1. LIPIDS Biochemistry Department

2. Fatty Acids And Triacylglycerol Metabolism

3. © I L O Intended Learning Outcomes By the end of this lecture, the student should be capable of: 1.Recognizing fatty acid and TAG chemistry 2.Recognizing the different types of fatty acid synthesis and oxidation. 3.Finding the role of storage of fatty acid as TAG 4.Identifying the TAG synthesis and degradation 5.summarizing the synthesis and oxidation processes of fatty acids. 6.explaining the modes of regulation for TG & FA metabolism 7.Describing some abnormalities in lipid metabolism

4. *It is the simplest lipid unit; it consists of a hydophobic hydrocarbon chain with terminal carboxyl group (hydrophilic). CH3-CH2-CH2----------

5. *Its monocarboxylic, straight and even mostly, its pKa at physiologic pH is 4.8, the COOH ionizes. *> 90% FAs are esters (TAG, CE, and PL) in lipoproteins. Unesterified (free) are transported with albumin. Structure of Fatty Acids

6. (Delta numbering) and )Greek lettering) 6 5 4 3 2 1 CH 3 – CH 2 – CH 2 - CH 2 – CH 2 – COOH (  ) (  ) (  ) (  ) There are methods of naming of fatty acid carbons: Omega (  ) or n numbering CH 3 – CH 2 – CH 2 - CH 2 – CH 2 – COOH (  1 ) (  2 ) (  3) *From the carboxyl end * Or from the methyl carbon (CH3)

7. 1-Saturated FA (No Double bond) General formula: CH3 - (CH2) n - COOH n represent number of methylene (CH2) groups A. Saturation of fatty acids

8. B. Unsaturation of fatty acids Bonds are cis rather than trans, causing FA “kink” (120  bend) at bond position which is important for packing in biological membranes. Cis Trans

9. Classification according to length of F.A: Short<6 carbons Middle 6-12 carbons Long >12 carbons hydroxyl group containing FAs :  - hydroxy FA of Sphingolipids (Cerebronic acid =24:0) Hydrophobicity of the FA increases as the chain length increases. Short chain saturated FAs are liquid at room temp. While long ones Addition of double bonds decreases the melting temperature ( Tm ) of FAs. are solid at room temp.

10. 18 C Double bond(en ) are always separated by methylene bridges (–CH=CH– CH2–CH=CH–) (i.e. neither adjacent nor conjugated) that provide greater protection against auto-oxidation (peroxidation) of unsaturated fatty acids when exposed to environment containing oxygen.

11. Essential Fatty acids EFA These are fatty acids with double bonds beyond  9 (9=10). They must be supplied in diet because they can not be synthesized in the body.

12. HydrocarbonFatty acid nameCommon name 1C Methane MethanoicFormic HCOOH 2C EthaneEthanoicAcetic CH 3 -COOH 3C PropanePropanoicPropionic CH 3 -CH 2 -COOH 4C ButaneButanoicButyric CH 3 -[CH 2 ] 2 -COOH 16C HexadecaneHexadecanoicPalmitic CH 3 -[CH 2 ] 14 -COOH 18C OctadecaneOctadecanoicStearic CH 3 -[CH 2 ] 16 -COOH 20C EicosaneEicosanoicArachidic CH 3 -[CH 2 ] 18 -COOH 24C TetracosaneTetracosanoicLignoceric CH 3 -[CH 2 ] 22 -COOH Fatty acids are named after the hydrocarbons of the same number.

13. ClassParent Fatty acid Omega Numbering Delta Numbering ω-7Palmitoleic16:1ω-716:1∆9 ω-9Oleic18:1ω-918:1∆9 ω-6Linoleic18:2ω-618:∆9,12 ω-3Linolenic18:3ω-318:∆9,12,15 Four classes of unsaturated fatty acids are common: Double bonds are always separated from each other by a methylene group i.e. They are neither conjugated nor adjacent which protects them from auto-oxidation.

14. Fatty acid synthesis Storage of FA as TAG (Lipogenesis) Mobilization of stored fat (lipolysis) Fatty Acid Oxidation Metabolism of ketone bodies Fatty Acids And Triacylglycerol Metabolism

15. Fatty acid synthesis  Extramitochondrial(De novo) synthesis  Site of synthesis (tissues with active FA synthesis)  Provision of acetyl-CoA & NADPH  Synthesis of malonyl CoA by acetyl CoA carboxylase  FA synthase complex  Steps of synthesis  Synthesis of FA shorter than palmitic in mammary gland  Regulation of fatty acid synthesis(Short and long term)  Microsomal and mitochondrial FA elongation  Desaturation of FA.

