Lipid Metabolism PDF

METABOLISM OF LIPIDS DIGESTION AND ABSORPTION OF LIPIDS OVERVIEW OF LIPID METABOLISM COMMON DIEATRY LIPIDS Mainly Triacylglycerol (TAG) (~95%) Remaining 5%:  Sterols(mainly cholesterol)  Mostly as free cholesterol  10-15% are cholesterol esters  Phospholipids  DIG...

METABOLISM OF LIPIDS DIGESTION AND ABSORPTION OF LIPIDS OVERVIEW OF LIPID METABOLISM COMMON DIEATRY LIPIDS Mainly Triacylglycerol (TAG) (~95%) Remaining 5%:  Sterols(mainly cholesterol)  Mostly as free cholesterol  10-15% are cholesterol esters  Phospholipids  DIGESTION OF LIPIDS Lingual lipase released in mouth Primary target: fatty acids of short or medium chain length (milk fat ). Activity decreases with age and highly active in neonates Removes sn-1 and sn-3 on TAG containing short and medium chain fatty acid Product of lingual lipase: fatty acids+ 2-monoacylglycerol Optimal work at pH: 4 to 6 ,it is acid stable  DIGESTION OF LIPIDS Gastric lipase released in stomach Lipids (non polar) –need to be emulsified in order to allow digestion in stomach  Polysaccharides, phospholipids, digested proteins in chyme act as emulsifiers  Optimal pH: 4 to 6 Removes sn-1 and sn-3 on TAG containing short and medium chain fatty acid Product of gastric lipase is: fatty acids+ 2-monoacylglycerol  DIGESTION OF LIPIDS  Presence of fat in stomach causes release of hormones  CCK  Secretin Chyme enters SI  GIP makes release  GLP-1  Lipids have a high satiety value CCK Secretin Contract Contract Contract pancreases gallbladder pancreases EMULSIFICATION OF LIPIDS IN SMALL INTESTINE E mul si f ic at i o n i nc re ase s t he sur fac e are a o f t he hydrophobic fat droplets so that digestive enzymes can act effectively. Done by  Peristalsis: Mechanical mixing  Bile salts: Detergent properties as they decrease the surface tension and cause fat emulsification.  Phospholipids  BILE ACIDS/BILE SALTS  Made in liver from cholesterol  Bile acids are oxygenated derivatives of cholesterol that have several hydroxyl groups on the sterol rings  Stored in the gallbladder EMULSIFICATION BY BILE ACIDS/SALTS Bile acids and salts  DIGESTION OF LIPIDS 1- Degradation of triacylglycerol (TAG):-  Pancreatic Lipase : Released from pancreases into duodenum  Activated by calcium and Colipase, bile salt inhibit PL Removes sn-1 and sn -3 on TAG containing long chain fatty acid Product of pancreatic lipase is: fatty acids+ 2- monoacylglycerol 2- DEGRADATION OF CHOLESTERYL ESTERS  Cholesteryl estrase produces cholesterol and free fatty acids.  Activity is increased in presence of Bile salts. Cholesteryl estrase Products of esterase digestion: free fatty acids + cholesterol + 3- DEGRADATION OF PHOSPHOLIPIDS Phospholipase A2 in proenzyme form,  Activated by Trypsin,  Requires bile salts for activity.  Removes one fatty acid from carbon 2 of a phospholipid lysophospholipid. Lysophospholipase A1  Rem oves fat t y acid at carbon 1 and form s glycerylphosphoryl base that is excreted, degraded or absorbed. Phospholipase A2 Product of phospholipase A2 is free fatty acid and lysophopholipids Digestion of a phosphoglyceride by pancreatic phospholipase. Summary of digestion of lipids ABSORPTION OF LIPIDS BY INTESTINAL MUCOSAL CELLS Jejunum gets : Free fatty acids Free cholesterol Combine with bile and fat 2-monoacylglycerol soluble vitamins Lysophospholipid Form micelles : Soluble in aqueous intestinal environment, absorbed at the brush border of enterocytes. Notes: Fatty acids with short and medium chain length do not need micelles for absorption. Absorption of fat into enterocytes is mainly done through diffusion and by FAT/CD36 RESYNTHESIS OF TRIACYLGLYCEROL The conversion of free fatty acids to fatty acyl-CoA in enterocytes utilizes the ubiquitous fatty acyl-CoA synthetase reaction fatty acid + ATP + CoASH acyl-CoA + AMP + PPi In enterocytes, synthesis of triacylglycerol occurs through the sequential action of monoacylglycerol acyltransferase and diacylglycerol acyltransferase, for a net reaction 2-monoacylglycerol+ 2 fatty acyl-CoA triacylglycerol + 2CoASH This pathway is distinct from the triacylglycerol synthesis pathway in other cells, such as hepatocytes and adipocytes, which utilizes glycerol 3-phosphate as the acceptor of acyl groups. RESYNTHESIS OF PHOSPHOLIPIDS/ CHOLESTERYL ESTER  R e a c yl a t i o n o f l yso ph o sph o l i pi d s c a u se d by acyltransferases forms phospholipids.  Cholesteryl ester formation by Acyl CoA: cholesterol acyltransferase (ACAT).  ACAT and ACAT , each with different expression 1 2 patterns and unique physiological functions. ACAT is 2 expressed exclusively in the liver and intestine, where it participates in lipoprotein assembly. ACAT is present 1 in most tissues, and is the primary ACAT isoform in macrophages and steroidogenic tissues. Note: Free fatty acids with short and medium chains are released into portal circulation. Glycerol: diffuses to portal vein and then reach liver. SUMMARY OF RESYNTHESIS OF LIPIDS IN ENTEROCYTES microsomal triglyceride transfer protein(MTP)  ABSORPTION OF BILE SALTS  Bilesalts are not absorbed together with the products of hydrolysis of dietary triglycerides, phospholipids, and cholesteryl esters, and they are not incorporated into chylomicrons.  Instead, bile salts remain in the intestinal lumen until they reach the distal ileum, where most are absorbed by an active transport mechanism that utilizes a Na+-bile salt co-transport  LIPID MALABSORPTION  Steatorrhea: increased lipid and fat soluble vitamin excretion in feces (> 5g/day).  Caused by defects in lipid digestion and/or l i p i d absorption  SECRETION OF LIPIDS FROM ENTEROCYTES  Formation of Chylomicrons:- Aggregates of TAG and cholesteryl esters are formed, surrounded by a thin layer p h o s p h o l i p i d s , f r e e c ho le ste ro l and spe c ial pro te in Apolipoprotein B-48. Apo A1  Chylomicrons are only synthesized in small intestine released into lacteals by exocytosis.  After a lipid rich meal , lymph is called chyle.  From lymph, chylomicrons f inally enter blood.  CHYLOMICRONS  A ssembled in intestinal mucosal cells  Lowest density  Largest size  Highest % of lipids and lowest % proteins  Highest triacylglycerol (dietary origin)  Carry dietary lipids to peripheral tissues  Responsible for physiological milky appearance of plasma (up to 2 hours after meal) METABOLISM OF CHYLOMICRONS  These n a s c e n t chylomicrons are released in bl o o d t hro ugh l ym phat i c system.  In plasma it receives from HDL two more apopreoteins i.e. apo C-II and apo-E.  Apo C-II activates the enzyme Lipoprotein lipase, located on the capillary walls.  Lipoprotein lipase degrades TAG in c hylo mic ro ns and forms free fatty acids and glycerol.  LIPOPROTEIN LIPASE  Extracellular enzyme, anchored b y he p aran s ul f ate to the capillary walls of most tissues  Pred o m i n an tl y p res en t i n ad i p o s e ti s s ue, c ard i ac & skeletal muscle  Requires ApoC-II for activation  Degrades TG into glycerol and free fatty acids  Insulin stimulates its synthesis an d tran s f e r to the l um i n al surface of the capillary  Duri n g starvati on acti vi ty declines in adipocytes while increases in cardiac muscles. METABOLISM OF CHYLOMICRONS  Degradation of TAG leads to decrease in the size of chylomicron particles and increases its density.  apoC-II is returned to HDL leaving behind chylomicron remnant which has apoE and apoB48.  Liver c ells rec ognize apoE and rapid ly take up chylomicron remnants.  In liver cells they are acted upon by lysosomal enzymes and degradation of all the components take place , releasing amino acids, free cholesterol and fatty acids.  The receptor is recycled. USE OF DIETARY LIPIDS BY THE TISSUES  Fate of fatty acids: oxidized by various tissues for energy production and  Fate of Glycerol : used by liver to form glycerol 3- phosphate which can enter glycolysis or gluconeogenesis. OXIDATION OF FATTY ACIDS  FUNCTIONS OF FATTY ACID OXIDATION 1. Fatty Acid Oxidation Provides Energy for Cellular and Metabolic Work  Generating 9 kcal/g compared to 4 kcal/g from glucose  Recommended fat in diet (29 -31%)  Current , American diets typically contain 35 to 50% of calories as fat  Ethiopian diet ? Oxidation of fatty acids takes place in 3 stages FUNCTIONS OF FATTY ACID OXIDATION 2. Fatty Acid Oxidation Provides Fuel to the Brain during Starvation  Brain preferably use glucose.  During starvation, where is scarcity of glucose. it can also use ketone bodies as source of energy  Oxidation of ketones by the brain reduces the brain’s dependence on glucose and thus decreases the body’s need to catabolize muscle proteins to provide amino acid carbon skeletons for gluconeogenesis. FUNCTIONS OF FATTY ACID OXIDATION 3. Fatty Acid Oxidation Generates Heat  Brown fat is a spec ialized tissue that has a high metabolic rate although it does not produce very much ATP from the NADH and FADH2 generated during the oxidation of fatty acids. FUNCTIONS OF FATTY ACID OXIDATION The presence of thermogenin or “uncoupling protein” in the inner membrane of the mitochondria of brown fat results in the generation of heat rather than ATP from electron f lo w through the electron-transport chain FUNCTIONS OF FATTY ACID OXIDATION 4. Fatty Acid Oxidation G e n e r a t e s Intermediates in Synthetic processes  Examples include: Phospholipids (in membranes).  E i c o sa no i d s, i nc l ud i ng pro st a gl a nd i ns a nd leucotrienes, which play a role in physiological regulation.  Acetyl CoA  Propionyl CoA TISSUES IN WHICH FATTY ACID OXIDATION IS ACTIVE  All cells and tissues except red blood cells and the brain oxidize fatty acids to generate ATP.  Fatty acid oxidation is most active in tissues that are highly active metabolically.  Thus, skeletal and heart muscle in particular have a large capacity for oxidizing fatty acids.  Normally, 60 to 90% of the energy required for contraction of the heart is derived from the oxidation of fatty acids PHYSIOLOGICAL CONDITIONS IN WHICH FATTY ACID OXIDATION IS MOST ACTIVE  Fatty Acid Oxidation Increases in the Fasted State  Depletion of glycogen in the liver/gluconeogensis  Fatty Acid Oxidation Increases During Exercise  TRANSPORT OF FATTY ACIDS Fatty acids FAT/CD36 is the major fatty acid tra nspor te r in he a r t m uscle , skeletal muscle, adipocytes, and intestine FAT/CD36, Fatty acid translocase in the plasma me mbrane , MM. FATP, fatty acid transport protein; FABP, fatty acid-binding protein of the plasma membrane. ACTIVATION OF FREE FATTY ACIDS  Once inside cells, fatty acids must be activated before they can be metabolized. COASH + ATP + R-COOH R- C-SCOA + AMP + PPi  Acyl-CoA synthetases are localized to three different sites in cells:  The cytosolic face of the endoplasmic reticulum  The outer mitochondrial membrane  The peroxisomal membrane.  