Fatty Acid Synthesis

1. Fatty Acid Synthesis Lecture 16 Modified from internet sources, journals and books

2. Fatty Acid Synthesis • Prediction: the pathway for the synthesis of fatty acids would be the reversal of the oxidation pathway • this would not allow distinct regulation of the two pathways to occur even given the fact that the pathways are separated within different cellular compartments • pathway for fatty acid synthesis occurs in the cytoplasm (oxidation occurs in the mitochondria) • the essential chemistry of the two processes  reversals of each other

3. continued • oxidation and synthesis of fats utilize an activated two carbon intermediate  acetyl-CoA • acetyl-CoA in fat synthesis  exists temporarily bound to the enzyme complex as malonyl-CoA • synthesis of malonyl-CoA  the first committed step of fatty acid synthesis • the enzyme that catalyzes this reaction  acetyl-CoA carboxylase (ACC) = the major site of regulation of fatty acid synthesis

5. The rate of fatty acid synthesis • controlled by the equilibrium between monomeric ACC and polymeric ACC • activity of ACC requires polymerization  the conformational change is enhanced by citrate and inhibited by long-chain fatty acids • ACC is also controlled through hormone mediated phosphorylation (see below). • The acetyl-CoA and malonyl-CoA are transferred to ACP (acetyl-CoA phosphatase) by the action of acetyl-CoA transacylase and malonyl-CoA transacylase, respectively

6. continued • attachment of these carbon atoms to ACP allows them to enter the fatty acid synthesis cycle. • The synthesis of fatty acids from acetyl-CoA and malonyl-CoA  carried out by fatty acid synthase (FAS)

7. continued • All of the reactions of fatty acid synthesis are carried out by the multiple enzymatic activities of FAS (fatty acid synthase) • like fat oxidation  fat synthesis involves 4 enzymatic activities: • β-keto-ACP synthase, β-keto-ACP reductase, 3-OH acyl-ACP dehydratase and enoyl-CoA reductase (the two reduction reactions require NADPH oxidation to NADP+) • the primary fatty acid synthesized by FAS is palmitate; then released from the enzyme and can then undergo separate elongation and/or unsaturation to yield other fatty acid molecules

8. Origin of Cytoplasmic Acetyl-CoA • Acetyl-CoA  generated in the mitochondria primarily from two sources: • the pyruvate dehydrogenase (PDH) reaction • fatty acid oxidation • these acetyl units to be utilized for fatty acid synthesis  they must be present in the cytoplasm • shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energy diminishes • This results in  reduced oxidation of acetyl-CoA in the TCA cycle and the oxidative phosphorylation pathway • Under these conditions  the mitochondrial acetyl units can be stored as fat for future energy demands

9. continued • Acetyl-CoA  enters the cytoplasm in the form of citrate via the tricarboxylate transport system • In the cytoplasm  citrate is converted to oxaloacetate and acetyl-CoA (by the ATP driven ATP-citrate lyase reaction) • resultant oxaloacetate  is converted to malate by malate dehydrogenase (MDH)

11. continued • The malate produced by this pathway  can undergo oxidative decarboxylation by malic enzyme • co-enzyme for this reaction is NADP+ generating NADPH • advantage of this series of reactions for converting mitochondrial acetyl-CoA into cytoplasmic acetyl-CoA  the NADPH produced by the malic enzyme reaction can be a major source of reducing co-factor for the fatty acid synthase activities

12. Regulation of Fatty Acid Metabolism • must consider the global organismal energy requirements in order to effectively understand how the synthesis and degradation of fats (and also carbohydrates) needs to be exquisitely regulated • blood  is the carrier of triacylglycerols in the form of VLDLs and chylomicrons, fatty acids bound to albumin, amino acids, lactate, ketone bodies and glucose • The pancreas  is the primary organ involved in sensing the organisms dietary and energetic states via glucose concentrations in the blood

13. continued • The regulation of fat metabolism occurs via distinct mechanisms: • short term regulation  regulation effected by events such as substrate availability, allosteric effectors and/or enzyme modification • ACC (acetyl-CoA carboxylase)  the rate-limiting (committed) step in fatty acid synthesis

14. continued • two major isoforms of ACC in mammalian tissues: • ACC1 and ACC2 • ACC1  is strictly cytosolic and is enriched in liver, adipose tissue and lactating mammary tissue • ACC2  originally discovered in rat heart but is also expressed in liver and skeletal muscle • Both isoforms of ACC  allosterically activated by citrate and inhibited by palmitoyl-CoA and other short- and long-chain fatty acyl-CoAs

15. continued • Citrate  triggers the polymerization of ACC1 which leads to significant increases in its activity • ACC2  does not undergo significant polymerization (presumably due to its mitochondrial association), is allosterically activated by citrate • Glutamate and other dicarboxylic acids can also allosterically activate both ACC isoforms

