Carbohydrate Metabolism II: Glycolysis & Gluconeogenesis PDF

Comenius University in Bratislava Jessenius Faculty of Medicine in Martin Department of Medical Biochemistry Carbohydrate metabolism II. Glycolysis and gluconeogenesis BIOCHEMISTRY, 2nd COURSE, Winter semester  Metabolism of glucose in liver...

Comenius University in Bratislava Jessenius Faculty of Medicine in Martin Department of Medical Biochemistry Carbohydrate metabolism II. Glycolysis and gluconeogenesis BIOCHEMISTRY, 2nd COURSE, Winter semester  Metabolism of glucose in liver GLUT 2 Liver GLUCOSE GLU PC GLU 6 P GLYCOGEN GLYCOLYSIS GLUCONEO GENESIS GLUCURONIDE PYRUVATE LACTATE ACETYL CoA H+ TUKY TCA ATP Release of congested energy in Glu: - step by step of glucose degradation/oxidation - the primary point is the metabolic pathway of glycolysis → products: - in aerobic conditions: pyruvate , principally all tissues - in anaerobic conditions lactate, Er, anaerobic muscles CAVE! Hypoxic/aneerobic tissue conditions - organism is utilizing preferentially Glu - from diet – GIT- the liver - From liver synthetized from non-saccharide substances = gluconeogenesis Two metabolic conditions: a) aerobic glycolysis - cells with mitochondria and adequate oxygen supply- all tissues - oxygen is necessary to reoxidation the NADH - pyruvate (Pyr) – end product - oxidative decarboxylation of Pyr to acetyl-CoA → Krebs Cycle- energy b) anaerobic glycolysis - cells without mitochondria or non-adequate oxygen supply- Er, anaer. muscles,hypoxia - lactate formation – reoxidation of the NADH by lactate dehydrogenase  Glycolysis or GLUCOLYSIS - located in cell cytosol → Glu breakdown- all cells a) energy production – release a small amount of Glu energy - substrate-level phosphorylation = ATP production b) production of intermediates for other metabolic pathways - main importance: pyruvate production → conversion to acetyl-CoA → citric acid cycle → remaining Glu energy is contained in reduced coenzymes → electron and proton transport to terminal oxidation process- production of ATP by oxidative phosphorylation Initiation of Glc metabolism : Glu transport from the blood into the tissue cells (or intracellularly from ) - transport into muscle and adipose: insulin dependent GLUT 4 - transport into Er (Glut1), brain and liver is insulin non-dependent GLUT1,2,3 Glycolysis: oxidation and breakdown of glucose production of ATP (aerobic and anaerobic conditions) all cells (no exeption) in cytosol (transport of reduced equivalents ( NADH via shuttles) Production of ATP: 1. Substrate phosphorylation 2. NADH- oxid. phosphorylation 3. Oxidation of pyruvate- NADH+FADH2- OPH Regulation: 1. Hexokinase 2. Phosphofructokinase 3. Pyruvatekinase Production of precursors for synthesis: fatty acids, cholesterol Aminoacids and ribose-5-P and heptoses Two phases of glycolysis metabolic pathway: The first phase – energy consumption (investment) a) formation of phosphorylated forms of the saccharides ( - 2 ATP) b) trioses formation - starts with glucose-6-P and ends with synthesis of fructose-1,6-bisP glucose → glucose-6-P → fructose-6-P → fructose-1,6-bis-P ATP ATP - phosphokinases: phosphoric acid transfer from ATP onto substrates or from substrates onto ADP - Phosphofructokinase 1: the most important enzyme in glycolysis - catalyzes fructose-1,6-bis-P formation - regulatory enzyme - enzyme activation or inhibition: according to requirement of tissues - accelerate or decelerate pyruvate formation 1. reaction: Glu phosphorylation → Glu-6-P formation - Glu- 6 P is not able to penetrate plasma membrane - hexokinase - regulatory enzyme (first one)- all tissues except liver - broad specificity – phosphorylation of the several hexoses - inhibited by Glu-6-P and high ratio of ATP/ADP - low Km - lower Glu level - glucokinase = hexokinase D - liver and ß cells of the pancreas - Glu phosphorylation in the post resorption period → high Glui concentration → fast Glu metabolism (high Km and Vmax) - not inhibited by Glu-6-P 2. reaction: Isomerization of Glu-6-P → Fru-6-P - enzyme: phosphoglucose isomerase Michaelis-Menten for hexokinase