Lec 8,9 Biochemistry II Fatty Acids, Triacylglycerol & Ketone Bodies PDF

Biochemistry II (PB 503) Dr. Nourhan El Samaloty Associate Professor of Biochemistry 1  Outline ❑De novo synthesis of fatty acids ❑TAG Synthesis (lipogenesis). ❑Lipolysis (Mobilization of stored fats) ❑Carnitine shuttle ❑β-ox...

Biochemistry II (PB 503) Dr. Nourhan El Samaloty Associate Professor of Biochemistry 1  Outline ❑De novo synthesis of fatty acids ❑TAG Synthesis (lipogenesis). ❑Lipolysis (Mobilization of stored fats) ❑Carnitine shuttle ❑β-oxidation of fatty acids ❑Oxidation of ODD chain fatty acids. ❑Ketogenesis and ketolysis 2 I. Fatty acid and Triacylglycerol Synthesis 3  De novo Synthesis of Fatty Acids Overview of the pathway ❑ When: fed state (insulin) ❑ Where: ▪ Cellular tissue (site): liver, lactating mammary glands and to lesser extent adipose tissue ▪ Intracellular location: cytosol ❑ Initial substrate: Acetyl CoA “from glucose excess of the body’s need for energy” Glucose pyruvate Acetyl CoA FA synthesis ❑ Final product: palmitic acid (palmitate) ❖ Requirements: Acetyl CoA, NADPH, CO2, ATP, biotin 4 Steps of de novo synthesis of FA: Step 1: Transport of Acetyl CoA from mitochondria to cytosol by “citrate shuttle” FA ❑ Translocation of citrate occurs as coenzyme A portion cannot pass the mitochondrial membrane. ❑ Cytosolic citrate is viewed as a high-energy signal. ❑ High ATP and high citrate enhance this pathway 5  Step 2: Carboxylation of acetyl CoA to malonyl CoA By acetyl CoA carboxylase (ACC) (Regulatory enzyme and the rate- limiting step of FA de novo synthesis). biotin Step 3: Palmitate synthesis by Fatty acid synthase multienzyme complex We need 8 acetyl CoA to synthesize palmitate (16 carbons) 1 Acetyl CoA 7 Acetyl CoA are converted to 7 malonyl CoA by ACC, using 7 ATP FA synthase multienzyme complex Acetyl CoA + 7 Malonyl CoA + 14 NADPH.H+ Palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 CO2 7 cycles 6 Major sources of NADPH H+ required for fatty acid synthesis ❑ Each glucose molecule entering HMP shunt yields 2 NADPH.H+ ❑ Cytosolic conversion of malate to pyruvate by malic enzyme. ❑ Cytosolic (extramitochondrial) isocitrate dehydrogenase. 7  Regulation of FA synthesis Acetyl CoA carboxylase (ACC) is the rate-limiting step 1. Allosteric regulation (Short-term regulation) Allosteric activator: citrate Allosteric inhibitor: palmitoyl CoA (long chain fatty acid) 8 2. Covalent modification via hormonal actions (short-term regulation) Acetyl CoA carboxylase is active in the dephosphorylated form by insulin and inactive in the phosphorylated form by glucagon and epinephrine. Acetyl CoA carboxylase is inactivated by phosphorylation through AMP– activated protein kinase (AMPK). 9  3. Hormonal regulation (long-term regulation) Prolonged consumption of a diet containing excess calories “high calorie-high CHO diet, low-fat diet” → ↑ acetyl CoA carboxylase synthesis as its gene expression is increased by glucose and insulin actions→ ↑ FA synthesis. Conversely, a low-calorie, low-CHO or a high-fat diet →↓FA synthesis by decreasing acetyl CoA carboxylase synthesis. 10  Fate of Palmitate 1- Elongation: by the addition of two-carbon units primarily in the smooth endoplasmic reticulum (microsomal elongation). ❑ The microsomal elongation system uses separate enzymic processes rather than a multifunctional enzyme. ❑ Uses Malonyl CoA (2 carbon source) and NADPH (hydrogen donor). ❑ The brain has additional elongation capabilities, allowing it to produce the very long chain fatty acids (> 22 carbons) that are required for synthesis of brain lipids. 2- Desaturation: (adding cis double bonds) by mixed-function oxidases (desaturases) that require NADH and oxygen. 3- Esterification into triacylglycerol (TAG) (Lipogenesis). 