There are at least f iv e genetically distinct acyl-CoA synthetase (ACS) isoforms, each having its own specificity with regard to the fatty acid substrate. For example, ACS4 prefers polyunsaturated fatty acids such as arachidonic acid Activation of a fatty acid by a fatty acyl CoA synthetase. The fatty acid is activated by reacting with ATP to form a high-energy fatty acyl AMP and pyrophosphate. The AMP is then exchanged for CoA. Pyrophosphate is cleaved by a pyrophosphatase. TYPES OF FATTY ACID OXIDATION Fatty acid oxidation in the cell can be done in three ways  α-oxidation  β- oxidation  ω-oxidation 1-TRANSPORT OF LONG CHAIN FATTY ACIDS INTO MITOCHONDRIA  β- oxidation occurs in mitochondrial matrix  Inner mitochondrial membrane is impermeable to CoA  Specialized carrier is required to transport long chain acyl groups from cytosol to mitochondria  This carrier is CARNITINE  It is a rate-limiting transport process and is called CARNITINE SHUTTLE. STEPS IN LCFA TRANSPORT 1- In the inter-membrane space of the mitochondria, fatty acyl CoA reacts with carnitine in a reaction catalyzed by carnitine acyltransferase I (CAT-I), yielding CoA and fatty acyl carnitine. The resulting acyl carnitine crosses the inner mitochondrial membrane.  ATP + CoA AMP + PPi palmitate palmitoyl-CoA Cytoplasm CAT-I OUTER ACS MITOCHONDRIAL CPT-I MEMBRANE CoA palmitoyl-CoA Intermembrane palmitoyl-carnitine carnitine Space CPT-I defects cause severe muscle weakness because fatty acids are an important muscle fuel during exercise. REGULATION OF CAT-I  CAT-I is associated with the outer mitochondrial membrane.  CAT-I reaction is rate-limiting;  The enzyme is allosterically inhibited by malonyl CoA.  Malonyl CoA concentration would be high during fatty acid synthesis.  I nhi bi t i o n o f C AT- I by m a l o nyl C o A pre v e nt s simultaneous synthesis and degradation of fatty acids.  CAT-I CPT-I palmitoyl-CoA CoA Intermembrane Space carnitine palmitoyl-carnitine INNER MITOCHONDRIAL Translocase MEMBRANE CPT-II Matrix carnitine palmitoyl-carnitine palmitoyl-CoA CoA Mitochondrial uptake via of palmitoyl-carnitine via the carnitine- acylcarnitine translocase. STEPS IN LCFA TRANSPORT  2-Fatty acyl carnitine is transported across the inner mitochondrial membrane in exchange for carnitine by carnitine-acylcarnitine translocase.  In the mitochondrial matrix fatty acyl carnitine reacts with C o A in a reac tio n c atalyzed by c arnitine acyltransferase II (CAT-II), yielding fatty acyl CoA and carnitine.  The fatty acyl CoA is now ready to undergo beta- oxidation. CARNITINE SHUTTLE SOURCES OF CARNITINE C arnitine is o btaine d fro m the d ie t o r c an be synthesized in liver and kidney from amino acids lysine and methionine.  Skeletal and heart muscles cannot synthesize c arni t i ne and d e pe nd o n d i e t o r e nd o ge no us synthesis. The reactions use S-adenosylmethionine (SAM) to donate methyl groups, and vitamin C (ascorbic acid) is also required for these reactions CARNITINE DEFICIENCIES PRIMARY CAUSES:-  Genetic CAT-I def ic iency: Mainly affects liver. Liver cannot synthesize glucose in a fast , results in hypoglycemia, coma and death.  CAT-II def ic iency: Mainly affects skeletal and cardiac muscles.  Defect in renal tubular reabsorption of carnitine.  Defect in carnitine uptake by cells.  CARNITINE DEFICIENCIES SECONDARY CAUSES :  Liver diseases; decreased endogenous synthesis.  Malnutrition or strict vegetarian diet  Increased metabolic demands  Hemodialysis ENTRY OF SHORT AND MEDIUM CHAIN F.A INTO MITOCHONDRIA  Carnitine and CAT system not required for fatty acids shorter than 12 carbon length.  They are activated to their CoA form inside mitochondrial matrix.  Not inhibited by malonyl CoA. REACTIONS OF ΒETA -OXIDATION β-oxidation is the process by which the fatty a c id s und e r g o o x id a t iv e r e m o v a l o f successive two-carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acyl chain. The products of β -oxidation are:  Acetyl CoA  FADH2, NADH and H+ FOUR STEPS- FOUR ENZYMES There are four individual reactions of β -oxidation, each catalyzed by a separate enzyme. 1-Dehydrogenation between carbon 2 and 3 in a FAD- linked reaction. Enzyme is acyl CoA dehydrogenase. 2-Hydration of the double bond by enoyl CoA hydratase. 3-A second dehydrogenation in a NAD-linked reaction. Enzyme is 3-hydroxyacyl CoA dehydrogenase. 4-Thiolytic cleavage of the thioester by beta-ketoacyl CoA thiolase. This sequence of four steps is repeated until the fatty acyl chain is completely degraded to acetyl CoA FOUR STEPS- FOUR ENZYMES Each round in fatty acid degradation involves four reactions 1. Oxidation to trans-Δ2-Enoyl-CoA by acyl CoA dehydrogenase FOUR STEPS- FOUR ENZYMES 2. Hydration to L–3–Hydroxylacyl CoA enoyl CoA hydratase FOUR STEPS- FOUR ENZYMES 3. Oxidation to 3–Ketoacyl CoA 3-hydroxyacyl CoA dehydrogenase FOUR STEPS- FOUR ENZYMES 4. Thiolysis to produce Acetyl–CoA beta-ketoacyl CoA thiolase. Transfer of electrons from acyl CoA dehydrogenase to the electron transport c h a i n. A b b r e v i a t i o n s : E T F, e l e c t r o n - transferring f la voprotein; ETF-QO, electron- transfe rring f la voprote in–C oe nzy me Q oxidoreductase. TYPES OF FATTY ACYL COA DEHYDROGENASES  Long chain fatty acyl CoA dehydrogenase (LCAD) acts on chains greater than C12.  Medium chain fatty acyl CoA dehydrogenase (MCAD) acts on chains of C6 to C12.  Short chain fatty acyl CoA dehydrogenase (SCAD) acts on chains of C4 to C6. MCAD def ic iency is thought to be one of the most common inborn errors of metabolism.  REACTION PRODUCTS Fate of acetyl CoA  Oxidation by the citric acid cycle to CO2 and H2O.  In liver only, acetyl CoA may be used for ketone body synthesis. Fate of the FADH2 and NADH + H+  FADH2 and NADH + H+ are oxidized by the mitochondrial electron transport system, yielding ATP. Fate of acetyl CoA FATE OF THE FADH2 AND NADH + H+ What is the total ATP yield for the oxidation of 1 mole of palmitic acid to carbon dioxide and water? ENERGY YIELD FROM BETA OXIDATION OF PALMITIC ACID Oxidation of one molecule of palmitoyl CoA to CO 2 and water produces  8 acetyl CoA  7 NADH  7 FADH2  Palmitoylcarnitine inner mitochondrial Carnitine membrane respiratory chain translocase Palmitoylcarnitine matrix side 2 ATP 3 ATP Palmitoyl-CoA FAD oxidation FADH2 hydration H2O recycle NAD+ oxidation 6 times NADH thiolase CoA CH3-(CH)12-C-S-CoA + Acetyl CoA Citric O acid cycle 2 CO2 NET ENERGY PRODUCTION  7 FADH2 = 2X 7 = 14 ATP  7 NADH = 3 X 7 = 21 ATP  8 Acetyl CoA = 12 x 8 = 96 ATP Total ATP = 131 ATP  2 ATP are utilized during the formation of acyl CoA. Therefore net yield is 129 ATP. OXIDATION OF UNSATURATED FATTY ACIDS  Undergoes beta oxidation  Less energy yield  L e ss f o r m a t i o n o f r e d u c i n g e q u i v a l e n t s a s unsaturated F.A are not highly reduced.  One or two additional enzymes are required based on the number of double bonds in the fatty acid backbone. UNSATURATED FATTY ACIDS (MONOUNSATURATED) If there is a double bond at an odd-numbered carbon (e.g., 16:1 9), the action of enoyl CoA isomerase is required to move the naturally occurring cis- bond and conve r t it to t he t rans- bond use d in be t a- oxidation. The product , wit h a trans- double bond, is a substrate for e n oyl C oA h ydra t a se , t h e se cond e nzym e of be t a- oxidation UNSATURATED FATTY ACIDS (POLYUNSATURATED) I n c a s e o f poly unsa t ura t e d fatty acids, e.g linoleic acid that is 18:2(9,12), NADPH- dependent Dienoyl CoA Reductase is required in addition to isomerase Oxidation of linoleate. After three spirals of β -oxidation (dashed lines), there is now a 3,4 cis double bond and a 6,7 cis double bond. The 3,4 cis double bond is isomerized to a 2,3-trans double bond, which is in the proper conf iguration for the normal enzymes to act. One spiral of -oxidation occurs, plus the f irst step of a second spiral. A reductase that uses NADPH now converts these two double bonds (between carbons 2 and 3 and carbons 4 and 5) to one double bond between carbons 3 and 4 in a t r a n s c o n f ig u r a t i o n. T h e isomerase (which can act on double bonds that are in either the cis or the trans conf ig ura t ion) m ov e s t h is double bond to the 2,3-trans position, and -oxidation can resume. BETA OXIDATION IN PEROXISOME  Fatty acids with 20 or more carbons ( VLCFA ) are first oxidized in the peroxisomes.  The shor tened fatty ac id then goes to the mitochondria.  The enzyme for initial dehydrogenation is FAD containing Acyl CoA oxidase.  H2O2 is produced during the process which is toxic to cells and is therefore converted to H2O by Catalase.  Very-long-chain fatty acids of 24 to 26 carbons are oxidized exclusively in peroxisomes by a sequence of reactions similar to mitochondrial -oxidation in that they generate acetyl CoA and NADH Very-long-chain fatty acids of 20 to 26 carbons are oxidized exclusively in peroxisomes by a sequence of reactions similar to mitochondrial oxidation in that they generate acetyl CoA and NADH ZELLWEGER SYNDROME  Rare inherited disorder.  Absence of peroxisomes.  VLCFA cannot be oxidized  Accumulation of VLCFA in brain, blood and other tissues like liver and kidney. BETA OXIDATION OF ODD CHAIN FATTY ACIDS  Oxidation of fatty acids with odd number of carbons yield acetyl CoA and one molecule of propionyl CoA.  Propionyl CoA is converted to Methylmalonyl CoA by carboxylase (a biotin requiring enzyme).  MMCoA is moved within the molecule by MMCoA mutase (vit.B12 coenzyme) to form succinyl CoA… gluconeogenic.  I n h e rit a ble disorde r of propion a t e m e t a bolism is due to a de fe ct in propionyl-CoA carboxylase, resulting in propionic acidemia (and aciduria). Such in d iv id u a ls, a s w e ll a s t h ose w it h methylmalonic acidemia, are capable of oxidizing some propionate to CO2, even in the a bse nce of propiony l-C oA carboxylase. Two inheritable types of methylmalonic acidemia (and aciduria) are associated in young children with failure to grow and mental retardness.  