16. Synthesis Elongation Unsaturation FA

17. *Occurs 1ry in liver + lactating mammary glands & brain, to a lesser extent, in adipose tissue and lung. *Occurs in the cytoplasm (Extra-mitochondrial); Requiring Acetyl-CoA (from mitochondria by citrate shuttle)& NADPH. Site of De Novo Synthesis of FA

18. O || CH3-C-S-COA Acetyl-CoA= Active acetate= 2C Oxidation in TCA for energy (12ATP/mol) synthesis of : -Cytoplasmic FA -HMG-COA for Ketone bodies & Cholesterol synthesis Acetyl CoA is Used For: Glucose oxidation  pyruvate  PDH FAs oxidation (Beta oxidation) Amino Acids Ketone bodies

19. Provision of cytosolic acetyl CoA Sources: -Oxidation of pyruvate. -Catabolism of FAs, KBs,AAs. The increase in both ATP and citrate enhances this pathway.

20. 1-HMP Pathway * ( 2 NADPH /one glucose). 2-Cytosolic Malic* 3-Cytosolic isocitrate dehydrogenase(minor) Isocitrate   keto gluterate Provision of NADPH

21. Remember! Fatty acid synthesis needs! NADPH- ATP-Mn++-Biotin and Bicarbonate(as a source of CO2). The immediate substrate for synthesis is acetyl CoA while, the final product is free palmitate.

22. Remember!! First Step is Carboxylation of Acetyl CoA to Malonyl CoA is by Acetyl CoA Carboxylase First step: CH 3 O S CoA CH 2 O S CoA O HO CO 2, Biotin Acetyl CoA Carboxylase Acetyl CoA Malonyl CoA Mn2+

23. Remember! The rest of steps are by Fatty Acid Synthase: a multifunctional enzyme in eukaryotes Head to Tail Dimeric multiprotein complex with7 enzymatic activities for FA synthesis, except Acetyl-CoA carboxylase It has 2-SH groups, of cysteine & ACP (acyl carrier protein). CYS ACP SH

24. AT KS MTDE ER KR ACPTE Cys Palmitate release Reduction Condensation Translocation SH HS Substrate entry KS AT MTACP KR ER DETE Cys Palmitate release Reduction Substrate entry Translocation SH HS Condensation 1 2 AT: Acetyl transferase DE: Dehydratase ACP: ACYL carrier protein MT: Malonyl transferase ER: Enoyl reductase TE: Thioestrase KS :βketoacyl synthase KR:β-ketoacyl ACP reductase SH: Thiol group Steps are:

25. [1] Acetate is transferred from acetyl CoA to the –SH group of the ACP. Domain: Acetyl CoA-ACP acetyl transacylase. Steps are:

26. [2] Next, this two-carbon fragment is transferred to a temporary holding site, the thiol group of a cysteine residue on the enzyme.

27. [3] The now-vacant ACP accepts a three- carbon malonate unit from malonyl CoA (from step of acetyl CoA carboxylase). Domain: Malonyl CoA-ACP transacylase.

28. [4] The acetyl group condenses with malonyl group on ACP losing originally added CO2. So, a 4C unit is attached to ACP. Decarboxylation with loss of free energy drives the reaction. Domain: 3- Ketoacyl-ACP synthase. CONDENSATION 3-Keto Acyl Synthase (CE)

29. [5] The keto group is reduced to an alcohol. Domain: 3-Ketoacyl-ACP reductase. REDUCTION Keto-Acyl Reductase

30. [6] A molecule of water is removed to introduce a double bond between carbons 2 and 3 (the α- and β- carbons). Domain: 3-Hydroxyacyl-ACP dehydratase. DEHYDRATION  -hydroxyacyl-dehydratase

31. [7] The double bond is reduced. Domain: Enoyl- ACP reductase. REDUCTION (4C) Enoyl Reductase

32. The acyl (Butyryl) transfered to SH-cys. Keeping SH- ACP free for malonyl to continue from step3*. Then, Palmitoyl thioesterase cleaves the thioester bond giving saturated 16C. 4 6 16

33. the original acetyl CoA, which are found at the methyl-group end All the carbons in palmitic acid have come from malonyl CoA except the two donated by the original acetyl CoA, which are found at the methyl-group end of the fatty acid. This underscores the rate-limiting nature of the acetyl CoA carboxylase reaction. QUESTION Did all palmitic acid carbons come all from Malonyl coA?

34. Where are the short fatty acids formed? Shorter-length FAs shorter than palmitic are important end-products in the lactating mammary gland. As milk is rich in these FAs. What happens if propionyl CoA used as a primer instade of Acetyl Coa ? Odd number FA will be formed

35. Regulation of Fatty Acid Synthesis: -Short term regulation of acetyl CoA carboxylase: 1) Allosteric: Acyl CoA inhibits both: -Citate transport to the mitochondria -Pyrvate Dehydrogenase allosterically Thus affecting the availability for Acetyl CoA needed for lipogenesis process.