11  TAG Synthesis “Lipogenesis” Lipogenesis occurs during the fed state in the cytosol of the liver, adipose tissue and lactating mammary gland. Steps of Lipogenesis: Step 1: Conversion of a free fatty acid to its activated form (fatty acyl CoA) FA thiokinase = 12  Steps of Lipogenesis Step 2: Synthesis of glycerol phosphate in liver and adipose tissue Q. Liver can synthesize glycerol-3-P from both DHAP and glycerol, while adipose tissues can synthesize glycerol-3-P from DHAP only?? 13  Steps of Lipogenesis Step 3: Synthesis of TAG molecule from glycerol phosphate and 3 fatty acyl CoA in liver and adipose tissue 1-Esterification R1 is typically saturated, R2 typically 3-Dephosphorylation unsaturated, while R3 is either. ❑Fate of TAG: ✓ In adipose tissue, TAG molecules (fats) are stored 2-Esterification 4-Esterification in the cytosol of adipocytes and serve as depot fat ready to be mobilized when the body requires it for energy. ✓ In the liver, little TAG is stored. Instead, TAG is packaged on VLDL and is carried in blood to tissues. 14 II. Fatty acid and Triacylglycerol Degradation 15  Lipolysis Lipolysis is the mobilization of stored fats in adipose tissue (hydrolysis of TAG into glycerol and 3 FAs). Overview of the pathway ❑ When: fasting state ❑ Where: ❑ Cellular tissue (site): adipose tissue ❑ Intracellular location: cytosol ❑ Initial substrate: TAG ❑ Final product: 3 FAs and glycerol from each TAG molecule. FAs are then oxidized in tissues to yield energy. N.B. The energy yield from oxidation of fats is 9 Kcal/g as compared to 4 Kcal/g protein or carbohydrate. 16  Steps of Lipolysis N.B. Hormone-sensitive TAG lipase (HSL) has both triglyceride and diglyceride 1- Adipose triglyceride lipase (ATGL) hydrolase activities, but with much greater affinity for diglycerides than DAG FA triglycerides. = diglyceride lipase = Hormone- sensitive lipase (HSL) MAG FA = monoglyceride lipase (MGL) FA Glycerol 17  Glucagon Regulation of Lipolysis Covalent modification via hormonal actions Hormone-sensitive lipase (HSL) is the rate limiting step of lipolysis. ✓ HSL is activated when phosphorylated by PKA (protein kinase A = cAMP-dependent protein kinase A) through the actions of glucagon and epinephrine, and inactivated by dephosphorylation through the action of insulin. + Insulin + Lipogenesis - Insulin Glucagon, - Epinephrine - Lipolysis + 18 - Fate of Glycerol and Free FAs Lipolysis Lipolysis in blood carried on albumin to tissues β-oxidation DHAP Krebs cycle Gluconeogenesis for energy 19  β-Oxidation of Fatty Acids ✓ It is the most common pathway for fatty acid oxidation. ✓ When: fasting state. ✓ Where: in the mitochondrial matrix of most tissues. ✓ Don’t occur in RBCs?? ✓ FA oxidation is very low or poor in the brain? Due to slow transport of FFA across the blood-brain barrier + low levels of FA degrading enzymes in the brain. FA oxidation occurs in 3 steps: 1- Activation of FA to fatty acyl CoA by acyl CoA synthetase (FA thiokinase) in the cytosol. Consumes 2 ATP 2- Transport of fatty acyl CoA from cytosol to mitochondria by carnitine shuttle (only for long-chain FAs) 3- β-oxidation in the mitochondrial matrix 20 2. Transport of long chain fatty acyl CoA from cytosol to mitochondria via the Carnitine shuttle (CPT-I) (CPT-II) CPT-I is also called carnitine acyl transferase-I (CAT-I) CPT-II is also called carnitine acyl transferase-II (CAT-II) 21 Carnitine shuttle (cont.) ❑ The use of carnitine shuttle ▪ ONLY for the transport of long chain fatty acid (LCFA) across the inner mitochondrial membrane. ▪ SCFA and MCFA can cross the inner mitochondrial membrane without the aid of carnitine or the CPT system. Once inside the mitochondria, they are activated to their CoA derivatives by matrix enzymes, then oxidized. ❑ Regulation ▪ Regulatory enzyme: