1 Ó Department of Biochemistry 2013 (E.T.) Gluconeogenesis Glycogen metabolism 2 Resorption phase Postresorption phase, fasting Saccharides from food Glycogenolysis (liver) Gluconeogenesis (liver, kidney) 3,1-5,0 mmol/l Concentration of glucose in blood Glucose in blood 3 Hormone Source Effect on the level of glucose Insulin b-cells of pancreas ¯ Glucagon a-cells of pancreas Adrenaline Cortisol Adrenal medulla Adrenal cortex Main hormones in metabolism of glucose 4 Gluconeogenesis - synthesis of glucose de novo • Organ: liver (kidney) • Location: cytoplasma • Substrates for synthesis: non-saccharide compounds (lactate, pyruvate, glucogenic amino acid, glycerol) • Reactions: enzymes of glycolysis are used for gluconeogenesis, only 3 irreversible reactions are circumvented by alternate reactions that energetically favor synthesis of glucose Enzymes are regulated so that either glycolysis or gluconeogenesis predominates, depending on physiologic conditions 5 glucose Glc-6-P Fru-6-P Fru-1,6-bisP Glyceraldehyde-3-P Dihydroxyaceton -2-P 1,3-bis-P-glycerate 3-P-glycerate 2-P-glycerate phosphoenolpyruvate pyruvate Irreversible reactions of glycolysis Glycolysis x gluconeogenesis 6 1. Glc + ATP ® Glc-6-P + ADP (reverse reaction is catalyzed by different enzyme) 2. Fru-6-P + ATP ® Fru-1,6-bisP (reverse reaction is catalyzed by different enzyme) 3. PEP + ADP ® pyruvate + ATP (reverse reaction is replaced by „by-pass“) Irreversible reactions of glycolysis (kinase reactions) 7 Reactions unique to gluconeogenesis 1.Synthesis of phosphoenolpyruvate Why the reverse reaction cannot proceed? D Go = -61,9 kJ/mol Cleavage of ATP does not provide energy sufficient for reverse reaction ATP ADP 8 Formation of phosphoenolpyruvate occurs in two steps: 1. Formation of oxalacetate by carboxylation of pyruvate * enzyme: pyruvate carboxylase energy: consumption of 1 ATP location: mitochondria 2. Conversion of oxalacetate to phosphoenolpyruvate enzyme: phosphoenolpyruvate carboxykinase energy: consumption of 1 GTP location: cytoplasma *note.: carboxylation of pyruvate is also anaplerotic reaction of citric acid cycle 9 1. Conversion of pyruvate to phosphoenolpyruvate (reaction) •carboxylation pyruvate Carboxybiotin CH3 C=O COOH pyruvate carboxylase biotin Pyruvate Oxaloacetate 10 • decarboxylation of oxalacetate PEP carboxykinase phosphoenolpyruvate (PEP) PEP enters reversible reactions of glycolysis - O O C - C - C H 2 - C O O - O + G T P H 2 C =C H - C O O - O P O 3 2 - + G D P CO2 11 11 • Carboxylation of pyruvate is located in mitochondrial matrix – at the same time it can serve as anaplerotic reaction of citric acid cycle (se lecture citric acid cycle) • Oxaloacetate cannot be transported across mitochondrial membrane – it must be transported in form of malate or aspartate • malate ans aspartate are again converted to oxaloacetate in cytoplasma Compartmentation of reactions at phosphoenolpyruvate formation 12 12 pyruvate pyruvate oxalacetate Glucogenic amino acids malate acetylCoA citrate mitochondria cytoplasma oxalacetate malate alanin lactate aspartate aspartate C.C. Kompartmentation of reactions 13 Synthesis phosphoenolpyruvate from pyruvate or lactate requires consumption of 2 ATP Pairing of carboxylation and decarboxylation drives the reaction that would be otherwise energetically unfavorable. (see also the synthesis of fatty acids) 14 14 glucose Glc-6-P Fru-6-P Fru-1,6-bisP Glyceraldehyde-3-P Dihydroxyaceton -2-P 1,3-bis-P-glycerate 3-P-glycerate 2-P-glycerate phosphoenolpyruvate pyruvate Irreversible reactions of glycolysis Glycolysis x gluconeogenesis > 15 3-Phosphoglycerate kinase 3-phosphoglycerate 1,3-bisphosphoglycerate ADP ATP Further consumption of ATP at gluconeogesis Reversal proces of substrate phosphorylation in glycolysis reversible 16 16 glucosa Glc-6-P Fru-6-P Fru-1,6-bisP Glyceraldehyde-3-P Dihydroxyaceton -2-P 1,3-bis-P-glycerát 3-P-glycerate + ATP 2-P-glycerate fosfoenolpyruvát Pyruvát + ATP Glycolysis x gluconeogenesis Substr.fosforylace Irreversible reactions of glycolysis > 17 2. Dephosphorylation of fructose-1,6-bisphosphate fructose-1,6-bisphosphatase allosteric inhibition