ALPHA OXIDATION  α oxidation is the removal of one carbon atom (i.e., αcarbon) at a time from the carboxyl end of the BRANCHED FATTY ACIDS molecule.  α oxidation of long-chain fatty acids to 2-hydroxy acids and then to fatty acids with one carbon atom l e s s t h a n t h e o r i gi n a l s u bs t ra t e h a v e be e n demonstrated in the microsomes of brain and other tissues also.  Long-chain α hydroxy fatty acids are constituents of brain lipids, e.g. , the C24 cerebronic acid ( = 2 hydroxylignoceric acid), CH3 (CH2)21. CH(OH). COOH.  These hydroxy fatty acids can be converted to the 2- keto acids, followed by oxidative decarboxylation, resulting in the formation of long-chain fatty acids with an odd number of carbon atoms :  ALPHA OXIDATION  The initial hydroxylation reaction is catalyzed by a mitochondrial enzyme, monoxygenase that requires O2, Mg2+ , NADPH.  Conversion of the α hydroxy fatty acid to CO2 and the next lower unsubstituted acid appears to occur in the endoplasmic reticulum and to require O2, Fe2+ and ascorbate.  PHYTANIC ACID  The α-oxidation system plays a key role in the capacity of mammalian tissues to oxidize phytanic acid.  Phytanic acid is an oxidation product of phytol and is present in animal fat, cow’s milk and foods derived from milk.  The phytol presumably originates from plant sources, as it is a substituent of chlorophyll and the side chain of vitamin K2.  Normally, phytanic acid is rarely found in serum lipids because of the ability of normal tissue to degrade (or oxidize) the acid very rapidly.  REFSUM’S DISEASE  Large amounts of phytanic acid accumulate in the tissues and serum of individuals with Refsum’s disease, a rare inheritable autosomal recessive disorder affecting the nervous system because of an inability to oxidize this acid.  Diets low in animal fat and milk products appear to relieve some of the symptoms of Refsum’s disease.  In Refsum’s disease, there is a lack of the enzyme, phytanate α hydroxylase OMEGA -OXIDATION OF FATTY ACIDS  Fatty acids also may be oxidized at the ω- c arbo n o f the c hain by enzymes in the endoplasmic reticulum.  The terminal-methyl group is f ir st oxidized to an alcohol by an enzyme that uses cytochrome P450, mo l e c ul ar o x yge n, and N A D PH. Dehydrogenases convert the alcohol group to a carboxylic acid.  The dicarboxylic acids produced by ω -oxidation can undergo β-oxidation, forming compounds with 6 to 10 carbons that are water-soluble.  Such compounds may then enter blood, be oxidized as medium-chain fatty acids, or be excreted in urine as medium-chain dicarboxylic acids. REGULATION OF BETA OXIDATION Beta-oxidation is regulated as a whole primarily by fatty acid availability; once fatty acids are in the mitochondria they are oxidized as long as there is adequate NAD+ and CoA. FATTY ACID OXIDATION DURING FASTING Mobilization of lipids from storage sites  Fatty acids are stored primarily in adipocytes as triacylglycerol.  Triacylglycerol must be hydrolyzed to release the fatty acids.  Adipocytes are found mostly in the abdominal cavity and subcutaneous tissue.  Adipocytes are metabolically very active; their stored triacylglycerol is constantly hydrolyzed and resynthesized. Adipose tissues  MOBILIZATION OF STORED FATS: LIPOLYSIS Non-esterif ied fatty acid release from the adipocytes is initiated by the action of horm one se nsit ive lipase (HSL), which begins to hydrolyze the stored triglyceride. The f in al products of t r i a cyl gl yce rol hydrolysis are glycerol and nonesterif ie d fatty acids. HSL is activated by epinephrine, norepinephrine, ACTH and glucagon, acting via phosphorylation of the enzyme.  It is inhibited by insulin. Non-esterified fatty acids are bound to serum albumin for transport to other tissues, where they are used. KETONE BODIES  KETONE BODIES  Ketone bodies are water-soluble, that are generated from acetyl CoA  There are three different types of ketone bodies.  Acetoacetate  Beta hydroxybutyrate  Acetone 93 CELLULAR AND TISSUE LOCATION Ketone bodies are purely synthesized in  Liver  Synthesis takes place in the mitochondria of liver cells.  The primary sources of ketone bodies are  Fatty acids (Acetyl CoA)  Ketogenic amino acids (leu, Ile, Lys, Trp, Phe, Tyr (Acetyl CoA or AcetoacetylCoA)  CONDITIONS WHERE KETONE BODIES ARE SYNTHESIZED Rate of Ketone bodies synthesis increases during  Fasting or starvation  Heavy or Prolonged physical exercises  Extremely low carbohydrates diet (as with the high-protein diets).  Uncontrolled diabetes mellitus (Type I)  KETOGENESIS  Two molecules of acetyl CoA condense to form acetoacetyl CoA, catalyzed by thiolase, is the reverse of the thiolysis step in the oxidation of fatty acids.  Acetoacetyl CoA then reacts with ac e t yl C o A and wat e r to gi v e 3-hydroxy-3- methylglutaryl CoA (HMG-CoA) and CoASH.  The reaction is catalyzed by HMG CoA synthase. Thi s e nz ym e i s e xc l usi v e l y pre se nt i n l i v e r mitochondria.  There are two isoforms, the mitochondrial enzyme is needed for ketogenesis while the cytosolic form is associated with cholesterol biosynthesis.  Acetoacetic acid is the primary ketone body , which will be reduced to beta hydroxy butyrate and spontaneously decarboxylated to acetone 96 Peculiarity of acetone  Acetone is formed by decarboxylation in the presence of decarboxylase enzyme and undergoes a slow, spontaneous decarboxylation to acetone.  Not used as metabolic fuel  Do not cause acidosis  Lost via expired air  Produce characteristic odor of acetone may be detected in the breath of a person who has a high level of acetoacetate in the blood. “Acetone-breath” has been used as a crude methodof diagnosing individuals with un treated Type I diabetes mellitus.  FORMATION OF Β-HYDROXY BUTYRATE  β- Hyd ro x y B ut yrat e i s fo rm e d by t he re d uc t i o n o f ac e to ac e tate in the mito c ho nd rial matrix by D (- )3- hydroxybutyrate dehydrogenase.  The β-hydroxybutyrate dehydrogenase reaction has two functions: 1) It stores energy equivalent to an NADH in the ketone body for export to the tissues 2) It produces a more stable molecule (3:1).  The ratio of β hydroxybutyrate to acetoacetate depends on the NADH/NAD+ ratio inside mitochondria.  If NADH concentration is high, the liver releases a higher proportion of β-hydroxybutyrate. 99  BIOLOGICAL SIGNIFICANCE OF KETONE BODIES Ketone bodies serve as a fuel for extra hepatic tissues Brain, heart, skeletal muscles Brain:  It is metabolically active and metabolically privileged.  The brain generally uses 60 -70% of total body glucose requirements.  As glucose availability decreases, the brain is forced to use either amino acids or ketone bodies for fuel.  In pro lo nged starvatio n,30 -40% of the fuel needs of the b ra i n a r e m e t b y k e t o n e bodies.  Heart muscle and the renal cortex use acetoacetate in 100 preference to glucose  TISSUES THAT USE KETONE BODIES AS FUEL  During starvation  Brain  Intestinal mucosa  Adipocytes  Developing fetus use ketone bodies  Almost all tissues except liver and RBC 101  KETOLYSIS Utilization of Beta- hydroxybutyrate 1.Beta-hydroxybutyrate, is f ir st oxidized to acetoacetate with the production of one NADH. Under conditions where tissues are utilizing ketones for energy p ro d u c t i o n t h e i r NAD /NADH ratios + are go ing to be relatively high, thus driving the β- h y d r o x y b u t y ra t e de hydro ge nase catalyzed reaction in the direction of acetoacetate. 102  UTILIZATION OF KETONE BODIES 2) Coenzyme A must be added to the acetoacetate.  The thioester bond is a high energy bond, so ATP equivalents must be used.  In this case the energy comes from a trans esterif ication of the CoAS from Succinyl CoA to acetoacetate by Coenzyme A transferase, also called Succinyl co A : Acetoacetate co A transferase, also known as Thiophorase.  The Succinyl CoA comes from the TCA cycle. 103  REGULATION OF KETOGENESIS Ketogenesis is regulated at three steps- 1) Lipolysis in Adipose tissue  Ketogenesis does not occur unless there is an FA that arise from lipolysis of triacylglycerol in adipose tissue.  When glucose levels fall, lipolysis induced by glucagon secretion causes increased hepatic ketogenesis due to increased substrate (free fatty acids) delivery from adipose tissue.  Conversely, insulin, released in the well-fed state, inhibits ketogenesis via the triggering dephosphorylation and inactivation of adipose tissue hormone sensitive lipase (HSL). 104  LIPOLYSIS IN ADIPOSE TISSUE Horm one se nsit ive lipase e xist s in t wo form s inact ive de phosphor ylat e d (brought by Insulin) and act ive phosphorylated form (brought by glucagon, catecholamines). Insulin promotes lipogenesis105while the other hormones promote lipolysis.  REGULATION OF KETOGENESIS 2)Transport of fatty acid-There is regulation of entry of fatty ac id s into the oxid ative pathway by c arnitine A c yl transferase-I (CAT-I)  Malo nyl- C o A , the initial inte rme d iate in fatty ac id biosynthesis formed by acetyl-CoA carboxylase in the fed state, is a potent inhibitor of CAT-I.  Under these conditions, free fatty acids enter the liver cell in low concentrations and are nearly all esterif ie d to acylglycerols and transported out of the liver in very low density lipoproteins (VLDL). 106  REGULATION OF CAT-1 ACTIVITY CAT-I activity is low in the fed state, leading to depression of fatty acid oxidation. However, CAT-1 activity is higher in starvation, allowing fatty acid oxidation to increase. 107  REGULATION OF KETOGENESIS 3) Fate of Acetyl co A  The acetyl-CoA formed in beta-oxidation is oxidized in the citric acid cycle, or it enters the pathway of ketogenesis to form ketone bodies.  A s t h e l e v e l o f se r u m fre e fa t t y a c i d s i s ra i se d , proportionately more free fatty acids are converted to ketone bodies and less are oxidized via the citric acid cycle to CO2.  Entry of acetyl CoA into the citric acid cycle depends on the availability of Oxaloacetate for the formation of citrate, but the concentration of Oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. 