and glucokinase: Hexokinase Glucokinase Glucose mmol/l 3. reaction: - irreversible phosphorylation of Fru-6-P → Fru-1,6-bis-P - enzyme: phosphofructokinase 1 (PFK 1) !!! - rate limited reaction - the most important regulatory step Regulation: 1. Energetic status of the cell a) ↑ ATP and ↑ citrate → - PFK 1 inhibition b) ↑ AMP → + PPK 1 activation 2. Regulation by Fru-2,6-bis-P- produced by isoenzyme P fructo Kinase 2 a) strong activator of PFK 1 b) inhibitor of fructose 1,6-bisphosphatase (gluconeogenesis) Metabolism of PFK 2 – bifunctional enzyme: 1. production → phosphofructokinase 2 (PFK 2) activity---- Fru2,6bisP 2. breakdown → fructose bisphosphatase 2 (FBP 2) activity !!! - kinase and phosphatase activity → 2 domains in one bifunctional enzyme molecule 4. reaction: - cleavage of Fru-1,6-bisphosphate - enzyme: aldolase A – reverse aldol condensation → glyceraldehyde-3-P + dihydroxyacetonephosphate - reversible reaction 5. reaction: - isomerization: dihydroxyacetonephosphate → glyceraldehyde-3-P - enzyme: triose phosphate isomerase 1. 2.  3. ! 4. 5. The second phase – energy production –utilization substrate phosphorylation a/ production of 4 ATP b/ production of 2 NADH c/ production of 2 Pyruvates (3 carbons) molecules from one Glu (6 carbons) - starts with reverse aldole condensation of fructose-1,6-bis-P (6 C, 2 P) on two trioses (3 C, 1 P) - conversion to pyruvate → on intermediates with bound phosphoric acid – high energy P group –substrate-level phosphorylation ATP production (phosphoric acid transfer onto ADP) 6. reaction: - oxidation: conversion of glyceraldehyde-3-P → 1,3-bisphosphoglycerate - enzyme: glyceraldehyde 3-P-dehydrogenase - coenzyme: NAD+ - oxidation of produced NADH: Pyr → Lac (anaerobic) - oxidation of produced NADH in the respiratory chain (aerobic) - cofactor: Pi - substrate-level phosphorylation - aldehyde group oxidation → carboxyl group - attachment of Pi to the carboxyl group (high energy P group) Side chain reaction in Er: - synthesis of 2,3-bisphosphoglycerate (2,3-BPG) - enzyme: bisphosphoglycerate mutase - high concentration in Ery- regulation of oxygen binding to the Hb + ABR - cleavage: phosphatase → 3-phosphoglycerate 7. reaction: - ATP production (2x-2 molecules): 1,3-bisphosphoglycerate → 3- phosphoglycerate - enzyme: phosphoglycerate kinase 8. reaction: - intramolecular shift of phosphate: 3-phosphoglycerate → 2-phosphoglycerate - enzyme: phosphoglycerate mutase 9. reaction: - dehydratation: intramolecular energy redistribution → high-energy enolphosphate: conversion 2-phosphoglycerate → phosphoenolpyruvate (PEP) - enzyme: enolase 10. reaction: - pyruvate formation: PEP → pyruvate - enzyme: pyruvate kinase (PK) - 2 ATP production by substrate-level phosphorylation (2 molecules) !!! - „feed-forward„ regulation: liver → Fru-1,6-bis-P activate PK- not in muscles (Two kinases activation: i) ↑ PFK 1 → ii) ↑ PK) !!! - covalent modification of PK - phosphorylation → cAMP dependent protein kinase - liver: ↑ glucagon (low Glu level) → ↑ c AMP → phosphorylation of PK (inactive) - dephosphorylation: phosphatase → PK (active)- insulin - PEP → common intermediate also for gluconeogenesis Conversion: pyruvate → lactate - enzyme: lactate dehydrogenase (LD) - anaerobic glycolysis in the eukaryotic cells - Ery, Leu, lens, cornea, kidney medulla, testes 1. Lac formation in the muscle (Er) - working anaerobic skeletal muscle, energy production during anaerobic conditions - ↑ production of NADH (glyceraldehyde-3- phosphate dehydrogenase)- used in LD reaction - lactate-H+--- ↓ pH → muscle pain- fewer → Lac diffusion into blood –skel.muscles, Er 2. Lac consumption – alternative metabolic substrate for heart - LD metabolic function liver: a) Lac → Pyr → Glu (gluconeogenesis) (high in low Glu) b) Lac → Pyr → Krebs cycle (low) myocard: Lac → Pyr → Krebs cycle (alternative source of energy)  6. ! 7.  8. 