by AMP, activation by ATP inhibition by fructose-2,6-bisphosphate (its level is decreased by glucagon) H2O + Pi hydrolytic cleavage Like its glycolytic counterpart phosphofructokinase-1, it participates in the regulation of gluconeogenesis. The second unique reaction on gluconeogesis 18 3. Dephosphorylation of glucose-6-P It is present only in liver. Not present in muscle!!! glucose-6-phosphatase + Pi H2O Enzyme is located in lumen of ER The third unique reaction on gluconeogesis 19 Energetic requirements for gluconeogenis reaction ATP/glucose 2 pyruvate → 2 oxalacetate -2 2 oxalacetate → 2 phosphoenolpyruvate -2 (GTP) 2 3-phosphoglycerate → 2 1,3-bisphosphoglycerate -2 -6 ATP/glucose Source of energy is mainly b-oxidation of fatty acids 20 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2H+® glucose + 2NAD+ + 4 ADP + 2 GDP + 6 Pi Sumary equation of gluconeogenesis -6 ATP Consumption: Gluconeogenesis is energy demanding process 21 Lactate formation in tissues, transport by blood to the liver lactate + NAD+ ® pyruvate + NADH + H+ (cytoplasma) (Cori cycle) Origin of substrates for gluconeogenesis Pyruvate E.g. from transamination of alanine, dehydrogenation of lactate 22 Glycerol • formation in adipocytes at cleavage of triacylglycerols • transport by blood to the liver • in liver (cytoplasma): glycerol + ATP ® glycerol-3-P + ADP glycerol-3-P + NAD+ D dihydroxyaceton-P + NADH + H+ What is the energy requirement for synthesis of 1 mol of glucose from glycerol? 23 Glucogenic amino acids They provide pyruvate or intermediates of citric acid cycle, that can be converted to oxalacetate Acetyl CoA – is not the substrate for gluconeogenesis !!! It is metabolised to CO2 in citric acid cycle. Fatty acid cannot be converted to glucose in animals! 24 The most important amino acid for gluconeogenesis is alanin It is formed mainly in muscle by transamination of pyruvate and is transported by blood to the liver. Here is again converted to pyruvate by reverse transamination liver muscle glucose pyruvate lactate alanin lactate alanin pyruvate glucose amino acids 2-oxo acid glutamate 2-oxoglutarate 25 Gluconeogenesis from lactate and glycerol requires NAD+ The ratio NADH/NAD+ may by high at some metabolic conditions – gluconeogenesis can not occur The ratio NADH/NAD+ is increased e.g. at ethanol metabolism (alcohol dehydrogenase). Therefore intake of alcohol can decrease gluconeogenesis Þ hypoglycemia at alcoholics 26 The main features of gluconeogenesis regulation Availability of substrates. Allosteric and hormonal regulation of irreversible reactions. Allosteric effects are rapid (they affect the reaction immediately) Hormons can act through • direct inhibition or activation by a second messenger (rapid effect) • induction or repression of enzyme synthesis (slow effect – hours - days) 27 Enzyme Activator Inhibitor Hexokinase glucose-6-phosphate Phosphofructo kinase 5´AMP, fructose-6-phosphate, fructose-2,6-bisphosphate Citrate, ATP, glucagon Pyruvate kinase fructose-1,6-bisphosphate, ATP, alanin Pyruvate dehydrogenase CoA, NAD+, ADP, pyruvate acetylCoA, NADH, ATP Pyruvate carboxylase acetylCoA ADP Activation and inhibition of enzymes involved in glycolysis and gluconeogenesis 28 Enzyme Inductor Represor glucokinase insulin glucagon phosphofructokinase insulin glucagon Pyruvate kinase insulin glucagon Pyruvate carboxylase glucokortikoids glucagon Adrenalin insulin phosphoenolpyruvate carboxykinase glucocorticoids glucagon adrenalin insulin glucose-6-phosphatase glucocorticoids glucagon adrenalin insulin Effects of hormones on enzyme expression 29 29 Conversions of pyruvate at different conditions pyruvate acetylCoA Lactate, alanine oxaloacetate Pyruvate dehydrogenase Pyruvate carboxylase Aktivation:CoA, NAD+, insulin, ADP, pyruvate Inhibition:acetylCoA, NADH, ATP Activation: acetylCoA Inhibition: ADP 30 Gluconeogenesis in kidneys Substrates: mainly lactate, glycerol and glutamin Glucose can be released from kidneys – in post-resorptive state or during starvation, at acidosis 31 Glycogen - synthesis and degradation 32 • synthesis and degradation of glycogen occurs in most types of