108  REGULATION OF KETOGENESIS During high rates of fatty acid oxidation, primarily in the liver, large amounts of acetyl-Co A are generated. These exceed the capacity of the TCA cycle, and one result is the synthesis of ketone bodies. 109 NORMAL LEVELS OF KETONE BODIES  Normal plasma concentration : 1-3 mg/dL.  In urine … less than 125 mg in 24 hrs. CERTAIN TERMINOLOGIES  Ketosis: Accumulation of abnormal amounts of ketone bodies in tissues and body fluids  Ketonemia: level of ketone bodies in blood above normal level.  Ketonuria : Urinary excretion of ketone bodies exceeds the normal amounts.  Ketoacidosis: Acetoacetic acid and beta hydroxy butyric acid are moderately strong acids. When their synthesis exceeds their utilization, their amount exceed s in blood and tissues. They need to be buffered. There can be progressive loss of buffer cations and this results in ketoacidosis.  CAUSES OF KETOSIS  Uncontrolled diabetes mellitus (Type I)  Starvation  Chronic alcoholism  Von- Gierke’s disease  Heavy exercise  Low carbohydrate diet- For weight loss  Pyruvate carboxylase deficiency  STARVATION INDUCED KETOSIS Prolonged fasting may result: From an inability to obtain food, from the desire to lose weight rapi d l y, o r i n c l i ni c al situations in which an individual cannot eat be c ause o f trauma, su rge r y, n e o pl a sm s, burns etc.  In the absence of food t he pl asma l e v e l s o f glucose, amino acids and triacylglycerols fall, triggering a decline in insulin secretion and an inc rease in gluc agon release. 113  STARVATION INDUCED KETOSIS  The decreased insulin to glucagon ratio, makes this period of nutritional deprivation a catabolic state, characterized by degradation of glycogen, triacylglycerol and protein.  This sets in to motion an exchange of substrates between liver, adipose tissue, muscle and brain that is guided by two priorities- (i) the need to maintain glucose level to sustain the energy metabolism of brain ,red blood cells and other glucose requiring cells and (ii) to supply energy to other tissues by mobilizing fatty acids from adipose tissues and converting them to ketone bodies to supply energy to other cells of the body. 114 HEAVY EXERCISE INDUCED KETOSIS LOW CARBOHYDRATES DIET INDUCE KETOSIS  CLINICAL SIGNIFICANCE-KETOACIDOSIS Both β-hydroxybutyrate and acetoacetate are organic acids. and are released in the protonated form, to lower the pH of the blood. In normal individuals, other mechanisms compensate for the increased proton release.  When ketone bodies are released in large quantities the normal pH-buffering mechanisms are overloaded ; the reduced pH, in combination with a number of other metabolic abnormalities results in ketoacidosis.  In severe ketoacidosis, cells begin to lose ability to use ketone bodies also. 117  KETOGENESIS IN DIABETES  Insufficient insulin leads to.. Increase in lipolysis , i nc re ase fat t y ac i d s f lo od the liver Acetyl- CoA  I nc re ase d i nsul i n to glucagon ratio trigger more glucose synthesis,  oxaloacetate is needed to pro d uc e gl uc o se , c i t r i c A c i d C yc l e i s limited KETOACIDOSIS IN DIABETES MELLITUS  In uncontrolled type 1 diabetes mellitus ----- severe def ic iency or absence of insulin ---- lipolysis ---- very high levels of FFA ---- high levels of acetyl CoA ---- raised ketogenesis.  In severe ketosis ---- blood level above 90 mg/dl and urine level above 5000 mg/24 hrs.  With each ketone body , one hydrogen atom is released in blood --- lowering of pH…. Acidosis.  The body initially buffers these with the bicarbonate bu ffe r i n g syst e m , a n d o t h e r m e c h a n i sm s t o compensate for the acidosis, such as hyperventilation to lower the blood carbon dioxide levels.  This hyperventilation, in its extreme form, may be observed as Kussmaul respiration.  Ketones, too, participate in osmotic diuresis and lead to further electrolyte losses  DIABETIC KETO- ACIDOSIS  It happens predominantly in type 1 diabetes mellitus,  But can also occur in type 2 diabetes mellitus under certain circumstances.  This may be due to inter-current illness (pneumonia, inf lu enza, gastroenteritis, a urinary tract infection),  Young patients with recurrent episodes of DKA may have an underlying eating disorder, or may be using insuf fic ient insulin for fear that it will cause weight 120 gain.  DIABETIC KETO- ACIDOSIS Diabetic Ketoacidosis may be diagnosed when the combination of hyperglycemia (high blood sugars), 121 ketones on urinalysis and acidosis are demonstrated.  ALCOHOLIC KETOACIDOSIS(AKA)  A l t ho ugh t he ge ne ral physi o l o gi c al fac to rs and mechanisms leading to AKA are understood, the precise factors have not been fully defined. The following are the 3 main predisposing events:  Delay and decrease in insulin secretion and excess glucagon secretion, induced by starvation  Elevated ratio of (NADH)/NAD + ) secondary to alcohol metabolism  Volume depletion resulting from vomiting and poor oral intake of fluids 122  ALCOHOLIC KETOACIDOSIS(AKA)  The metabolism of alcohol itself is a probable contributor to the ketotic state.  A l c o h o l d e h yd ro ge n a se m e t a bo l i z e s a l c o h o l t o acetaldehyde in the cytoplasm of hepatocyte.  Acetaldehyde is metabolized further to acetic acid by aldehyde dehydrogenase mitochondria.  Both steps require (NAD+) and produce (NADH). 123  ALCOHOLIC KETOACIDOSIS(AKA) The decreased ratio of NAD+/ NADH has the following implications:  Impaired conversion of lactate to pyruvate with an increase in serum lactic acid levels  Impaired gluconeogenesis because pyruvate is not available as a substrate for glucose production  A shift in the hydroxybutyrate (β-OH) to acetoacetate (AcAc) equilibrium toward β-OH butyrate  In contrast to diabetic ketoacidosis, the predominant ketone body in AKA is β-OH. Routine clinical assays for ketonemia test for AcAc and acetone but not for β-OH.  Clinicians underestimate the degree of ketonemia if they rely solely on the results of laboratory testing. 124  ALCOHOLIC KETOACIDOSIS(AKA)  Prolonged vomiting leads to dehydration, which decreases renal perfusion, thereby limiting urinary excretion of ketoacids.  M o r e o v e r, v o l u m e d e p l e t i o n i n c r e a s e s t h e concentration of counter-regulatory hormones, further stimulating lipolysis and ketogenesis.  The pivotal variable appears to be a relative def ic iency of insulin.  Individuals with higher insulin levels are more likely to pre se nt wi t h t he synd ro me o f al c o ho l - i nd uc e d hypoglycemia without ketoacidosis 125  ALCOHOLIC KETOACIDOSIS(AKA)  Most cases of AKA occur when a person with poor nutritional status due to long-standing alcohol abuse who has been on a drinking binge suddenly decreases energy intake because of abdominal pain, nausea, or vomiting.  In addition, AKA is often precipitated by another medical illness such as infection or pancreatitis.  AKA results from the accumulation of the ketoacids, hydroxybutyric acid, and acetoacetic acid.  S u c h a c c u m u l a t i o n i s c a u se d by t h e c o m pl e x i nt e ra c t i o n st e m m i ng fro m a l c o ho l c e ssa t i o n, decreased energy intake, volume depletion, and the metabolic effects of hormonal imbalance. 126 DE NOVO SYNTHESIS OF FATTY ACID FATTY ACID SYNTHESIS SERVES TWO MAIN FUNCTIONS Glucose ketogenic amino acids Acetyl CoA Fatty acid synthesis Eicosanoid , Sterol Energy/ TAG/PL lipid hormones.  TISSUE AND CELLULAR LOCATIONS Fatty acid synthesis occurs primarily in  Liver  Adipose tissue (fat)  Lactating mammary glands Fatty acid synthesis takes places in the cytosol of the cells CONDITIONS AT WHICH FATTY ACID SYNTHESIS OCCURS  Excess energy following a meal ( insulin/glucagon ratio)  High-carbohydrate diet.  Low fat diet  FA synthesis is very high during embryogenesis and in fetal lungs.  Fatty acid synthesis is also greatly increased in cancer cells.  PRECURSORS FOR FATTY ACID SYNTHESIS  Metabolites required  NADPH  Acetyl-CoA  ATP, Mn2+  Biotin  HCO 3– (as a source of CO 2).  Enzymes required  Acetyl CoA Carboxylase  Fatty acid synthase complex  PPP -SOURCES OF NADPH  The oxidative reactions of the pentose phosphate pathway are the chief source of the hydrogen required for the reductive synthesis of fatty acids.  NADPH is involved as donor of reducing equivalents  In hepatocytes, adipose tissue and the lactating mammary glands, the NADPH is supplied primarily by the pentose phosphate pathway THE MALIC ENZYME- SOURCE OF NADPH The second important NADPH source, is produced by reversible reaction of Malate to pyruvate produced in the reaction reenters the mitochondrion for further utilization CYTOSOLIC ISOCITRATE DEHYDROGENASE- SOURCE OF NADPH There are three iso enzymes o f iso c itrate dehydrogenase. One, which uses NAD+, is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. SOURCES OF ACETYL COA FOR FA SYNTHESIS TAG/PL/Fatty acid Pyruvate Amino acids oxi/alcohol Acetyl CoA Fatty acid TCA Sterol synthesis TRANSPORTATION OF ACETYL CO A  Fatty acid synthesis requires considerable amounts of acetyl-CoA  Nearly all acetyl-CoA used in fatty acid synthesis is formed in mitochondria  Acetyl co A has to move out from the mitochondria to the cytosol Cytosol – Site of acetate utilization Mitochondria – Site of acetate synthesis TRANSPORTATION OF ACETYL CO A  Acetate is shuttled out of mitochondria as citrate  The m itochondrial inne r membrane is impermeable to acetyl-CoA  Intra-mitochondrial acetyl- C o A f ir s t r e a c t s w i t h oxaloacetate to form citrate, in the TCA cycle catalyzed by citrate synthase  Citrate then passes into the cytosol through the mitochondrial inner membrane on the citrate transporter.  In t he cytosol, cit rat e is cle ave d by cit rat e lyase regenerating acetyl-CoA.  FATE OF OXALOACETATE The product of Citrate cleavage, oxaloacetate can be-  Channeled towards glucose production  C o n v e r t e d t o mal at e b y malate dehydrogenase  Converted to Pyruvate by Malic enzyme, producing more NADPH, that can be used for fatty acid synthesis  Pyruvate and Malate pass through special transporters present in the inner mitochondrial membrane ENZYMES INVOLVED IN FATTY ACID SYNTHESIS Two main enzymes-  Acetyl co A carboxylase  Fatty acid Synthase Both the enzymes are multienzyme complexes  ACETYL-COA CARBOXYLASE  The production of malonyl-CoA is the initial committed step in the fatty acid synthesis  The reaction is catalyzed by the enzyme, acetyl-CoA carboxylase.  