LIPID METABOLISMAbdul Salam M. SofroFaculty of Medicine YARSI University &Research Center for BiotechnologyGadjah Mada University

Teaching aimsTo highlight utilization and storage of energy in lipid form and understand the individual biochemical pathways of lipid metabolism

Scope of lipid metabolismBiosynthesis of fatty acidsOxydation of fatty acids: ketogenesisUFA & Eicosanoids MetabolismCholesterol synthesis, transport & excretionLipid transport & storageMetabolism of acylglycerols & sphingolipids

IntroductionAs biomolecule, lipids are important for structure, obtain and storage of energyTheir characteristics are nonpolar & hidrofobicMostly contain or derived from fatty acidsStored in the form of triacylglycerol more efficient and quantitativey more important compared with carbohydrate storage as glycogenMore important functions such as: integrity of alveoli, solubilization of nonpolar compounds, metabolic processes etc.s

FoodCarbohydrateLipidProteinOthers (Nucleic acids, water, minerals, vitamins etc.)Lumen of GI tractMucosal cellsDigestion of TAG to: glycerol & fatty acidsRe-esterification of TAG from : glycerol & fatty acidsLympatic system and then blood circulationOrgans & tissuesBiosynthesis of fatty acidsOxydation of fatty acids: ketogenesisUFA & Eicosanoids MetabolismCholesterol synthesis, transport & excretionLipid transport & storageMetabolism of acylglycerols & sphingolipidsLipid transport

Lipids are a major source of energy during rest and exercise. Approximately half of the lipidsstored as triglyceridesthat are used for energy come from adipose tissue with the other half from intramuscular stores. There are several steps in the mitochondrial oxidation of lipids that begin with the mobilization of the triglycerides

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(Lipogenesis)

Biosynthesis of fatty acids (cont.)Fatty acid synthesis or lipogenesis may vary between individualsHighly active extramitochondrial system is responsible for the complete synthesis of palmitate from acetyl-CoA (in the cytosol)Another system for fatty acid elongation is also present in liver endoplasmic reticulum

The main pathway for de novo lipogenesisPresent in many tissues, including liver, kidney, brain, lung, mammary gland, and adipose tissueIn most mammals, glucose is the primary substrate for lipogenesis, but in ruminants acetate.Requires cofactors, including NADPH, ATP, Mn2+, biotin & HCO3- (as a source of CO2)Acetyl-CoA is the immediate substrate and free palmitate is the end product

Important notes in lipogenesisThe initial & controlling step in fatty acid synthesis is the production of Malonyl-CoA

Fatty acid synthase complex is a multienzyme polypeptide complex with acyl carrier protein (ACP) as its part containing seven enzyme activitiesAcetyl-CoA Malonyl-CoAEnz-biotin-COO- Enz-biotinADP + Pi ATP + HCO3-Enz = acetyl-CoA carboxylase

Bicarbonate as a source of CO2 is required in the initial reaction for carboxylation of acetyl-CoA to malonyl-CoA in the presence of ATP & Acetyl-CoA carboxylase that requires vitamin Biotin.

Important notes in lipogenesis (cont.)The main source of NADPH for lipogenesis is the Pentose Phosphate Pathway (PPP)Acetyl-CoA is the principal building block of fatty acidsElongation of fatty acid chains occurs in the endoplasmic reticulumChain elongation also occurs in mitochondria but less active and uses acetyl-CoA as acetyl donor (its function is speculative)

Important notes in lipogenesis (cont.)Nutritional state regulates lipogenesis the rate is higher in the well-fed animal whose diet contains a high proportion of carbohydrate (Nutritional state of organism is the main factor regulating the rate of lipogenesis)There is inverse relationship between hepatic lipogenesis & concentration of serum FFA => decreased lipogenesis in increased serum FFA (which is associated with restricted caloric intake, on a high-fat diet or insulin deficiency as in DM)

Important notes in lipogenesis (cont.)High fat in the diet causes depression of lipogenesis, or when there is more than 10% of fat in the diet, there is little conversion of dietary carbohydrate to fatInsulin stimulate lipogenesis by several mechanism increases glucose transp. into the cell (e.g. in adipose tissue)Insulin inhibits lipolysis (lipid degradation) in adipose tissueFatty acid synthase complex & acetyl-CoA carboxylase are adaptive enzymes

Short term & long term mechanisms regulate lipogenesisShort term by allosteric & covalent modification of enzymesLong term by changes in gene expression governing rates of enzymes synthesis (FA Synthase Complex & Acetyl-CoA Carboxylase are adaptive enzymes)

Oxidation of Fatty Acids: (& Ketogenesis) Oxidized to Acetyl-CoA and synthesized from Acetyl-CoAStarting material of one process is identical to the product of the other and the chemical stages involved are comparable, the fatty acid (FA) oxidation is not the simple reverse of FA biosynthesis. It is entirely different process taking place in a separate cell compartment => oxidation in mitochondria, synthesis in cytosol. Fatty acid.

