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AV. Lipid metabolism 1-2. 2011 Fatty acid oxidation Ketone bodies Fatty acid synthesis Triacylglycerols Major energy reserve Oxidation: 9 kcal/g (for carbohydrates: 4 kcal/g) 11 kg of 70 kg total body weight Site of accumulation: cytoplasm of ADIPOSE CELLS Adipose tissue -specialized for synthesis, storage, mobilization of lipids Lipases The most important fatty acids Number of carbons Number of double bonds Name 16 0 Palmitate 18 0 Stearate 20 0 Arachidate 16 1 Palmitoleate Cis D9 18 1 Oleate Cis D9 18 2 Linoleate Cis D9, D12 18 3 Linolenate Cis D9, D12, D15 20 4 Arachidonate Cis D5, D8, D11, D14 Mobilization of triacylglycerols stored in adipose tissue Glukagon, adrenalin, ACTH ↓ receptor activation ↓ protein kinase A ↓ phosphorylation of Perilipin (when dephosphorylated it inhibits the access of lipases to TG and DG + phosphorylation of HSL (hormon-sensitive lipase) activation ↓ CGI dissociates from Perilipin then associates with ATGL (adipocyte trigliceride lipase) ↓ TG→DG + fatty acid ↓ HSL: DG→MG + fatty acid ↓ MGL (monoacylglycerol lipase): MG→G + fatty acid Lehninger, Principals of Biochemistry, 2013 ADIPOSE TISSUE TG CIRCULATION Fatty acids Fatty acids - bound to albumin (10 fatty acids/albumin monomer) (free fatty acids, FFA) MUSCLE, HEART MUSCLE, RENAL CORTEX Fatty acid activation, transport into the mitochondria, β-oxidation Site of β-oxidation: mitochondria Activation of fatty acids: Fatty acid + ATP + CoA <------> Acyl-CoA + PPi + AMP Acyl-CoA synthetase O 1. R-COOH + ATP R C AMP + PPi O O R C P O Ribose Adenin O acyl-adenylate O O 2. R C AMP + HS-CoA R C S CoA + AMP acyl-CoA Fast PPi hydrolysis reaction is irreversible in vivo Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane Triacylglycerols Major energy reserve Oxidation: 9 kcal/g (for carbohydrates: 4 kcal/g) 11 kg of 70 kg total body weight Site of accumulation: cytoplasm of ADIPOSE CELLS Adipose tissue -specialized for synthesis, storage, mobilization of lipids Lipases The most important fatty acids Number of carbons Number of double bonds Name 16 0 Palmitate 18 0 Stearate 20 0 Arachidate 16 1 Palmitoleate Cis D9 18 1 Oleate Cis D9 18 2 Linoleate Cis D9, D12 18 3 Linolenate Cis D9, D12, D15 20 4 Arachidonate Cis D5, D8, D11, D14 Mobilization of fatty acids from the adipose tissue Glukagon, adrenalin, ACTH Receptor activation Protein kináz A Phosphorylation of Perilipin (when dephosphorylated it inhibits the access of lipase to TG, phosphorylated perilipin has no such effect) + Phosphorylation of Hormon-sensitive lipase (activation) Mobilization of fatty acids from TG Lipid droplet ADIPOSE TISSUE TG CIRCULATION Fatty acids Fatty acids - bound to albumin (10 fatty acids/albumin monomer) (free fatty acids, FFA) MUSCLE, HEART MUSCLE, RENAL CORTEX Fatty acid activation, transport into the mitochondria, β-oxidation Site of β-oxidation: mitochondria Activation of fatty acids: Fatty acid + ATP + CoA <------> Acyl-CoA + PPi + AMP Acyl-CoA synthetase O 1. R-COOH + ATP R C AMP + PPi O O R C P O Ribose Adenin O acyl-adenylate O O 2. R C AMP + HS-CoA R C S CoA + AMP acyl-CoA Fast PPi hydrolysis reaction is irreversible in vivo Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane Transport of fatty acids into the mitochondria Transport of Long-chain (12-18 C atom) fatty acids with carnitine Transport of fatty acids into the mitochondria carnitine-acyltransferase I (CPT I) Transport of carnitine-acylcarnitine is the rate-limiting step and most important control point in fatty acid oxidation carnitine-acyltransferase II (CPT II) Fatty acids with <12 C enter mitochondria without carnitine and are activated in the mitochondria Three isoenzymes: -long-chain fatty