AcetylCoA carboxylase -Is the initial & rate controlling step in fatty acid synthesis  It is a multienzyme complex containing- Biotin  Biotin Carboxylase  Biotin carboxyl carrier protein  Transcarboxylase  FORMATION OF MALONYL COA  ATP-dependent carboxylation provides energy input.  The CO i s l ost l a t e r dur i ng 2 condensation with the growing fatty acid.  The s p o n t a n e o u s de ca r box yl a t i on dr i v e s t he condensation reaction.  As wit h ot he r carboxylat ion reactions, the enzyme prosthetic group is biotin.  The reaction takes place in two steps: carboxylation of biotin (involving ATP) and transfer of the carboxyl to acetyl-CoA to form malonyl-CoA. FATTY ACID SYNTHASE ENZYME COMPLEX  The Fatty Acid Synthase Complex is a polypeptide containing seven enzyme activities  In bacteria and plants, the individual enzymes of the f atty aci d syn thase system are separate, and the acyl radicals are found i n com b i n ati on wi th a protein called the acyl carrier protein (ACP).  In yeast, mammals, and b i rd s , the s yn thas e system is a multienzyme polypeptide complex that incorporates ACP, which takes over the role of CoA. SUBSTRATE CHANNELING The organized structure of the fatty acid synthases of higher organisms enhances the ef fic iency of the overall process because of the following reasons : 1. The intermediates are directly transferred from one active site to the next. 2. The intermediates are not diluted in the cytosol. 3. The intermediates do not have to f ind each other by random diffusion. 4. The covalently-bound intermediates are secluded and protected from competing reactions.  FATTY ACID SYNTHASE COMPLEX  Each segment of the disk represents one of the six enzymatic activities of the complex  At the center is the ACP – acyl carrier protein - with its phosphopantetheine arm ending in –SH. Fa t t y Aci d S ynt ha se pr ost he t i c groups: The thiol (-SH)of the side-chain of a cysteine residue of keto acyl synthase enzyme(also called condensing enzyme) The thiol (-SH) of phosphopantetheine, equivalent in structure to part of coenzyme A. It is a component of Acyl carrier protein INTERMEDIATES IN FATTY ACID SYNTHESIS AND THE ACP  The intermediates in fatty acid synthesis are linked to an acyl carrier protein. Specif ically, the intermediates are attached to the sulfhydryl(–SH) terminus of a phosphopantetheine group.  This single polypeptide chain of 77 residues can be regarded as a giant prosthetic group, a “macro-CoA”.  STRUCTURE/FUNCTION OF PHOSPHOPANTETHEINE  Phosphopantetheine (Pant) i s c o val e n t l y i n k e d vi a a phosphate ester to a serine OH of the acyl carrier protein domain of Fatty Acid Synthase.  T h e lon g f le xib le arm of phosphopantetheine helps its thiol to move from one active site to another within the complex.  Carrying the reaction in termediates from on e enzyme active site to the next.  THE FIRST ROUND OF FA BIOSYNTHESIS  To initiate FA biosynthesis, malonyl and acetyl groups are activated on to the enzyme fatty acid synthase.  Initially, a priming molecule of acetyl-CoA combines with a cysteine —SH group catalyzed by acetyl transacylase  Malonyl-CoA combines with the adjacent —SH on the 4'-phosphopantetheine of ACP of the other monomer, catalyzed by malonyl transacylase (to form acetyl (acyl)-malonyl enzyme.  THE ACTIVATION OF THE ACETYL GROUP  The acetyl group from acetyl- CoA is transferred to the Cys- SH group of the -ketoacyl ACP synthase  This reaction is catalyzed by acetyl-CoA transacetylase.  THE ACTIVATION OF THE MALONYL GROUP  Transfer of the malonyl group to the –SH group of the ACP is catalyzed by malonyl-CoA ACP transferase.  The charged acetyl and malonyl groups are now in close proximity to each other  SERIES OF REACTIONS After activation, the processes involved are- 1. Condensation 2. Reduction 3. Dehydration 4. Reduction These steps are repeated till a fatty acid with 16 carbon atoms is synthesized STEP-1(CONDENSATION) 1.Condensation –  The acetyl group attacks the methylene group of the ma l onyl resi due to form Acetoacetyl-ACP  The reaction is catalyzed by β-ketoacyl-ACP synthase.  Decarboxylation allows the reaction to go to completion, pulling the whole sequence of reactions in the forward direction.  STEP-2 (REDUCTION) 2. Reduction-  The Acetoacetyl-ACP is reduced to b- hydroxybutyryl-ACP, catalyzed by β- ketoacyl-ACP reductase  NADPH + H+ are required f o r reduction STEP-3 (DEHYDRATION) 3. Dehydration –  Dehydration yields a double bond in the product, trans-Δ2- butenoyl-ACP,  Reaction is catalyzed by β-hydroxybutyryl- ACP dehydratase. STEP-4 (REDUCTION) 4. Reduction  R e d uc t i o n o f t he d o ubl e bond takes place to form bu t y r y l - A C P, R e a c t i o n i s catalyzed by enoyl-reductase.  Another NADPH dependent reaction. THE GROWING CHAIN IS TRANSFERRED FROM THE ACYL CARRIER PROTEIN This reaction make s way fo r t h e n e x t i n c o m i n g malonyl group. The enzyme involved is acetyl - C o A transacetylase BEGINNING OF THE SECOND ROUND OF THE FA SYNTHESIS CYCLE  The butyryl group is on the Cys-SH group  The incoming malonyl group is first attached to ACP.  In the condensation step, the entire butyryl group is exchanged for the carboxyl group on the malonyl residue REPETITION OF THESE FOUR STEPS LEADS TO FATTY ACID SYNTHESIS  The 3-ketoacyl group is reduced, dehydrated, and re d uc e d agai n (re ac t i o ns 2, 3, 4) to fo rm t he corresponding saturated acyl-S-enzyme.  A new malonyl-CoA molecule combines with the —SH of 4'-phosphopantetheine, displacing the saturated acyl residue onto the free cysteine —SH group.  The sequence of reactions are repeated until a saturated 16-carbon acyl radical (Palmityl) has been assembled.  It is liberated from the enzyme complex by the ac t i v i t y o f a se v e nt h e nz yme i n t he c o mpl e x , Thioesterase (deacylase). REPETITION OF THESE FOUR STEPS LEADS TO FATTY ACID SYNTHESIS THE RESULT OF FATTY ACID SYNTHASE ACTIVITY  Seven cycles of condensation and reduction produce the 16-carbon saturated palmitoyl group, still bound to ACP.  Chain elongation usually stops at this point, and free palmitate is released from the ACP molecule by hydrolytic activity in the synthase complex.  Smaller amounts of longer fatty acids such as stearate (18:0) are also formed  In mammary gland, there is a separate Thioesterase specif ic for acyl residues of C 8, C 10, or C 12, which are subsequently found in milk lipids. STOICHIOMETRY OF FATTY ACID SYNTHESIS The overall reaction for the synthesis of palmitate from acetyl- CoA can be broken down into 2 parts : (a) the formation of 7 malonyl-CoA molecules 7 Acetyl-CoA + 7 CO2 + 7ATP → 7 Malonyl-CoA + 7 ADP + 7 Pi...(i) (b) the 7 cycles of condensation and reduction Acetyl-CoA + 7 Malonyl–CoA + 14 NADPH + 14 H+ → Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O...(ii) Hence, the overall process for the synthesis of palmitate is : 8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H+ → Palmitate + 8 CoA + 7 ADP + 7 Pi + 14 NADP+ + 6 H2O...(iii) Note that the CO2 utilized (formation of malonyl-CoA) and the CO2 produced (condensation reaction) cancel each other when the overall stoichiometry is tabulated.  REGULATION OF FATTY ACID SYNTHESIS When a cell has more Glycerol-P energy, the excess is Glucose generally converted to Fatty Acids and stored Triacylglycerol as lipids such as triacylglycerol. Fatty acyl CoA Malonyl CoA Pyruvate Acetyl CoA TCA cycle  REGULATION OF FATTY ACID SYNTHESIS  The reaction catalyzed by acetyl-CoA carboxylase is the rate limiting step in the biosynthesis of fatty acids. The mammalian enzyme is regulated, by Alloste ric control by local O Acetyl-CoA metabolites = Phosphorylation CH3-C-S-CoA HCO3- Conformational changes associated with regulation: In the active conformation, Acetyl- CoA Carboxylase associates to O form m ultim e ric f il a m e ntous complexes. = -OOC-CH -C-S-CoA 2 Transition to the inactive conformation is associated with Malonyl-CoA dissociation to yield the monomeric form of the enzyme (protomer).  REGULATION OF ACETYL-COA CARBOXYLASE Allosteric control Palmitoyl-CoA acts as a feedback inhibitor of the enzyme, and citrate is an activator. When there is an increase in mitochondrial acetyl-CoA and ATP, citrate is transported out of mitochondria Citrate becomes both the precursor of cytosolic acetyl-CoA and a signal for the activation of acetyl-CoA carboxylase. REGULATION OF ACETYL-COA CARBOXYLASE Phosphorylation A c e t y l - C o A carboxylase is also regulated by hormones such as g l u c a g o n , epinephrine, and insulin via changes i n i t s pho spho r yl at i o n state REGULATION OF ACETYL-COA CARBOXYLASE  Additionally, these pathways are regulated at the level of gene expression  Long-chain fatty acid synthesis is controlled in the short term by allosteric and covalent modif ic ation of enzymes and in the long term by changes in gene expression governing rates of synthesis of enzymes. NUTRITIONAL STATE REGULATES LIPOGENESIS  Excess carbohydrates is stored as fat in many animals in anticipation of periods of caloric def iciency such as starvation, hibernation, etc, and to provide energy for use between meals in animals, including humans, that take their food at spaced intervals.  The nutritional state of the organism is the main factor regulating the rate of lipogenesis. FATTY ACID SYNTHESIS DURING FED STATE  The rate is higher in the well-fed state if the diet contains a high proportion of carbohydrate  Lipogenesis c onver ts surplus gluc ose and intermediates such as pyruvate, lactate, and acetyl- CoA to fat, assisting the anabolic phase of this feeding cycle  Lipogenesis is increased when sucrose is fed instead o f gl uc o se be c ause fruc to se bypasse s the phosphofructokinase control point in glycolysis and floods the lipogenic pathway FATTY ACID SYNTHESIS DURING FASTING  It is depressed by restricted caloric intake, high fat diet, or a deficiency of insulin, as in diabetes mellitus  These conditions are associated with increased concentrations of plasma free fatty acids  An inverse relationship has been demonstrated between hepatic lipogenesis and the concentration of serum-free fatty acids. ROLE OF INSULIN IN FATTY ACID SYNTHESIS  I nsulin stimulates lipogenesis by several other mechanisms as well as by increasing acetyl-CoA carboxylase activity.  