently bound biotin); ATP; CO2; product is malonyl CoA (3 carbons) H) Regulation of acetyl CoA carboxylase (rate-limiting, committed enzyme) 1) short term - polymerization of dimer (protomer) is stimulated by citrate; inhibited by malonyl CoA and palmitoyl CoA(Fig. 16.7)

2) phosphorylation – glucagon and epinephrine stimulate phosphorylation = inactive; high insulin and carbohydrates promote dephosphorylation = active(Fig. 16.8) 3) long term - enzyme levels; increased by high carbohydrate, low fat diet; inhibited by low carbohydrate, high fat diet. Same is true forfatty acid synthase (orsynthetase)

I) Fatty acid synthase – complex, multi-activity enzyme; seven different enzyme activities; binding site for 4’- phosphopantetheine in acyl carrier protein (ACP) portion J) Reaction sequence – “enzymes” are activities of the same protein (Fig. 16.9) 1) acetyl CoA-ACP transacylase 2) intramolecular transfer 3) malonyl-CoA-ACP transacylase 4) b-ketoacyl-ACP synthase 5) b-ketoacyl-ACP reductase 6) b-hydroxyacyl-CoA dehydratase 7) enoyl-ACP reductase

End result – have synthesized a four carbon, saturated, fatty acyl ACP starting with a two carbon compound (acetyl CoA) and a three carbon compound (malonyl CoA) 8) steps 3-6 are repeated until palmitoyl (16:0) unit is attached to the enzyme 9) palmitoyl thioesterase hydrolyzes palmitic acid from the enzyme

10) overall reaction 8 acetyl CoA + 14 NADPH + 14 H+ + 7 ATP  palmitic acid (16:0) + 8 CoA + 14 NADP+ + 7 ADP + 7 Pi + 7 H20 K) Sources of NADPH - hexose monophosphate shunt and cytoplasmicmalicenzyme (Fig. 16.10) NADH + H+ NAD+ cytoplasmic

L) Interrelationship with glucose metabolism(Fig. 16.11) – glycolysis (pyruvate & NADH); gluconeogenesis (OAA production); pyruvatedehydrogenase (acetyl CoA); citric acid cycle (citrate) M) Further chain elongation –Mitochondria - Acetyl (2C) units are added, followed by reduction by NADH and NADPH. Endoplasmic reticulum - successive condensation of malonyl CoA and acyl CoA, and NADPH reductions (similar to fatty acid synthase reactions)

N) Desaturation (introduction of double bonds) – Mammals have D9, D6,D5, and D4 desaturases. Desaturation past carbon 9 in humans does not occur. This is why linoleic acid and linolenic acid are essential fatty acids in humans. O) Storage as triacylglycerol (Fig. 16.12) 1) carboxyl group is esterified to hydroxyl on glycerol 2) fat = solid at room temperature; oil = liquid at room temperature 3) fatty acids in triacylglycerol - #1 is usually saturated, #2 is usually unsaturated, #3 is either

4) glycerol phosphate - acceptor for triacylglycerol synthesis(Fig. 16.13) a) liver - from NADH-dependent reduction of DHAP by glycerolphosphatedehydrogenase and from phosphorylation of glycerol by glycerol kinase b) adipose tissue - only glycerol phosphate dehydrogenase path

5) fatty acid activation - by thiokinase to fatty acyl CoA (costs 2 ATP “equivalents”) 6) reactions in triacylglycerol synthesis (4)(Fig. 16.14) a) acyltransferase produces lysophosphatidic acid (position #1) b) acyltransferase produces phosphatidic acid (position # 2) c) phosphatase removes phosphate to form diacylglycerol d) acyltransferase produces triacylglycerol 7) fates of triacylglycerol - liver: packaged into very low density lipoprotein (VLDL) particles and transported to other tissues; adipocytes: stored as lipid droplets (“depot fat”)