16. continued • ACC activity can also be affected by phosphorylation • Glucagon-stimulation  increases in cAMP and subsequently increasing PKA activity also lead to phosphorylation of ACC and ACC2 • This insulin-mediated effect  has not been observed in hepatocytes or adipose tissues cells • Activation of α-adrenergic receptors in liver and skeletal muscle cells  inhibits ACC activity as a result of phosphorylation (undetermined kinase)

17. continued • Control of a given pathways' regulatory enzymes can also occur by alteration of enzyme synthesis and turn-over rates  these changes are long term regulatory effects • Insulin  stimulates ACC and FAS synthesis, whereas, starvation leads to decreased synthesis of these enzymes • Adipose tissue lipoprotein lipase levels  also are increased by insulin and decreased by starvation

18. continued • in contrast to the effects of insulin and starvation on adipose tissue  their effects on heart lipoprotein lipase are just the inverse • this allows the heart to absorb any available fatty acids in the blood in order to oxidize them for energy production • Adipose tissue  contains hormone-sensitive lipase (HSL), that is activated by PKA-dependent phosphorylation leading to increased fatty acid release to the blood

19. continued • In the liver  the net result of activation of HSL (due to increased acetyl-CoA levels) is the production of ketone bodies • This would occur under conditions where insufficient carbohydrate stores and gluconeogenic precursors were available in liver for increased glucose production • Insulin  has the opposite effect to glucagon and epi leading to increased glycogen and triacylglyceride synthesis • One of the many effects of insulin  to lower cAMP levels which leads to increased dephosphorylation through the enhanced activity of protein phosphatases

20. ChREBP: Master Lipid Regulator in the Liver • ChREBP = helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor, carbohydrate-responsive element-binding protein  has emerged as a central regulator of lipid synthesis in liver • ChREBP  identified as a major glucose-responsive transcription factor and it is required for glucose-induced expression of the hepatic isozyme of the glycolytic enzyme pyruvate kinase (identified as L-PK) • ChREBP  acts to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)

21. continued • Expression of the ChREBP gene  induced in the liver in response to increased glucose uptake • Under conditions of low (basal) glucose concentration  ChREBP is phosphorylated and resides in the cytosol •  An emerging model of the role of ChREBP in overall glucose and lipid metabolism  indicates it a master regulator of glucose-mediated lipid homeostasis not only in the liver but also in adipose tissue

22. Elongation and Desaturation • The fatty acid product released from FAS is palmitate (a 16:0 fatty acid, i.e. 16 carbons and no sites of unsaturation) • Elongation and unsaturation of fatty acids  occurs in both the mitochondria and endoplasmic reticulum • The predominant site of these processes  the ER membranes • Elongation  involves condensation of acyl-CoA groups with malonyl-CoA • resultant product  two carbons longer (CO2 is released from malonyl-CoA as in the FAS reaction) which undergoes reduction, dehydration and reduction yielding a saturated fatty acid • Mitochondrial elongation  involves acetyl-CoA units and is a reversal of oxidation

23. continued • Desaturation occurs in the ER membranes • involves 4 broad specificity fatty acyl-CoA desaturases (non-heme iron containing enzymes) • These enzymes  introduce unsaturation at C4, C5, C6 or C9 • electrons transferred from the oxidized fatty acids during desaturation  are transferred from the desaturases to cytochrome b5 and then NADH-cytochrome b5 reductase • These electrons  are un-coupled from mitochondrial oxidative-phosphorylation and do not yield ATP

24. Since these enzymes cannot introduce sites of unsaturation beyond C9  they cannot synthesize either linoleate (18:2Δ9,12) or linolenate (18:3Δ9,12,15) • These fatty acids must be acquired from the diet  referred to as essential fatty acids • Linoleic  especially important in that it is required for the synthesis of arachidonic acid • arachindonate  a precursor for the eicosanoids (the prostaglandins and thromboxanes)

25. continued • role of fatty acids in eicosanoid synthesis  that leads to poor growth, wound healing and dermatitis in persons on fat free diets • linoleic acid  a constituent of epidermal cell sphingolipids that function as the skins water permeability barrier

26. Synthesis of Triglycerides • Fatty acids  stored for future use as triacylglycerols in all cells, but primarily in adipocytes of adipose tissue • fatty acids present in triacylglycerols  predominantly saturated • major building block for the synthesis of triacylglycerols, in tissues other than adipose tissue, = glycerol • Adipocytes lack glycerol kinase  dihydroxyacetone phosphate (DHAP), produced during glycolysis, is the precursor for triacylglycerol synthesis in adipose tissue • adipoctes must have glucose to oxidize in order to store fatty acids in the form of triacylglycerols

29. continued • The glycerol backbone of triacylglycerols  activated by phosphorylation at the C-3 position by glycerol kinase • The fatty acids incorporated into triacylglycerols  activated to acyl-CoAs through the action of acyl-CoA synthetases • Two molecules of acyl-CoA  esterified to glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid).