It increases the transport of glucose into the cell (eg, in adipose tissue),  Increases the availability of both pyruvate for fatty acid synthesis and glycerol 3-phosphate for esterification of the newly formed fatty acids,  I nsul i n c o nv e r t s t he i nac t i v e fo rm o f pyruv at e dehydrogenase to the active form in adipose tissue but not in liver, thus provides more of Acetyl co A  Insulin also acts by inhibiting c AMP mediated lipolysis in adipose tissue and thereby reduces the concentration of plasma free fatty acids (long-chain fatty acids are inhibitors of lipogenesis. Insulin is a powerful anabolic signal, particularly in hepatocytes and adipocytes, where it induces synthesis of the lipogenic family of enzymes, which includes acetyl- CoA carboxylase, citrate lyase, the malic enzyme, glucose 6-phosphate dehydrogenase, pyruvate kinase, and the FAS complex. The mechanism underlying this action by insulin involves activation of the sterol regulatory element- binding protein- 1 (SREBP- l), a membrane-bound transcription factor that enhances transcription of the genes encoding proteins required for fatty acid synthesis. Glucagon, on the other hand, represses de novo synthesis of these enzymes in adipocytes and liver, and stimulates degradation of the lipogenic family of enzyme proteins. AMP-activated protein kinase also suppresses expression of fatty acid synthase, acetyl-CoA carboxylase, and citrate lyase.  FATTY ACID ELONGATION  Palmitate in animal cells is the precursor of other long- chained FAs.  Elongation of fatty acids occurs primarily in the endoplasmic reticulum and utilizes malonyl-CoA to add two-carbon units to long-chain fatty acyl-CoAs. (microsomal chain elongation system, elongase)  There is a minor, secondary chain elongation system (elongase) in mitochondria that utilizes acetyl-CoA as the two-carbon donor BIOSYNTHESIS OF UNSATURATED FATTY ACIDS  Palmitate and stearate serve as precursors of the two most common monounsaturated fatty acids of animal cells: palmitoleate (16:19), and Oleate (18:19).  The double bond is introduced by fatty acyl-CoA desaturase in the smooth endoplasmic reticulum. BIOSYNTHESIS OF UNSATURATED FATTY ACIDS  Palmitate and stearate serve as precursors of the two most common monounsaturated fatty acids of animal tissues, palmitoleate and oleate.  The double bond is introduced into the fatty acid chain by an oxidative reaction catalyzed by fatty acyl-CoA desaturase. The enzyme is an example of a mixed- function oxidase ESSENTIAL FATTY ACIDS  EICOSANOIDS Compounds containing a 20-carbon core Comprise:  prostaglandins  thromboxanes prostanoids  leukotrienes  lipoxins  hydroxyeicosatetraenoic acids (HETEs)  hepoxilins  In h u m a n s , arac hi d o ni c ac i d i s formed from linoleic acid:  In humans, the double bonds cannot be introduced beyond the Δ9 position  linoleic and linolenic acids are e sse ntial: must be supplied in food (plant oils, peanut, soybean, corn)  EICOSANOID PRODUCTION FROM PUFAS food linoleic acid dihomo-γ-linolenic acid arachidonic (8,11,14-eicosatrienoic) acid 1…cyclooxygenase pathway linolenic acid 2…lipoxygenase pathway eicosapentaenoic acid food 3..cytochrome P450s (monooxygenases) food – mainly fish oils MAIN SITES OF EICOSANOID BIOSYNTHESIS  Endothelial cells  Leukocytes  Platelets  Kidney Eicosanoids are NOT synthesized in advance and stored in granules – when needed, they can be produced very quickly from arachidonate released from membranes MAIN STEPS OF EICOSANOID BIOSYNTHESIS 1) Activation of phospholipase A2 (PLA2) 2)Release of arachidonate from membrane phospholipids by PLA2 3)Eicosanoid synthesis: COX or LO pathway + subsequent cell-specif ic modif ic ations by synthases / isomerases (c c onversion of the prec ursor PGH 2 to another prostanoid, conversion of LTA4…)  1) PHOSPHOLIPASE A2 ACTIVATION Ligand binding to a receptor induces phospholipase C (PLC) activation → PLC cleaves PIP2 to DAG and IP3 that opens the Ca2+ channels in the ER. PLA2, activated by Ca2+ and probably also by phosphorylation (MAPK), translocates to membranes of GA, ER, or nucleus from which it releases arachidonate for here residing COX/LO. plasma GA, ER, or nuclear membrane membrane NOS synthesis/ activation on i cat slo activ n tra ation Ca PLA2 expression / activity is stimulated by:  Interleukin-1  Angiotensin II  Bradykinin  EGF  Thrombin  Epinephrine… 2) ARACHIDONATE RELEASE FOR EICOSANOID SYNTHESIS From membrane phospholipids – mainly by the action of phospholipase A2: Arachidonate release from phospholipids can be blocked by the anti-inflammatory steroids!  3) EICOSANOID BIOSYNTHESIS In almost all cell types (except for red blood cells) 3 pathways: A) Cyclooxygenase (COX) – produces prostaglandins and thromboxanes B) Lipoxygenase (LO) – produces leukotrienes, lipoxins, 12- and 15-HETEs, and hepoxilins C) Cytochrome P450s (monooxygenases) – produce the other HETEs (20-HETE); principal pathway in the proximal tubules  A) CYCLOOXYGENASE (COX) PATHWAY Prostaglandin H synthase, present as two isoenzymes (PGHS-1/COX-1, PGHS-2/COX-2), each possessing two activities:  cyclooxygenase – catalyzes addition of two molecules of O2 to the arachidonic acid molecule, forming PGG2  hydroperoxidase – converts the hydroperoxy function of PGG2 to an OH group (of PGH2) Mostly, a given cell type produces 1 type of prostanoids: platelets produce almost exclusively thromboxanes, vascular endothelial cells prostacyclins, heart muscle makes PGI2, PGE2, PGF2 PROSTAGLANDIN H SYNTHASE PGH = precursor of all 2 series 2 prostaglandins and thromboxanes cyclic 9,11-endoperoxide, 15 -hydroperoxide is formed  PRODUCTS OF THE COX PATHWAY  Platelets contain thromboxane synthase producing TXA2, TXB2  Vascular endothelial cells contain prostacyclin synthase which converts PGH2 to prostacyclin PGI2  INHIBITION OF THE COX PATHWAY Aspirin inhibits the COX activity of both PGHS-1 and PGHS-2 (by acetylation of a distinct Ser of the enzyme) Other nonsteroidal anti- inflammatory drugs (NSAIDs) also inhibit the COX activity (ibuprofen competes with arachidonate) Transcription of PGHS-2 can be blocked by anti-inflammatory corticosteroids  B) LIPOXYGENASE (LO) PATHWAY 12-lipoxygenase 15-lipoxygenase 3 different lipoxy- genases insert oxygen into the 5, 12, or 15 position of Hepoxilins 5-lipoxygenase arachidonate; the (HXA3) first product is the hydroperoxy- eicosatetraenoic acid (HPETE) 5-lipoxygenase Only 5-lipoxygenase produces leukotri- enes; requires se protein FLAP 15-lipoxygena Gly–Cys–Glu Leukotriene D 4 Leukotriene E4 peptidoleukotrienes -Glu -Gly PEPTIDOLEUKOTRIENE BIOSYNTHESIS: Requires glutathione!!! Peptido-leukotrienes are short- lived organic molecules known to have potent biological effects as mediators of inflammation, hypersensitivity and respiratory  C) EICOSANOID SYNTHESIS BY CYP450S Cytochrome P450s – monooxygenases: RH + O2 + NADPH + H+ ROH + H2O + NADP+ Two main classes of compounds are formed:  Epoxygenases catalyze the formation of epoxyeicosatrienoic acids (EETs) that are further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DiHETEs) which are almost inactive:  Hydroxylases catalyze the formation of HETEs (20-HETE, 13- HETE…)  SUMMARY OF THE PRODUCTS arachidonic acid CYP450s cyclooxygenases lipoxygenases 19-, 20-, 8-, EETs prostacyclin thromboxanes lipoxins 9-, 10-, 11-, 12-, 13-, 15-, s 16-, 17-, DiHETEs 18-HETE prostaglandins leukotrienes hepoxilins 5-, 8-, 12-, 15-HETE  STRUCTURAL FEATURES  Prostaglandins – cyclopentane ring  Thromboxanes – six-membered oxygen-containing ring  Leukotrienes – 3 conjugated double bonds + one more unconjugated  Lipoxins – conjugated trihydroxytetraenes  PROSTAGLANDIN NOMENCLATURE  The three classes A, E, F (third letter) are distinguished on the basis of the functional groups about the cyclopentane ring  The subscript numerals refer to the number of double bonds in the side chains  The subscript  refers to the configuration of the 9–OH group (projects down from the plane of the ring) PGE2 E…β-hydroxyketone 2 double bonds BIOLOGICAL EFFECTS OF EICOSANOIDS  Eicosanoids, like hormones, display profound effects at extremely low concentrations (10-9 TO 10-14)  They have a very short half-life; thus, they act in an autocrine or paracrine manner (unlike hormones)  Biological effects depend not only on the particular eicosanoid but also on the local availability of receptors that it can bind to IN GENERAL, EICOSANOIDS MEDIATE  Inflammatory response, notably as it involves the joints (rheumatoid arthritis), skin (psoriasis), and eyes  Production of pain and fever  Regulation of blood pressure  Regulation of blood clotting  Regulation of renal function  Control of several reproductive functions, such as the induction of labor  MECHANISMS OF ACTION Via the G protein-coupled receptors:  a) Gs stimulate adenylate cyclase (AC)  b) Gi inhibit adenylate cyclase (e.g. PKA)  c) Gq activates phospholipase C that cleaves phosphatidylinositol -4,5-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and diacylgly-cerol (DAG); DAG together with Ca2+ activates protein kinase C, IP3 opens Ca2+ channels of the ER +  EFFECTS OF PROSTAGLANDINS  Mediate inflammation:  cause vasodilation  redness, heat (PGE1, PGE2, PGD2, PGI2)  increase vascular permeability  swelling (PGE2, PGD2, PGI2)  Regulate pain and fever (PGE2)  PGE2, PGF2 stimulate uterine muscle contractions during labor  Prostaglandins of the PGE series inhibit gastric acid secretions (synthetic analogs are used to treat gastric ulcers)  Regulate blood pressure: vasodilator prostaglandins PGE, PGA, and PGI2 lower systemic arterial pressure  Regulate platelet aggregation: PGI2 = potent inhibitor of platelet aggregation  PGE2 inhibits reabsorption of Na+ and water in the collecting duct. PGI2: vasodilatation and regulation of glomerular filtration rate.  