carnitine palmitoyl transferase-I (CPT-I)= carnitine acyl transferase I (CAT-I). ▪ Activator: Long chain fatty acyl CoA ▪ Inhibitor: Malonyl CoA 22 3. β-oxidation of fatty acids (n) ❖ β-oxidation of an even numbered fatty acid: β-oxidation process involves 4 main steps: 1. Dehydrogenation (Oxidation) 2. Hydration 3. Dehydrogenation (Oxidation) 4. Thiolysis (Cleavage) This cycle repeats until the fatty acid has been completely degraded to acetyl-CoA (n - 2) 23  Energetics of FA β-oxidation Calculation of the energy yield from an even numbered fatty acid oxidation: 1- Number of acetyl CoA molecules= Total number of carbons / 2 2- Number of cycles= number of acetyl CoA molecules – 1 ATP yield from each acetyl CoA in Krebs cycle: 12 ATP ATP yield from each cycle: 2 ATP (FADH2) + 3 ATP (NADH) = 5 ATP e.g. β-oxidation of a molecule of palmitoyl CoA (16 carbon) to CO 2 and H2O Number of Acetyl CoA molecules: 16/ 2 = 8 Acetyl CoA Number of cycles: 8 – 1 = 7 cycles ATP yield from Acetyl CoA: 8 Acetyl CoA x 12 ATP = 96 ATP ATP yield from each cycle: 7 cycles x 5 ATP = 35 ATP Total yield of ATP: 96 + 35 = 131 ATP – 2 ATP (needed for fatty acid activation) Net = 129 ATP 24  Oxidation of fatty acids with an odd number of carbons ❖ The β-oxidation of a β-oxidation saturated fatty acid with an Odd chain FA odd number of carbon n acetyl CoA atoms proceeds by the Last 3 carbons same reaction steps as that of fatty acids with an even number, until the final three carbons are reached. This compound, propionyl CoA, is metabolized by a three-step pathway into succinyl CoA which enters Krebs cycle. 25 III. Ketone Bodies Metabolism 26  Characteristics of Ketone Bodies ❑ The 3 ketone bodies synthesized by the LIVER (ketogenesis) are: ✓ Acetoacetate ✓ 3-hydroxybutyrate (β-hydroxybutyrate) ✓ Acetone (released in the breath) ❑ They are soluble in aqueous solutions, therefore do not need to be incorporated into lipoproteins or carried by albumin. ❑ They are considered as an alternate fuel for tissues, as they are oxidized (ketolysis) by extrahepatic tissues-including brain (except cells lacking mitochondria). ❑ Ketone bodies spare glucose when used by brain during prolonged periods of fasting and starvation. 27  Ketogenesis Overview of pathway ❖ When: prolonged fasting/starvation ❖ Where: ▪ Cellular tissue (site): liver ▪ Intracellular location: mitochondrial matrix ❖ Initial substrate: excess Acetyl CoA (from FA oxidation) ❖ Final product: 3 ketone bodies 28 Steps of Ketogenesis Rate-limiting step 29  Cholesterol Synthesis versus Ketogenesis Steps: Steps 1 and 2 These 1st 2 reactions in the cholesterol synthetic pathway are similar to those in the pathway that produces ketone bodies; however the two pathways differ in: Item Ketone bodies synthesis Cholesterol synthesis State Fasting/starvation Fed Intracellular location Mitochondrial matrix Cytosol and ER Rate limiting step HMG CoA synthase HMG CoA reductase Fate of HMG CoA Converted to Converted to mevalonate acetoacetate by HMG by HMG CoA reductase CoA lyase 30  Ketolysis - Occurs in the mitochondrial matrix of extrahepatic tissues, including brain, kidney and heart (used as an alternative source of energy). All tissues, including brain are able to use ketone bodies as an alternate fuel EXCEPT: ✓ Liver: lacks thiophorase enzyme ✓ Tissues-lacking mitochondria Krebs Cycle 31  Ketone Bodies and Uncontrolled Diabetes Mellitus Insulin deficiency and increased glucagon levels lead to increased lipolysis, which in turn increases hepatic output of ketone bodies, resulting in: Diabetic ketoacidosis (DKA) characterized by: ✓ Ketonemia: increased levels of ketone bodies in the blood ✓ Ketonuria: increased levels of ketone bodies in the urine ✓ Fruity odor on the breath, as a result of increased acetone. 32 33