acids (C 12-18) -medium chain fatty acids (MCAD, 4-14) -short-chain fatty acids (4-8) MCAD deficiency is relatively frequent Specific for L-stereoisomere β-oxidation of fatty acids Oxidation of fatty acids with >12 C is carried out by a multienzyme bound to the mitochondrial inner membrane, in which the last three enzymes are tightly associated (trifunctional protein), when the chain is < 12 C soluble enzymes in the matrix continue the oxidation Conversion of glycerol to glycolysis intermediate – in the liver glycerol kinase Glycerol-P dehydrogenase Regulation of fatty acid oxidation hormones (adrenaline, glucagon) High energy state NADH Malonyl-CoA Inhibiton of Perilipin Activation of hormone-sensitive lipase Inhibition of Inhibition of carnitine 3-hydroxyacyl CoA acyltransferase I dehydrogenase Acetyl-CoA thiolase Increased level of free fatty acids Entry of fatty acids into mitochondria is inhibited Oxidation Inhibition of ß-oxidation Oxidation Long-term regulation: PPAR (peroxisome proliferator/activated receptors) nuclear receptor – transcription factors PPARα – muscle, adipose tissue, liver regulate the transcription of fatty acid transporters, CPT I és CPT II, and acyl-CoA dehydrogenase - energy need (fasting, between-meal periods) PPARα activation transcription of enzymes of fatty acid oxidation - fetus - principal fuels for heart: glucose and lactate -neonatal – fatty acid Regulation of metabolic transition by PPARα - sustained exercise – PPARα expression in muscles The most common genetic defect in fatty acid oxidation Acyl-CoA dehydrogenase deficiency For the medium length acyl-CoA dehydrogenase Prevalence: 1:40 – mutation in one of the chromosomes 1:10000 – two mutant copies – disease manifestation symptoms in the first years -hypoglycemia – with decreased ketone body formation (decreased fatty acid oxidation and gluconeogenesis in the liver) -accumulation of lipids in the liver -vomiting, drowsiness Therapy: frequent carbohydrate-rich meals + carnitine supply Deficiency of carnitine transport into the mitochondria Long-chain fatty acid transport Carnitine deficiency -high-affinity plasma membrane transporter (heart, kidney, muscle – but not liver) muscle cramps – weakness – death addition of carnitine -secondary carnitine deficiency due to deficiency on β-oxidation acyl-carnitine in the urine Carnititne acyltransferase deficiency most common - CPT II gene mutation – partial loss of enzyme activity muscle weakness when more serious – hypoglycemia with decreased ketone body formation FORMATION OF KETONE BODIES GLUCONEOGENESIS FATTY ACID GLUCOSE ß-oxidation PYRUVATE ACETYL-CoA ANAPLEROTIKUS REAKCIÓ OXALOACETATE Ketone bodies CITRATE Fatty acid oxidation + lack of oxaloacetate fasting untreated diabetes Synthesis of ketone bodies in the liver Ketone bodies as fuels Oxidation of ketone bodies in the extrahepatic tissues heart muscle striatal muscle kidney brain Ketone bodies can be regarded as a transport form of acetyl groups Important sources of energy: heart muscle, renal cortex (preference to glucose, 1/3 of the energy) brain - glucose is the major fuel but in starvation and diabetes brain uses acetoacetate Ketone bodies Fasting Diabetes high level of ketone bodies in the blood KETOSIS Formation in the liver exceeds the use in the periphery. Level of ketone bodies after an overnight fast: ~0.05 mM 2 days starvation: 2 mM (40-fold increase!) 