BIOLOGICAL ROLE OF THROMBOXANES  Thromboxanes are synthesized by platelets and, in general, cause vasoconstriction and platelet aggregation  TXA 2 is also produced in the kidney (by podocytes and other cells) where it causes vasoconstriction and mediates the response to ANGII  Thus, both thromboxanes and prostaglandins (PGI 2 ) regulate coagulation  In Eskimos, higher intake of eicosapentaenoic acid and group 3 prostanoids may be responsible for low incidence of heart diseases and prolonged clotting times since TXA3 is a weaker aggregator than TXA2 and both PG3 and TXA3 inhibit arachidonate release and TXA2 formation  BIOLOGICAL ROLE OF LEUKOTRIENES  LTs are produced mainly in leukocytes that also express receptors for LTs  Leukotrienes are very potent constrictors of the bronchial airway muscles: (LTC4, LTD4, and LTE4 = the slow-reacting substance of anaphylaxis)  They increase vascular permeability  They cause attraction (LTB4) and activation of leukocytes (primarily eosinophils and monocytes), promote diapedesis (increase expression of integrins on the leukocyte surface), enhance phagocytosis  They regulate vasoconstriction they regulate inflammatory reactions, host defense against infections as well as hyperreactivity (asthma…)  LIPOXINS Lipoxins are produced mainly by leukocytes and platelets stimulated by cytokines (IL-4, TGF-β):  a) 5-lipoxygenase (5-LO) of neutrophils produces leukotriene LTA4 which enters platelets where it is converted by 15-LO to LXA4 or LXB4  b) 15-LO of epithelial cells and monocytes forms 15-HPETE which becomes a substrate of 5-LO and epoxid hydrolase of leukocytes …transcellular biosynthesis Main products: LXA4, LXB4  BIOLOGICAL ROLES OF LIPOXINS  Unlike pro-inf lammatory eicosanoids, lipoxins attenuate the inf lammation and appear to facilitate the resolution of the acute inflammatory response  Hypothesis: in the f irst phase of the inf lammatory response, leukotrienes are produced (e.g. LTB4) → then, the level of PGs rises and PGs „switch“ the synthesis from leukotriene production to the pathway which, in the 2nd phase, produces lipoxins promoting the resolution of inflammation  Therefore, potential therapeutic use of LXs in the treatment of inf lammatory diseases (glomerulonephritis, asthma) is being extensively studied  EFFECTS OF LXS MEDIATING THE RESOLUTION OF INFLAMMATION  LXs inhibit chemotaxis of neutrophils and eosinophils and diapedesis  Inhibit formation of ROS (neutrophils, lymphocytes) and ONOO- (neutrophils)  Inhibit production of specific cytokines by leukocytes  Stimulate non-inflammatory phagocytosis (of apoptotic neutrophils…)  Antagonize LT receptors  Affect not only the cells of the myeloid line:  inhibit the contraction of the bronchial smooth muscle  inhibit production of cytokines by the cells of colon, fibroblasts…  inhibit the interaction between leukocytes and endothelial cells  BIOLOGICAL EFFECTS OF HETES  5-HETE participates in host defense against bacterial infection (chemotaxis and degranulation of neutrophils and eosinophils)  20-HETE causes vasoconstriction (by its effect on the smooth muscle of vessels); in kidney, it regulates Na+ excretion, diuresis, and blood pressure  12- and 15-HETE are produced in kidney and participate in the regulation of the renin-angiotensin system (12-HETE also mediates secretion of aldosteron induced by ANGII)  BIOLOGICAL ROLES OF HEPOXILINS  HXA3 stimulates glucose-induced insulin secretion by pancreatic β cells  Under oxidative stress, HXA3 formation is stimulated and HXA3 upregulates the expression of glutathione peroxidase…compensatory defense response to protect cell viability? BIOSYNTHESIS OF TRIACYLGLYCEROL  SYNTHESIS OF TAG  The pathway for triacylglycerol synthesis in most tissues, including liver and adipocytes, utilizes glycerol 3-phosphate and fatty acyl-CoA.  Synthesis of TAG takes place in endoplasmic reticulum.  The activated fatty acids (i.e., fatty acids attached to coenzyme A) are derived either from endogenous, de novo fatty acid synthesis or from dietary fats.  By contrast, triacylglycerol synthesis in the small intestine begins with 2-monoacylglycerol.  SYNTHESIS OF TRIACYLGLYCEROLS 1- Synthesis of glycerol phosphate 2- Formation of fatty acyl CoA 3- Formation of a molecule of TAG  SYNTHESIS OF GLYCEROL PHOSPHATE  In liver; this process depends on supply of glucose or free glycerol coming to liver is converted to glycerol phosphate by enzyme Glycerol kinase.  In adipose tissue glucose uptake is insulin dependent as it has GLUT-4 receptors. Low glucose--- low insulin ----- no synthesis of TAG in adipocytes. FORMATION OF ACTIVATED FREE FATTY ACID  Long chain fatty acids are converted to fatty acyl CoA. Enzyme required is Fatty acyl CoA synthase.  Fatty acyl CoA participates in TAG synthesis. SYNTHESIS OF A MOLECULE OF TAG G lyc e ro l ph o sph a t e combines with a fatty ac yl Co A and f o rms Lysophosphatidic acid. E n z y m e i s Acyltransferase which removes CoA. Lysophosphatidic acid combines with the second fatty acyl CoA to form DAG phosphate. E n z y m e i s Acyltransferase. Phosphatase removes phosphate and forms DAG. DAG combines with the third fatty acyl CoA and forms TAG. FATES OF TAG IN LIVER AND ADIPOSE TISSUE Adipose tissue ---- TAG stored in cytosol Liver --- very little stored. Exported out of liver in VLD L , which exports endogenous lipids to peripheral tissues.  SYNTHESIS OF PHOSPHOLIPIDS  All cells, with the possible exception of mature red blood cells, are capable of synthesizing one or more glycerophospholipids.  Most of the reac tions involved in phospholipid sy n t h e si s o c c u r o n t h e c y t o so l i c f a c e o f t h e endoplasmic reticulum and Golgi complex.  The liver is a major site of phospholipid synthesis  Two other tissues with a high capacity for phospholipid synthesis are intestinal enterocytes, type I1 cells of the lung, which synthesize pulmonary surfactant SYNTHESIS OF GLYCEROPHOSPHOLIPIDS Phosphatid ic ac id is the basic c omponent for glycerophospholipid synthesis which then combines with an alcohol. This may involve two processes, i.e 1. Phosphatidic acid may be donated from CDP- diacylglycerol to an alcohol 2. CDP- alcohol may donate its phosphomonoester to diacylglycerol. SYNTHESIS OF PC (LECITHIN) One of the most abundant PL in cells. Substrates required ar e :-  Cho line; preexisting obtained from diet or from turnover of PL.  Diacylglycerol; formed by removal of phosphate from pho sphatidic ac id. E n z y m e i s phosphatidate hydrolase. PC can also be formed from PS in the liver.  PC CAN ALSO BE FORMED FROM PS Takes place only in liver. Liver can make PC by this process even when free choline levels are low. PS is converted to PE.  Enzyme is PS decarboxylase. PE undergoes methylation. Enzyme is methyltransferase.  Re su l t i s t h e f or m a t i on of phosphatidylcholine. Choline is now recognized as an essential nutrient, especially for people with low dietary intakes of protein whose methionine pool may be i n a d e q u a t e. C h o l i n e d e f ic i e n c y compromises hepatic VLDL synthesis and secretion and can therefore result in fatty liver SYNTHESIS OF PE  F o r m e d f r o m p r e e x i s t i n g Ethanolamine.  Phosphorylation o f ethanolamine by Kinase.  Formation of CDP- Ethanolamine.  T r a n s f e r o f E t hano l ami ne Phosphate from CDP to Diacylglycerol forms PE.  PS can be converted to PE by reversal of decarboxylation. SYNTHESIS OF PS  Formed from PE.  PE reacts with serine to form PS. Enzyme is PE- Serine transferase.  This is a base exchange reaction in which ethanolamine of PE is exchanged for free serine. SYNTHESIS OF PI  Substrate s re q uire d are fre e ino sito l and C DP- diacylglycerol.  Diacylglycerol 3 phosphate (PA) reacts with CTP to form CDP-diacylglycerol. Enzyme is Diacylglycerol- CDP synthase.  CDP-Diacylglycerol reacts with inositol and forms Phosphatidyl inositol. Enzyme is PI Synthase. SYNTHESIS OF CARDIOLIPIN  Cardiolipin is di-phosphatidyl glycerol in nature.  It is composed of two molecules of phosphatidic acid connected by a molecule of glycerol. C D P - d i a c y l g l y c e r o l t r a n s f e r s diacylglycerophosphate to phosphatidylglycerol to form cardiolipin. SYNTHESIS OF PLASMALOGENS Plasmalogens are the PL in which F.A at C1 of glycrol is attached by an ether linkage. Substrates required are di- hydroxy acetone phosphate and acyl CoA. DEGRADATION OF PHOSPHOLIPIDS Phospholipase A1:- -found in many mammalian tissues. -removes fatty acid from C1 Phospholipase A2:- -found in many tissues and pancreatic juice -removes F.A at C2 -when acts on PI, releases arachidonic acid -inhibited by glucocorticoids DEGRADATION OF PHOSPHOLIPIDS Phospholipase C:- - cleaves phosphate group at C3 - found in liver lysosomes and some bacteria - role in producing second messengers. Phospholipase D:- - found primarily in plant tissues. - removes the compound with alcohol group on C3 DEGRADATION OF SPHINGOMYELIN Enzyme is Sphingomyelinase, a lysosomal enzyme. It removes phosphorylcholine hydrolytically and ceramide is produced. Ceramide is cleaved by ceramidase and leaves behind sphingosine and a free fatty acid. Sphingosine and ceramide act as intracellular messengers. DEGRADATION OF GLYCOSPHINGOLIPIDS Done by lysosomal enzymes D i f f e r e n t e n z y m e s a c t o n s p e c i f ic b o n d s hydrolytically ---- the groups added last are acted first. SPHINGOLIPIDOSES Lipid storage diseases Accumulation of sphingolipids in lysosomes Partial or total absence of a specific hydrolase Autosomal recessive disorders TYPES OF SPHINGOLOIPIDOSES Gaucher disease:- - most common lysosomal storage disease - absence of glucocerebrosidase enzyme - accumulation of glucocerebrosides - enlargement of liver and spleen - osteoprosis of long bones - CNS involvement in infants Krebbe disease:- - accumulation of galactocerebrosides - absence of galactosylceramidase - mental and motor function defect - blindness and deafness - loss of myelin Farber disease:- - accumulation of ceramide - absence of ceramidase - joints and skin involvement Niemann pick disease:- - accumulation of sphingomyelin - absence of sphingomyelinase - liver and spleen enlargement - neuronal degeneration Fabry disease:- - accumulation of globosides - ansence of alpha galactosidase - skin rash - kidney and heart failure - burning pain in legs METABOLISM OF CHOLESTEROL CHOLESTEROL BIOSYNTHESIS  Principal sterol synthesized by animals.  In vertebrates, hepatic cells typically produce more cholesterol than other cells.  