LIPID STRUCTURE AND METABOLISM - III 09/26/07 • LEARNING OBJECTIVES • To describe the role and regulation of hormone sensitive lipase in the mobilization of fatty acids from triacylglycerol (TAG) • 2) To identify the location and steps in the b-oxidization of fatty acids • 3) To distinguish how odd chain fatty acids are metabolized • 4) To explain the metabolism of unsaturated fatty acids • To differentiate a- and peroxisomal b-fatty acid oxidation. • To explain the biochemical pathways in ketone body synthesis and degradation, including the concept of tissue-restricted expression of different enzyme activities

Glucagon II. FAT MOBILIZATION A) fat - major energy store (9 kcal/g when metabolized to CO2 and H20) B) Hormone sensitive lipase - releases fatty acids (#1 and/or #3) from TAG (Fig. 16.15) 1) stimulation - epinephrine and glucagon through the elevation of cyclic AMP 2) inhibition - high insulin and glucose; high fat diet C) glycerol - cannot be rephosphorylated in adipocyte; released into blood, goes to liver and is used (glycerol 3-phosphate; glycolysis, gluconeogenesis) D) fatty acids - released, bind to serum albumin, transported to peripheral tissues for energy production (brain, nervous tissue, erythrocytes, and adrenal medulla cannot use fatty acids for fuel)

III. b-OXIDATION OF FATTY ACIDS A) Occurs in mitochondrion B) Two carbon fragments are successively removed from carboxyl end of fatty acid to yield acetyl CoA C) Carnitine shuttle – transports fatty acids into mitochondrion(Fig. 16.16) - free fatty acid enters cell and thiokinase activates it to fatty acyl CoA; condensed with carnitine by carnitine palmitoyltransferase I (CPT-I) (aka,carnitineacyltransferase I, CAT-I) to form O-acylcarnitine; moves into mitochondrion; released by carnitine palmitoyltransferaseII using CoA to produce fatty acyl CoA and carnitine (energy neutral reactions)

1) carnitine shuttle inhibited by malonyl CoA (FA synthesis, opposing pathway) 2) Synthesized from lysine or methionine by the liver. 3) carnitine deficiencies – long chain fatty acids (LCFA) not used well for fuel. a) congenital CPT-I deficiency - decreases liver’s ability to synthesize glucose during fasting (liver uses CHO for cell maintenance). Causes severe hypoglycemia, coma, and death b) CPT II deficiency – cardiomyopathy, muscle weakness 4) Short chain fatty acids (SCFA) and MCFA – do not use carnitine shuttle. Pass into mitochondria. Not inhibited by malonyl CoA

D) Reaction sequence - four sequential reactions are repeated (Fig. 16.17) 1) acyl CoA dehydrogenase - forms double bond 2) enoyl CoA hydratase - adds water across double bond 3) b-hydroxylacyl CoA dehydrogenase – oxidizes by removing 2 H+ 4) acyl CoA:acyltransferase (“thiolase”) – cleaves acetyl CoA from the end 5) repeats until the final reaction liberates two acetyl CoA molecules

6) total energy yield after further metabolism of the acetyl CoA = 131 ATP from one palmitoyl CoA (16:0) (Fig. 16.18). N.B. If you start with the free FA, you need to invest 2 ATP “equivalents” (high energy bonds) in the thiokinase reaction needed to activate the FA. Thus, the net yield of ATP starting with the free fatty acid for complete b-oxidation is 2 ATP less (129 in the case of palmitic acid). E) Medium chain-length acyl CoA dehydrogenase deficiency – decreased fatty acid oxidation; severe hypoglycemia; involved in sudden infant death syndrome (SIDS) and Reye’s syndrome

F) Comparison of fatty acid synthesis and degradation (Fig. 16-19) – this is an excellent resource for reviewing, comparing, and contrasting fatty acid biosynthesis and degradation

G) Odd chain fatty acid metabolism (Fig. 16.20) – at first, same as even chain. But propionyl CoA (3 carbons) is a product 1) Propionyl CoA is converted to methylmalonyl CoA by propionyl CoA carboxylase (biotin). 2) Methylmalonyl CoA is converted to succinyl CoA by methylmalonyl CoA mutase (cobalamin [vitamin B12] derivative). 3) Succinyl CoA is metabolized in the citric acid cycle. 4) Deficiency in methylmalonyl CoAmutase or ability to synthesize cobalamin derivative causes methylmalonic acidemia and aciduria (low blood pH) – developmental retardation in patients