Oxidation of Fatty Acids: KetogenesisIncreased in starvation & DM leading to ketone body production by the liver (ketosis) when produced in excess over long periods, as in DM, cause ketoacidosis (ultimately fatal)Gluconeogenesis is dependent upon fatty acid (FA) oxidation any impairment in this FA oxidation leads to hypoglycemia. This occurs in various states of carnitine deficiency or deficiency of essential enzymes in FA oxidation Biomedical importance:

Oxidation of fatty acids Occurs in mitochondriaOrigin of FA : blood & cells, transported in blood as free fatty acids (FFA = unesterified state)In plasma FFA of longer chain FA are combined with albumin and in the cell they are attached to a FA binding protein (protein Z).

Shorter-chain FA are more water soluble and exist as the un-ionized acid or as a FA anionFA are activated before being catabolized in the presence of ATP & CoA, acyl-CoA synthetase (thiokinase) catalyzes the conversion of FFA to active fatty acid or acyl-CoA:

FA + ATP + CoA Acyl-CoA + PPi + AMP

Oxidation of fatty acids (cont.)

Long chain FA penetrate the inner mitochondrial membrane as carnitine derivatives

Activation of lower FA & their oxidation within mitochondria may occur independently of carnitine

Acetyl-CoA + carnitine Acetylcarnitine + CoA.Carnitine acetyl transferase

Long-chain FA (Acyl-CoA cannot pass through the inner mitochondrial membrane, but its metabolic product, acylcarnitine, canRole of carnitine in the transport of long-chain FA through the inner mitochondrial membrane

Transport of fatty acids from the cytoplasm to the inner mitochondrial space for oxidation. Following activation to a fatty-CoA, the CoA is exchanged for carnitine by carnitine-palmitoyltransferase I. The fatty-carnitine is then transported to the inside of the mitochondrion where a reversal exchange takes place through the action of carnitine-palmitoyltransferase II. Once inside the mitochondrion the fatty-CoA is a substrate for the b-oxidation machinery.

Types of FA oxidation Alfa Oxidation : the removal of one carbon at a time from the carboxyl end of themolecule (have been detected in brain tissue => it does not require CoA intermediate and does not generate high energy-PBeta Oxidation : two carbon atoms are cleaved at a time from acyl-CoA molecules, starting at the carboxyl end to produce acetyl-CoA => produce a large quantity of ATP. It is the main FA oxidation

Types of FA oxidation (cont.)Omega Oxidation : normally a very minor pathway and is brought about by hydroxylase enzymes involving cytochrome P450 in endoplasmic reticulum CH3 group is converted to a H2OH group that subsequently is oxidized to -COOH thus forming dicarboxylic acid. This is usually -oxidized to adipic (C6) and suberic (C8) acids which are excreted in the urineOxidation of unsaturated FA occurs by a modified -oxidation pathway

10816 24146146 x 51.6* = 7533.6 kJ

Comparison of FA oxidation & FA biosynthesis (lipogenesis)Oxidation: * location mitochondria * uses NAD+ & FAD as coenzymes * generates ATP * involving acyl-CoA derivativesLipogenesis: * location cytosol * uses NADP+ as coenzymes & requires ATP + bicarbonate ion * involving acyl derivatives bound to the multienzyme complex

KetogenesisKetogenesis is the generation of ketone bodies (acetoacetate, D(-)-3-hydroxybutyrate = -OH-butyrate & acetone)Occurs when there is a high rate of FA oxidation in the liver The enzyme system is in mitochondria, with FFA precursor in the liver

Ketogenesis (cont.)Two molecules of acetyl-CoA condence to form acetoacetyl-CoA, and with the addition of another acetyl-CoA produce 3-OH-3-methyl-glutaryl-CoA (HMG-CoA) catalyzed by HMG-CoA synthaseHMG-CoA is an intermediate in the pathway of ketogenesis -> in the presence of HMG-CoA lyase is split into acetyl-CoA and acetoacetate

Ketogenesis (cont.)Acetoacetate is in equilibrium with D(-)-3-OH-butyrate (predominant keton body present in the blood and urine in ketosis) catalyzed by D(-)-3-OH butyrate DH & NADH, or spontaneously converted into acetone releasing CO2Liver is the only organ in non-ruminants to add significant quantities of ketone bodies to the blood

Ketogenesis (cont.)Ketone bodies serve as a fuel for extrahepatic tissues while acetoacetate & D(-)-3-OH butyrate are readily oxidized by extrahepatic tissues, acetone is difficult to oxidize in vivo and volatilized in the lungs Prolonged ketosis leads to ketoacidosis (such as in DM). In starvation simple ketosisMeasurement of ketonemia is preferred than that of ketonuria

Clinical aspects of impaired oxidation of FACarnitine deficiencyCarnitine palmitoyltransferase-I deficiencyCarnitine palmitoyltransferase-II deficiencyAcute fatty liver of pregnancyHMG-CoA lyase deficiencyJamaican vomiting sicknessDicarboxylic aciduriaRefsums diseaseZellwegers (cerebrohepatorenal) syndrome

Metabolism of Unsaturated FA Compared with plants, animal tissues have limited ability in desaturating FA need PUFA (polyunsaturated FA) from a plant source which are essential FA to give rise to eicosanoic (C20) FA, from which are derived families of compounds known as eicosanoids. Eicosanoids are physiologicaly & pharmacologically active compounds known as prostaglandins (PG), tromboxanes (TX), leukotrienes (LT) & lipoxins (LX)

Metabolism of Unsaturated FA (cont.)