Absent among prokaryotes. CHOLESTEROL BIOSYNTHESIS Cholesterol required by the body is derived from diet (200-300mg/day) and synthesis in body (700 mg/day is synthesized). Liver and Intestine accounts for larger proportion. NOTE: Virtually all cells containing nucleated cells are capable of synthesizing cholesterol which occurs in endoplasmic reticulum (ER) and cytosol of cells. Acetyl-CoA is the source of all carbon atoms in cholesterol. Coronary atherosclerosis correlates with high plasma LDL/HDL cholesterol ratio. DIVIDED INTO 5 STEPS 1. Synthesis of mevalonate occurs from Acetyl-CoA 2. Isoprenoid units are formed from MEVALONATE by loss of CO2 3. Six isoprenoid units condense to form SQUALENE 4. SQUALENE is cyclizes to give rise to parent steroid LANOSTEROL 5. Cholesterol is formed from LANOSTEROL STEP 1 2. FORMATION OF ISOPRENOID UNITS 3. FORMATION OF SQUALENE 4. FORMATION OF LANOSTEROL 5. FORMATION OF CHOLESTEROL REGULATION OF CHOLESTEROL SYNTHESIS IS CONTROLLED BY HMG-COA REDUCTASE REGULATION OF CHOLESTEROL SYNTHESIS IS CONTROLLED BY HMG-COA REDUCTASE 1.Decreased synthesis of cholesterol in starving animals is accompanied by reduced activity of the enzyme. 2. Hepatic synthesis is inhibited by dietary cholesterol. 3.HMG-CoA reductase in liver is inhibited by Cholesterol. 4. Cholesterol and metabolites represses transcription of HMG-CoA reductase transcription by Sterol Regulatory Element Binding Protein (SREBP). 5. Insulin or thyroid hormone increases HMG-CoA reductase activity, While, Glucagon or glucocorticoids decrease it. INCREASE AND DECREASE IN CHOLESTEROL Increased by 1. Uptake of cholesterol-containing lipoproteins by receptors. 2. Uptake of free cholesterol from cholesterol-rich lipoproteins to the cell membrane 3. Cholesterol synthesis 4. Hydrolysis of cholesterol ester by the enzyme cholesterylester hydrolase. INCREASE AND DECREASE IN CHOLESTEROL Decreased by 1. Efflux of cholesterol from the membrane to lipoproteins of low cholesterol potential 2. Esterification of cholesterol by ACAT (acyl-CoA: cholesterol acyltransferase) 3. Utilization of cholesterol for synthesis of other steroids such as hormones or bile acids in liver WHAT ARE THE FACTORS THAT INFLUENCE CHOLESTEROL BALANCE IN TISSUES? DECREASE OF CHOLESTEROL IN CELLS Efflux of cholesterol from cells to HDL Esterif ic ation of cholesterol by the enzyme “Acyl- CoA-cholesterol acyl transferase” (ACAT). Utilization of cholesterol for synthesis of steroid hormones, viz. glucocorticoids, mineralo-corticoids, gonadal hormones In liver cells: formation of cholic acid Formation of Vitamin D3 CHOLESTEROL BALANCE IN TISSUES Increase of cholesterol in cells Increased synthesis of cholesterol Hyd ro lysis o f c ho lestero l ester by the enzyme “cholesterol ester hydrolyase”. U pt ake and d e l i v e ry o f c ho l e st e ro l i n c e l l s by circulating LDL Uptake of cholesterol-containing lipoproteins by ‘non- receptor’ Uptake of free cholesterol by cell membranes CONSIDERATION OF OTHER FACTORS THAT INFLUENCE CHOLESTEROL LEVEL IN BLOOD Dietary fats (saturated FA vs Unsaturated FA) Dietary cholesterol Dietary carbohydrates Heredity Blood groups Calorie Intake Vitamin B-complex Minerals Dietary fibers Physical exercise Life style of an individual METABOLISM OF LIPOPROTEINS CLASSES OF LIPOPROTEINS  On the basis of density ;-ultracentrifugation - chylomicrons - VLDL - LDL - HDL  On electrophoresis– - chylomicrons at origin - LDL (beta lipoproteins) - VLDL ( pre-beta lipoproteins) -HDL ( alpha lipoproteins) APOLIPOPROTEINS Serve very important functions  recognition site for cell-surface receptors  ac tivators or c oenzymes for enzymes of lipoprotein metabolism  some are essential structural component of the lipoprotein particle  transfer between different types of lipoproteins and bring about changes.  Classes from A to E. Some have sub classes. METABOLISM OF LIPOPROTEINS  There are striking similarities in the mechanisms of formation of chylomicrons by intestinal cells and of VLDL by hepatic parenchymal cells  Apart from the mammary gland, the intestine and liver are the only tissues from which particulate lipid is secreted.  Newly secreted or "nascent" VLDL contain only a small amount of apolipoproteins C and E, and the full complement is acquired from HDL in the circulation  Apo B 100 is essential for VLDL formation METABOLISM OF VLDL VLDL are secreted into the space of Disse and then into the hepatic sinusoids through fenestrae in the endothelial lining.  CATABOLISM OF VLDL  Catabolism of VLDL is similar to chylomicrons  Both phospholipids and apo C-II are required as cofactors for lipoprotein lipase activity, while apo A- II and apo C-III act as inhibitors.  Hyd ro lysis take s plac e while the VLD Ls are attached to the enzyme on the endothelium.  Triacylglycerol is hydrolyzed progressively through a diacyl glycerol to a monoacylglycerol and f inally to free fatty acids plus glycerol.  Some of the released free fatty acids return to the circulation, attached to albumin, but the bulk is transported into the tissue. CATABOLISM OF VLDL  Reaction with lipoprotein lipase results in the loss of approximately 90% of the triacylglycerol of VLDLs and in the loss of apo C (which returns to HDL) but not apo E, which is retained.  These changes occurring to VLDL, lead to the formation of VLDL remnants or IDL (intermediate-density lipoprotein)  After metabolism to IDL, VLDL may be taken up by the liver directly via the LDL (apo B-100, E) receptor, or it may be converted to LDL.  Only one molecule of apo B-100 is present in each of these lipoprotein particles, and this is conserved during the transformations.  Thus, each LDL particle is derived from a single precursor VLDL particle.  In humans, a relatively large proportion of IDL forms LDL, accounting for the increased concentrations of LDL in humans compared with many other mammals. METABOLISM OF VLDL T h e liver and many e xt rahe pat ic t issue s express the LDL (apo B -100, E) receptor. I tis so designated because it is specif ic for apo B-100  b u t n ot B- 4 8 , w h i ch lacks t he carboxyl terminal domain of B-100 cont aining t he LDL receptor ligand,  a nd i t a l so t a ke s up lipoproteins rich in apo E. 1/24/2025 Biochemistry for medics 256 METABOLISM OF LDL  The liver and many extra hepatic tissues express the LDL (apo B-100, E) receptor.  It is so designated because it is specif ic for apo B -100 but not B-48, which lacks the carboxyl terminal domain of B-100 containing the LDL receptor ligand, and it also takes up lipoproteins rich in apo E.  This receptor is defective in familial hypercholesterolemia.  Approximately 30% of LDL is degraded in extra- hepatic tissues and 70% in the liver.  A positive correlation exists between the incidence of coronary atherosclerosis and the plasma concentration of LDL cholesterol. METABOLISM OF HDL Synthesis of HDL  HDL is synthesized and secreted from both liver and intestine.  However, apo C and apo E are synthesized in the liver and transferred from liver HDL to intestinal HDL when the latter enters the plasma.  A major function of HDL is to act as a repository for the apo C and apo E required in the metabolism of chylomicrons and VLDL.  Nascent HDL consists of discoid phospholipid bilayer containing apo A and free cholesterol. METABOLISM OF HDL  LCAT and the LCAT activator apo A-I—bind to the discoidal pa r t i c l e s, a nd t he sur fa c e pho spho l i pi d a nd fre e cholesterol are converted into cholesteryl esters and lysolecithin.  The nonpolar cholesteryl esters move into the hydrophobic interior of the bilayer, whereas lysolecithin is transferred to plasma albumin.  Thus, a nonpolar core is generated, forming a spherical, pseudomicellar HDL covered by a surface f il m of polar lipids and apolipoproteins.  This aids the removal of excess unesterif ie d cholesterol from lipoproteins and tissues. METABOLISM OF HDL Role of LCAT LCAT( Lecithin Cholesterol Acyl Transferase) enzyme catalyzes the esterification of cholesterol to form Cholesteryl ester. The reaction can be represented as follows- Lecithin + Cholesterol Lysolecithin + Cholesteryl Ester REVERSE CHOLESTEROL TRANSPORT The cholesterol efflux is brought about by esterification of cholesterol under the effect of LCAT. The cholesteryl ester rich HDL (HDL2) gains entry through Scavenger receptor (SR-B1) METABOLISM OF HDL The class B scavenger receptor B1 (SR-B1) has been identif ie d as an HDL receptor with a dual role in HDL metabolism. In the liver and in steroidogenic tissues, it binds HDL via apo A-I, and cholesteryl ester is selectively delivered to the cells, although the particle itself, including apo A-I, is not taken up. In the tissues, on the other hand, SR-B1 mediates the acceptance of cholesterol from the cells by HDL, which then transports it to the liver for excretion via the bile in the process known as reverse cholesterol transport METABOLISM OF HDL  A second important mechanism for reverse cholesterol transport involves the ATP-binding cassette transporter A1 (ABCA1).  ABCA1 is a member of a family of transporter proteins that couple the hydrolysis of ATP to the binding of a substrate, enabling it to be transported across the membrane.  ABCA1 preferentially transfer cholesterol from cells to poorly lipidated particles such as pre -HDL or apo A-1, which are then converted to HDL3 via discoidal HDL  Pre -HDL is the most potent form of HDL inducing cholesterol efflux from the tissues. FUNCTIONS OF HDL  Scavenging action- HDL scavenges extra cholesterol from peripheral tissues by reverse cholesterol transport  HDL, with the help of apo E competes with LDL for bi nd i ng si t e s o n t he me mbrane s and prev e nt s internalization of LDL cholesterol in the smooth cells of the arterial walls  HDL contributes its apo C and E to nascent VLDL and chylomicrons for receptor mediated endocytosis  HDL stimulated prostacyclin synthesis by the endothelial cells, which prevent thrombus formation  HDL also helps in the removal of macrophages from the arterial walls.  HDL prevent LDL from getting oxidized SUMMARY OF FORMATION AND FATE OF LIPOPROTEINS  C hylomic rons is a transporter of dietary lipids whereas VLDL is a transporter of e n d o g e n o u s lipids(mainly TGs).  LDL transports cholesterol to peripheral cells while HDL transports cholesterol from peripheral cells back to liver. 265 ROLE OF HDL IN RECEPTOR MEDIATED ENDOCYTOSIS HDL contributes its apo C and E to nascent VLDL and chylomicrons for re