cells, the largest stores are in liver and skeletal muscle. • glycogen is a storage form of glucose in cells, that is rapidly released • Muscle – the mass of glycogen is about 1-2% of muscle mass, glycogen is degraded during intensive muscle work or stress • Liver: about 5-10 % of liver mass (after the meal) Glycogen is degraded when glucose level in blood drops Glycogen storage 33 33 Storage of glucose in human (70 kg) Tisue % tissue mass Tissue mass (kg) Mass of glucose (g) Liver 5,0 1,8 90 (glycogen) Muscle 0,7 35 245 (glycogen) Extracelular glucose 0,1 10 10 34 Glycogen is deposited cytoplasma of cells in form of glycogen particles (10-40 nm) Enzymes od degradation and synthesis are on the surface of particles Glycogenolysis is not a reversal proces of synthesis. Location of synthesis and degradation of glycogen 35 Molecules of glycogen have Mr ~108 The branched structure permits rapid degradation and rapid synthesis, because enzymes can work on several chains simultaneously. It also increases the solubility in water. 36 a-1,4-glycosidic bond Non-reducing end O O H O H C H 2 O O O H O H C H 2 O H O O O O H O H O O H O H C H 2 O H O C H 2 O H O O C H 2 O H O C H 2 O H a-1,6-glycosidic bond Types of bonds in glycogen Non-reducing end 37 Synthesis of glycogen (glycogenesis) 1. Activation of glucose to UDP-glucose 2. Transfer of glucosyl units from UDP-glucose to the 4´ ends of glycogen chains or primers 3. Formation a -1,4 glycosidic bond 4. Branching It occurs after the meal, activation by insulin 38 1. Synthesis of UDP-glucose • glucose-6-P glucose -1-P phosphoglucomutase • glucose-1-P + UTP UDP-glucose + PPi PP i + H2O 2Pi 2 ATP are consumed 39 2. Primer is necessary for synthesis of glycogen Pre-existing fragment of glycogen When glycogen stores are totally depleted, specific protein glycogenin serves an acceptor of first glucose residue Autoglycosylation on serine residues 40 • Iniciation – glucosyl residue is added from UDP-glucose to the non-reducing terminal of the primer by glycogen synthase • Elongation by glycogensynthase - formation of linear chains with a-1,4 glycosidic bond UDP-glucose + [glucose]n ® [glucose]n+1 + UDP 3. Formation of a -1,4 glycosidic bonds glycogensynthase 41 4. Branching (branching enzyme) 5-8 glucosyl residues are transferred from non-reducing end to another residue of the chain and attached by 1,6-glycosidic bond G-G-G-G-G G-G-G-G-G-G-G-G-G-G-G-G-G ® -G-G-G-G-G-G-G-G a-1,6 bond Elongation of both non-reducing ends by glycogensynthase New branching by branching enzyme 42 Degradation of glycogen (phosphorolysis) 1.phosphorolytic cleavage of a-1,4 glycosidic bonds by phosphorylase 2.Removal of a-1,6 branching (debranching enzyme) Proceeds during fasting (liver), muscle work (muscle) or stress (liver and muscle). Compare: Hydrolysis x phosphorolysis MCj00787110000[1] 43 1. Phosphorylase - phosphorolytic cleavage of a-1,4 glycosidic bonds at the non-reducing ends glukosa-1-P glykogenn-1 The cleavage continues untill four glucosyl units remain on the chain before a branch point („limit dextrine“) 44 Phosphorylase can split α-1,4-links, its action ends with the production of limit dextrins : Degradation of glycogen 44 G-G-G-G-G-G-G-G G-G-G-G G-G--G-G-G-G-G-G-G-G- 8 Pi G-G-G-G-G-G-G-G + 8 G-P G-G-G-G--G-G-G-G-G- G-G-G-G-G-G-G- G debranching enzyme G-G-G-G-G-G-G-G-G-G-G G-G-G-G-G-G-G-G-G-G-G-G + G G-G-G-G-G-G-G- G-G-G-G-G-G-G Limit dextrin transglycosylase 45 2. Debranching enzyme transferase activity: enzyme transfers unit containing 3 from 4 glucose molecules remaining on the 1,6-branch and adds it to the end of a longer chain by a-1,4 glycosidic bond glucosidase activity: the one glucosyl residue remaining at the end of a-1,6 branch is hydrolyzed by the 1,6 –glucosidase activity of debranching enzyme Free glucose is released ! Not Glc-1-P 46 Further fates of glucose-1-phosphate formed from glycogen phosphoglucomutase Serve as a fuel source for generation of ATP Only liver (kidney) All tissues glucose-6-phosphatase Source of blood glucose glucose-6-P 47 glucose-6-P cannot permeate across the cellular membrane, only free glucose can diffuse Enzyme