Some PUFA cannot be synthesized by mammals and are therefore nutritionally essentialOther C20, C22 & C24 polyenoic FA may be detected in the tissues may be derived from oleic, linoleic, & -linoleic acids by chain elongation. The double bond present in naturally occuring unsaturated FA of mammals are the cis configuration

Metabolism of Unsaturated FA (cont.)Trans-UFA may compete with cis-UFAUp to 15% of tissue UFA have been found to be in trans configuration metabolized more like saturated than like cis-UFAtend to raise LDL levels & lower HDL levels and thus contraindicated with respect to the prevention of atherosclerosis & coronary heart disease do not possess essent. FA activity and may antagonize the metabolism of essent. FA & exacerbate essent. FA deficiency

Metabolism of Unsaturated FA (cont.)Monounsaturated FA are synthesized by a 9 desaturase system and synthesis of PUFA involves desaturase and elongase enzyme systemsEicosanoids are formed from C20 PUFA (arachidonate and some other C20 FA)Deficiency of essential PUFA (linoleic, linolenic & arachidonic acid) leads to a deficiency syndrome in experiment rats include scaly skin, necrosis of the tail, and lesion in the urinary system

Metabolism of Unsaturated FA (cont.)Essential PUFA functions are various, apart from prostaglandin & leukotriene formation. They are found in the structural lipids of the cell & are concerned with the structural integrity of the mitochondrial membrane

Metabolism of Unsaturated FA (cont.)Example : arachidonic acid is present in membrane, accounts for 5-15% of FA in phospholipids; docosahexanoic acid (DHA 3,2:6) synthesized from -linolenic acid or obtained directly from fish oils is present in high concentrations in retina, cerebral cortex, testis & sperm. DHA is particularly needed for development of the brain & retina and is supplied via the placenta & milk

Pathway from linoleic acid to arachidonic acid. Numbers in parentheses refer to the fatty acid length and the number and positions of unsaturations.

Biosynthetic pathways of EicosanoidsTwo pathways from arachidonate:* Cyclooxygenase pathway forms Prostanoids (PG2 & TX2 series : PGD2, PGE2, PGF2, PGI2, TXA2)* Lypoxygenase pathway forms Leucotrienes (LTA4, LTB4, LTC4, LTD4, LTE4) & Lipoxins (LXA4, LXB4, LXC4, LXD4, LXE4)

PGE2 TXA2 LTA4 The eicosanoids consist of the prostaglandins (PGs), thromboxanes (TXs) and leukotrienes (LTs). The PGs and TXs are collectively identified as prostanoids.

Biosynthetic pathways of Eicosanoids (cont.)Prostanoids are potent biologically active substances:* thromboxanes are synthesized in platelets and upon release cause vasoconstriction & platelet aggregation* prostacyclins (PGI2) are produced by blood vessel walls and are potent inhibitors of platelet aggregation

Biosynthetic pathways of Eicosanoids (cont.)* Leucotrienes & lipoxins are potent regulators of many disease processes a mixture of leucotrienes C4, D4 & E4 is a slow reacting reacting substance of anaphylaxis (SRA-A) which is 100-1000 times more potent than histamine or prostaglandins as a constrictor of the bronchial airway musculature

Synthesis of the clinically relevant prostaglandins and thromboxanes from arachidonic acid. Numerous stimuli (e.g. epinephrine, thrombin and bradykinin) activate phospholipase A2 which hydrolyzes arachidonic acid from membrane phospholipids. The prostaglandins are identified as PG and the thromboxanes as TX. Prostaglandin PGI2 is also known as prostacyclin. The subscript 2 in each molecule refers to the number of -C=C- present.

Synthesis of the clinically relevant leukotrienes from arachidonic acid. Numerous stimuli (e.g. epinephrine, thrombin and bradykinin) activate phospholipase A2 which hydrolyzes arachidonic acid from membrane phospholipids. The leukotrienes are identified as LT. The leukotrienes, LTC4, LTD4, LTE4 and LTF4 are known as the peptidoleukotrienes because of the presence of amino acids. The peptidoleukotrienes, LTC4, LTD4 and LTE4 are components of slow-reacting substance of anaphylaxis The subscript 4 in each molecule refers to the number of -C=C- present.

Biosynthetic pathways of Eicosanoids (cont.) together with LT B4 also cause vascular permeability, attraction & activation of leukocytes seems to be important regulators in many diseases involving inflammatory or immediate hypersensitivity reactions, such as asthma

Membrane phospholipidArachidonateLeukotrienesLipoxinsProstaglandinsThromboxanesPHOSPHOLIPASEA2LIPOXYGENASECYCLOOXYGENASEVarious stuimuli, e.g.Angiotensin II,bradykinin.,Epinephrinethrombin