36. 2-Covalent modification(Hormonal Regulation) =Reversable phosphorylation & dephosphorylation AMPK also cause phosphorylation of the enzyme. Its also activated covalently by another kinase called AMPK-kinase and allosterically by rise in AMP relative to ATP.

37. Long term Regulation of Fatty Acid Synthesis: ( Nutritional State): FA-synthase multi-complex ensures freedom from interference imposed by other pathways in cytoplasm. The synthesis of all enzymes in it is coordinate; since it is encoded by single gene High Calorie High Carbohydrate Low Calorie Low Carbohydrate Acetyl-CoA Carboxylase Synthesis Increase Decrease Fatty acid synthase  Insulin  Glucagon  PDH  Acetyl CoA

38. Further Elongation Of Fatty Acid Chains The brain has more elongation capabilities, to produce very-LCFA (22C &24 C) used for synthesis of brain lipids( during myelination). Remember: Low insulin activity due to fasting or diabetes, abolishes chain elongation.

39. Elongation=Palmitate (16:0)+2C units. Occur In: smooth endoplasmic reticulum (as a major system) and mitochondria (as a minor system). By: elongase similar to FA synthase but Its: not a multiprotein complex Enz. Uses fatty Acyl-CoA (not ACP) that condenses with one malonyl-CoA (2C donor) each time. Requires NADPH + +H + each time (as reductant)

40. In Smooth ER by Desaturases (for cis double bonds). They are mixed function oxidases (hydroxylation+dehydration), requiring NADH or NADPH and O2. Humans only have (  ) 9, 6, 5 & 4types. Desaturation of fatty acid chains Stearyl C0A. CH3-(CH2)7-CH2-CH2-(CH2)7-C0-SC0A NADPH+H Hydroxylase NADP NADPH+H Hydroxylase NADP 9-Hydroxy-Stearyl-C0A 10 9 CH3-(CH2)7-CH2-CH-(CH2)7-C0-SC0A CH3-(CH2)7-CH2-CH-(CH2)7-C0-SC0A 0H 0H Dehydrase Dehydrase - H20 - H20 Olyl-C0A. 10 9 CH3--(CH2)7--CH=CH- CH2)7--C0-SC0A CH3--(CH2)7--CH=CH- CH2)7--C0-SC0A

41. In Smooth ER by Desaturases (for cis double bonds). They are requiring NADH,cytochrome b5 and its FAD linked reductase. Humans only have (  ) 9, 6, 5 & 4types. Desaturation of fatty acid chains

42. In Human, Desaturases lack the ability to form double bonds from carbon 10 to the ω- end of the chain. This clears the nutritional essentiality of the polyunsaturated linoleic and linolenic acids.?????

43. Storage of FA as TAG (Lipogenesis)  Structure of TAG  Why TAG is the major energy reserve of the body?  Synthesis of TAG  Synthesis of glycerolphosphate  Activation of FA  Addition of FAs to active glycerol  Different fates of synthesized TAG in Liver and Adipose tissue. Mobilization of stored fat (lipolysis)  Activation of hormone sensitive lipase  Fate of Glycerol and FAs

44. As TAGs are only slightly soluble in water, so they form oily droplets that are nearly anhydrous (consuming less space). So, These cytosolic lipid droplets are the major energy reserve of the body. Storage of FAs as components of TAG Structure of (TAG): 3 FAs esterified by their carboxyl groups, to a glycerol. This result in loss of negative charge and formation of “neutral fat. Glycerol Sat. FA Either-FA Unsat. FA Unsaturated FA(s) decrease(s) the (Tm) of the lipid.  Why TAG is the major energy reserve of the body?

45. A fatty acid must be activated by fatty acyl CoA synthetases (thiokinases) needing ATP. Also glycerol as Glycerol-3P before both can participate in TAG synthesis. Glycerol-3P synthesis is formed more in liver* than adipocytes why?. As, Adipocytes can take up glucose only in the presence of insulin. Low plasma glucose and insulin—levels, will make adipocytes have only a limited ability to synthesize glycerol-3- phosphate, and cannot produce TAG. Synthesis of TAG 3FA + Glycerol = TAG

46. 2Pathways for production of glycerol-3P in liver and adipose tissue. Glycerokinase GlycerolGlycerol-3P ATP ADP Mg By the Liver Only Glycolysis Glucose Dihydroxyacetone phosphate NADH NAD+ By the Liver and Adipose tissues Gly cerol-P-dehydrogenase

47. Synthesize of TAG in 4 reactions sequentially: - addition of 2FAs (fatty acyl CoA), then removal of phosphate, & addition of the 3 rd FA.

48. In adipose tissue, TAG is usually stored ready for mobilization as fuel. Little TAG is stored in the liver. Instead, it is exported with other lipids (VLDL). Nascent VLDL are secreted directly into the blood where they mature and function to deliver the endogenously derived lipids to the peripheral tissues. Q=What are the different fates of TAG in Liver and Adipose tissue.