H) Unsaturated fatty acids produce less energy than saturated fatty acids 1) monounsaturated fatty acids [e.g., palmitoleic acid; 16:1(9)] first three cycles – same as saturated FA. Now have: H H H O | | | || --------C=C – C – C – S–CoA | H original C # 10 9 8 7 actual C # 4 3 2 1 D3-cis-enoyl CoA (NOT a substrate for enoyl CoA hydratase; Fig. 16.17) 3,2-enoyl-CoA isomerase H H O | | || --------C –C=C – C – S–CoA | | H H 4 3 2 1 D2-trans-enoyl CoA (a substrate for enoyl CoA hydratase)

2) polyunsaturated fatty acids – require an isomeraseplus an NADPH-dependent reductase to eliminate the double bonds that are separated by three carbons IV. OTHER TYPES OF FATTY ACID OXIDATION A) peroxisomal b-oxidation - can degrade very long chain fatty acids (VLCFA) fatty acids >20 carbons in length (mitochondrial b-oxidation cannot). Initial dehydrogenation uses an acyl CoA oxidase and FAD. Once the carbon chain is reduced to 18, mitochondrial b-oxidation is used. 1) Defect in peroxisome biosynthesis in all tissues – no metabolism of fatty acids >18 carbons in length, and these accumulate. Disease is called Zellweger (cerebrohepatorenal) Syndrome. 2) Defect in ability of peroxisome to activate VLCFA leads to X-linked adrenoleukodystrophy – mental impairment, motor problems, etc. (Lorenzo’s Oil)

B) a-oxidation - requires NADPH, molecular oxygen, and cytochromes (a fatty acid a-hydroxylase). Uses free fatty acid as substrate initially. The degradation of chlorophyll yields phytanic acid, a branched chain fatty acid (Fig. 16-21). Must use a-oxidation to metabolize it. Deficiency in a-hydroxylase causes Refsum's Disease. (retinitis pigmentosum, peripheral neuropathy, nerve deafness, cerebellar ataxia). Dietary restriction needed. V. KETONE BODIES A) Excess acetyl CoA - can be metabolized by liver mitochondria into ketone bodies (acetoacetate, b- (or 3-) hydroxybutyrate, acetone). B) Transported to other tissues, converted to acetyl CoA and metabolized by the citric acid cycle. C) Importance - water-soluble; produced in liver when excess acetyl CoA exceeds the oxidative capacity; can be used by skeletal and cardiac muscle, renal cortex, and even brain (when prolonged fasting causes large elevation in ketone bodies)

OVERALL METABOLIC CHANGES THAT OCCUR DURING STARVATION

D) Synthesis (Fig. 16.22) 1) production of acetoacetyl CoA a) incomplete breakdown of a fatty acid b) reversal of the thiolase reaction 2) Hydroxymethylglutaryl CoA (HMG CoA; 6 carbons) is synthesized by the condensation of acetoacetyl CoA and acetyl CoA by mitochondrialHMG CoAsynthase (rate-limiting, committed enzyme in ketone body synthesis; only found in liver). 3) acetoacetate is produced by HMG CoA cleavage 4) acetoacetate is reduced to b-hydroxybutyrate by b-hydroxybutyratedehydrogenase (NADH) or spontaneously decarboxylates to form acetone mitochondrial

E) Utilization by extrahepatic tissues (Fig. 16.23) - b-hydroxybutyrate is oxidized to acetoacetate by b-hydroxybutyrate dehydrogenase. Acetoacetate receives CoA from succinyl CoA (succinyl CoA:acetoacetate CoA transferase [thiophorase]; lacking in liver). Acetoacetyl CoA is converted into two acetyl CoA molecules. F) Because mitochondrialHMG CoAsynthase is only found in liver, only the liver can make ketone bodies. Because most other tissues except liver have thiophorase, the liver cannot use the ketone bodies that it synthesizes. Tissue-specific separation of opposing metabolic pathways.

Lipid Structure and Metabolism I & II