glucose-6-phosphatase is only in liver and kidneys – it is not present in muscle. Blood glucose can be maintened only by cleavage of liver glycogen but not by cleavage of muscle glycogen Cleavage of glycogen in muscle and other cells provides glucose-6-P that can be metabolized only within the given cell (by glycolysis) Significance of glucose-6-phosphatase 48 Lysosomal degradation of glycogen Lysosomal acidic glucosidase (pH optimum 4) Degradation of about 1-3% of cellular glycogen (glycogen particles are surrounded by membranes that then fuse with the lysosomal membrane -enzyme degrades a-1,4-bonds from non-reducing end - glucose is released (see also Pompe disease) 49 Regulation glycogen metabolism Glycogen synthase X glycogen phosphorylase Hormonal control Allosteric regulation 50 Hormons affecting synthesis and degradation of glycogen Hormon synthesis degradation Insulin Glucagon Adrenalin ¯ ¯ ¯ Hormons action is mediated by their second messengers. 51 Phosphorylation and dephosphorylation plays important role at regulation of glycogen metabolism • phosphorylation by kinases and ATP • dephosphorylation by phosphatases 52 H2O Pi Protein phosphatase OH O-P ATP ADP proteinkinase Non active enzyme Active enzyme H2O Pi Protein phosphatase OH O-P ATP ADP proteinkinase Active enzyme Non active enzyme Common examples of enzyme activity regulation by phosphorylation and dephosphorylation 53 Activation and inactivation of glycogen synthase Glycogen synthase a (dephosphorylated - active) Glycogen synthase b (phosphorylated - inactive) ATP ADP H2O Pi glycogensynthase kinase phosphatase 54 glycogensynthase a (dephosphorylated, active) Glycogen synthase b (phosphorylated, non active) Glucogensynthase phosphatase (activation by insulin, allosterically by glucose-6-P Inactivation by ↑ cAMP ) Activation and inactivation of glycogensynthase in liver ATP ADP Glycogene synthase kinase (activation by glucagon /cAMP/ or adrenalin /Ca-calmodulin/ inactivation activation Pi 55 Activation and inactivation of glycogen phosphorylase phosphorylase b (non phosphorylated form -low activity) phosphorylase a (phosphorylated form-active) ATP ADP H2O Pi Phosphorylases in liver and muscles are different phosphorylase kinase proteinphosphatase 56 Effect of hormons: Liver: glucagon (cAMP), adrenalin (cAMP, Ca2+calmodulin) Degradation of glycogen Muscle: adrenalin (cAMP) at the stress allosteric regulation AMP No effect of glucagon ! Ca2+ during muscle contraction Novinový papír Glucose, ATP, Glc-6P: allosteric inhibition 57 Glycogen storage diseases - enzyme deffects Inherited enzyme deficiences. They can be tissue specific, as in various tissues can be various isoenzymes. Typ Enzyme defect Organ Characteristics 0 I II III IV V VI VII Glycogen synthase Glc-6-phosphatase Lysosome α-glucosidase Debranching enzyme Branching enzyme Muscle phosphorylase Liver phosphorylase Phosphofructokinase Liver Liver, kidney All organs Liver, muscle, heart Liver Muscle Liver Muscle, ercs Hypoglycemia Enlarged liver, kidney. Hypoglykemia. Celly are overloaded by glycogen Accumulation of glycogen in lyzosomes Accumulation of branched polysaccharide. Accumulation of unbranched polysaccharide High content of glycogen in muscle, exercise induced muscular pain High content of glycogen in liver, mild hypoglycemia As in type V 58 Von Gierke disease (type I) Most common Deficit of glucose-6-phosphatase or transporter for glucose-6-P Concequences: Inability to provide glucose during fasting state •hypoglycemia at fasting •lactacidemia •(hyperlipidemia, hyperurikemia) Enlarged liver, increased glycogen store Growth reatardation, delayed puberty 59 Pompe disease (type II) Absence of a-1,4-glucosidase in lysosomes Acummulation of glycogen in lysosomes Loss of lysosomal function Damage of muscles®muscle weakness Infantile form: death before age 2 years Juvenile form: later –onset myopathy with variable cardiac involvment Adult form: limb-girdle muscular distrophy-like features. 60 McArdle disease (type V) Absence of muscle phosphorylase Stores of glycogen are not available for production of energy Muscle is not able to perform exercise or work