30. continued • The phosphate is then removed  to yield 1,2-diacylglycerol, the substrate for addition of the third fatty acid • Intestinal monoacylglycerols, derived from the hydrolysis of dietary fats, can also serve as substrates for the synthesis of 1,2-diacylglycerols

31. Phospholipid Structures • Phospholipids  synthesized by esterification of an alcohol to the phosphate of phosphatidic acid (1,2-diacylglycerol 3-phosphate) • Most phospholipids  a saturated fatty acid on C-1 and an unsaturated fatty acid on C-2 of the glycerol backbone • The most commonly added alcohols = serine, ethanolamine and choline • The major classifications of phospholipids are:

32. Phosphatidylcholine (PC)

33. PC • This class of phospholipids  also called the lecithins • At physiological pH  phosphatidylcholines are neutral • contain primarily palmitic or stearic acid at carbon 1 and primarily oleic, linoleic or linolenic acid at carbon 2 • lecithin dipalmitoyllecithin  a component of lung or pulmonary surfactant • the major (80%) phospholipid found in the extracellular lipid layer lining the pulmonary alveoli

34. Phosphatidylethanolamine (PE)

35. PE • These molecules are neutral at physiological pH • contain primarily palmitic or stearic acid on carbon 1 and a long chain unsaturated fatty acid (e.g. 18:2, 20:4 and 22:6) on carbon 2

36. Phosphatidylserine (PS)

37. PS • composed of fatty acids similar to the phosphatidyl-ethanol-amines • PE is in the lipid bilayer of the a membrane

38. Phosphatidylinositol (PI)

39. PI • contain almost exclusively stearic acid at carbon 1 and arachidonic acid at carbon 2 • molecules exist in membranes with various levels of phosphate esterified to the hydroxyls of the inositol • Molecules with phosphorylated inositol  polyphosphoinositides • polyphosphoinositides  important intracellular transducers of signals emanating from the plasma membrane

40. continued • One polyphosphoinositide (phosphatidylinositol 4,5-bisphosphate, PIP2)  a critically important membrane phospholipid involved in the transmission of signals for cell growth and differentiation from outside the cell to inside

41. Phosphatidylglycerol (PG)

42. PG • Phosphatidylglycerols  found in high concentration in mitochondrial membranes and as components of pulmonary surfactant • Phosphatidylglycerol  a precursor for the synthesis of cardiolipin (important component of the inner mitochondrial membrane, where it constitutes about 20% of the total lipid) • vital role of PG  serve as the precursor for the synthesis of diphosphatidylglycerols (DPGs)

43. Diphosphatidylglycerol (DPG)

44. DPG • These molecules  very acidic • primarily in the inner mitochondrial membrane and also as components of pulmonary surfactant

45. continued • The fatty acid distribution at the C-1 and C-2 positions of glycerol within phospholipids is continually in flux • phospholipid degradation and the continuous phospholipid remodeling that occurs while these molecules are in membranes (= highly dynamic systems) • Phospholipid degradation  results from the action of phospholipases • various phospholipases exhibiting substrate specificities for different positions in phospholipids • remodeling of acyl groups in phospholipids = the result of the action of phospholipase A1 (PLA1) and phospholipase A2 (PLA2)

46. Sites of Action of the Phospholipases A1, A2, C and D.

47. continued • products of these phospholipases  called lysophospholipids and can be substrates for acyl transferases utilizing different acyl-CoA groups • PLA2  an important enzyme, whose activity is responsible for the release of arachidonic acid from the C-2 position of membrane phospholipids • released arachidonate  a substrate for the synthesis of the eicosanoids • there is not just a single PLA2 enzyme; At least 19 enzymes have been identified with PLA2 activity  involved in numerous processes including modification of eicosanoid generation, host defense, and inflammation

48. The cytosolic PLA2 family (cPLA2)  essential component of the initiation of arachidonic acid metabolism • the sPLA2 enzymes  tightly regulated by Ca2+ and by phosphorylation

49. Plasmalogens • Plasmalogens are glycerol ether phospholipids • Three major classes of plasmalogens have been identified: • choline, ethanolamine and serine plasmalogens • Ethanolamine plasmalogen  prevalent in myelin • Choline plasmalogen  abundant in cardiac tissue. • One choline (1-O-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine)  identified as an extremely powerful biological mediator  is called platelet activating factor= PAF

50. continued • PAF functions as: • a mediator of hypersensitivity, acute inflammatory reactions and anaphylactic shock • PAF is synthesized in response to the formation of antigen-IgE complexes on the surfaces of basophils, neutrophils, eosinophils, macrophages and monocytes • synthesis and release of PAF from cells  leads to platelet aggregation and the release of serotonin from platelets • PAF also produces responses in liver, heart, smooth muscle, and uterine and lung tissues