40 days: 7 mM Fatty acid synthesis - repeated cycles – in each cycle the chain is extended by two carbons – four steps in each cycle Enzyme: fatty acid synthase Seven active site for different reactions in separate domains of a single large polypeptide Fatty acid synthesis –Lipogenesisnot a reversal of degradation SYNTHESIS BREAKDOWN Site Cytosol Mitochondrial matrix Intermediates bound to Acyl-carrier protein CoA Enzymes Joined in a single polypeptide chain (fatty acid synthase) Not associated Reducing equivalents NADPH NAD, FAD Units Malonyl-CoA Acetyl-CoA 3-hydroxyacyl-derivative D-enantiomer L-enantiomer Fatty acid synthesis: *Liver *Adipose tissue *Lactating mammary gland Committed step in fatty acid synthesis: formation of malonyl-CoA from acetylCoA acetyl-CoA carboxylase - prosthetic group: biotin Acetyl-CoA carboxylase has three activities in a single polypeptide Biotin – covalently bound to Lys έ-amino group 1. transfer of carboxyl group to biotin ATP-dependent move of activated CO2 from the biotin carboxylase region to the transcarboxylase active site 2. transfer of the activated carboxil group from biotin to acetyl-CoA Critical SH-groups carry the intermediates during the synthesis of fatty acids Acyl carrier protein β-ketoacyl-ACP synthase CH2 SH Condenzing enzyme - cys SH group is the site of ently of malonyl group during fatty acid synthesis SH Fatty acid synthase Acetyl group from Acetyl-CoA is transferred to Cys-SH of β-ketoacyl-ACP synthase (KS) by MAT Malonyl group is transferred to ACP-SH by MAT Acetyl group (from AcetylCoA) is transferred to the malonyl group on ACP (methyl terminal) Acetoacetyl-ACP Reduction by β-ketoacyl-ACP reductase Dehydration by β-hydroxyacyl-ACP dehydratase Reduction by enoyl-ACP reductase Second round of fatty acid synthesis cycle ACP is recharged with another malonyl group by MAT The overall process of palmitate synthesis Seven cycles for the synthesis of palmitate Palmitate is released from ACP by thioesterase (TE) STOICHIOMETRY Seven cycles for the synthesis of palmitate Ac-CoA + 7 malonyl-CoA + 14 NADPH + H+ Palmitate + 7 CO2 + 14 NADP+ + 8 CoA + 6 H2O 7 Ac-CoA + 7 CO2 + 7 ATP 7 malonyl-CoA + 7 ADP + 7 Pi Overall: 8 Ac-CoA + 7 ATP + 14 NADPH + H+ palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 ADP + 7 Pi Fatty acid synthase is present exclusively in the cytosol In adipocytes and hepatocytes cytosolic [NADPH]/[NADP] ratio is high (~75) – strongly reducing environment In hepatocytes and lactating mammary gland cytosolic NADPH is generated largely by pentose phosphate pathway but malic enzyme is also significant MITOCHONDRION CYTOSOL Citrate carries Acetyl-CoA from AcCoA CITRATE ATP:citrate lyase CITRATE AcCoA OXALOACETATE OXALOACETATE NADH MALATE malic enzyme PYRUVATE PYRUVATE NADPH mitochondria to the cytosol for fatty acid synthesis Source of NADPH for fatty acid synthesis Regulation of fatty acid synthesis -regulation of Acetyl-CoA carboxylase-negative feed-back inhibition by palmitate - allosteric stimulation by citrate -regulation by covalent modification Dephosphorylated form – active -Polymerizes into long filaments insulin Phosphorylation inactivates the enzyme Active dephosphorylated ACC Control of fatty acid synthesis Short term regulation • Ac-CoA • ATP isocitrate dehydrogenase inhibited citrate – stimulates Ac-CoA carboxylase +carries the substrate (Ac-CoA) (indicates that two-carbon units & ATP are available for synthesis) Control of fatty acid synthesis Glucagon cAMP activation of protein kinases phosphorylation of Ac-CoA carboxylase - switch off Palmitoyl-CoA *Inhibits Ac-CoA carboxylase *Inhibits translocation of citrate from mitochondria to cytosol *Inhibits glucose 6-P dehydrogenase NADPH Control of fatty acid synthesis Long term regulation – low fat, high carbohydrate diet the amount of Acetyl-CoA carboxylase & fatty acid synthase is increased - fasting and high fat diet the amount of Acetyl-CoA carboxylase is decreased High-fat low carbohydrate diet – Atkins diet fatty acid mobilization - ketone body formation (loss in the urine)