9. ! Liver 10. Metabolism of glucose-6-P in the erythrocyte, no mitochondria, Er shape- microvessel  Energetic yield of glycolysis 1. Anaerobic glycolysis Glu + 2 Pi + 2 ADP → 2 Lac´ + 2 ATP + 2 H2O a) ATP production - 2 molecules ATP on 1 molecule of Glu - small energetic yield - cells and tissues without or very limited amount of MIT → Ery, Leu, kidney medulla b) NADH production - no net NADH yield - 1x NADH + (glyceraldehyde dehydrogenase) - production - 1x NADH – (lactate dehydrogenase) - consumption  Main lactate producing tissues (in rest): Celková produkcia laktátu Total production 115 (g/d) Erytrocytes 29 Skin 20 Brain 17 Muscles 16 Kidney medulla 15 Intestinal mucose 8 Others(eye) 10 2. Aerobic glycolysis Glu + 2 Pi + 2 NAD+ + 2 ADP → 2 Pyr´ + 2 ATP + 2 NADH + 2 H+ + 2 H2O - 2 ATP consumption (phosphorylation in the first phase of glycolysis) - 4 ATP production (2 ATP per one triose) - net yield = 2 ATP - 2 x NADH → 2,5 ATP per one NADH Comparison of the yield from Glu after lactic acid production: glycolysis: 2 ATP (substrate level) Glu oxidation to CO2 and H2O in aerobic conditions: glycolysis + citrate cycle + terminal oxidation: 30- 32 ATP !!! Main pathway for energy production- brain, muscle, heart, kidney!!!!  Glycolysis regulation 1. short-term (min or hrs) - allosteric activation/inhibition - phosphorylation/dephosphorylation 2. long-term (hrs – days) - hormones (insulin+ , glucagon - ) - 10 – 20 x increase of the enzymatic activity 3. regulatory enzymes a/ hexo/glucokinase b/ phosphofructokinase 1 c/ pyruvate kinase phosphofructokinase - the main regulatory enzyme: - catalyzes fructose-6-P transformation to fructose-1,6-bis-P - the metabolic pathway rate depends on energetic charge of cell - if the cell has sufficient ATP → decrease pyruvic acid formation - high ATP concentration → allosteric inhibition of phosphofructokinase - high AMP concentration → signal for the glycolysis acceleration (ATP is used to cover the organism requirement) - AMP is allosteric activator of phosphofructokinase - activity of phosphofructokinase → accelerates Glu metabolism → allosteric activation by fructose-2,6-bis-P - high glucose concentration in the blood → secreting of insulin - insulin accelerates: a) Glu transport into cells b) glycolysis via stimulatory fructose-2,6-bis-P - low Glu concentration in the blood → glucagon from pancreas - glucagon decelerates glycolysis by decrease of fructose-2,6-bis-P (protection of low Glu levels in the blood for the brain function) - both conversions lead to normalization of blood glycaemia  Glucose hexokinase tissue specific isoenzymes (low Km, high affinity) Glc-6P Fructose 6P glucokinase (high Km) PhosphoFru kinase-1 Fru-2,6-bis-P Fru 1,6 bis P rate limiting, allosteric enzyme tissue specific izoenzymes Fructose-2,6-bis-P: is not intermediate of glycolysis! Phosphofructokinase-2: inhibited by phosphorylation – e.g.. cAMP-depend. proteinkinase in liver (inhibition of glycolysis during fasting ←glucagon)  Differences in the regulation of glycolysis in liver, heart and muscles by PPK-2 Liver – phosphorylation via glucagon- cAMP phosphorylation of phosphatase – stimulation of activity inhibition of glycolysis !!!!! Heart- phosphorylation via adrenalin and AMP – activation of kinase stimulation of glycolysis- positive clinical effect of adrenalin- inotropy- increase heart stroke Muscle- adrenalin no effect, kinase is activated by Fru6P and stimulates formation of Fru 2,6P activation of glycolysis !!!! arsenate  Liver isoenzymes- inhibited by cAMP-dep. proteinkinase (inhibition Pyruvatekinase of glycolysis during fasting by Fructose1,6P glucagon) lactate pyruvate Pyruvate dehydrogenase CLINICAL NOTES: Lactic acidosis: Increase of NADH/NAD+ ratio inhibition of pyruvate dehydrogense  LACTATE DEHYDROGENASE ETHANOL synthesis important in RBC, WBC ( other non- present in yeasts, bacteria (including mitochondria cells ), anaerobic sketal. muscle) gastrointestinal flora in humans) reversible in low NADH /NAD+, ratio, liver, depends on tiamine diP cardiac myocytes in cytosol cytosol CO2 (Thiamine-PP) Etanol Acetaldehyd PYRUVATE Lactate NAD+ NADH + H+ CO2 NADH + H+ NAD+ NAD+ CO2 NADH + H+ Oxálacetát Acetyl CoA PYRUVATE CARBOXYLASE PYRUVATE DEHYDROGENASE COMPLEX biotin as prosthetic group activated by acetyl CoA in liver tiamine -PP, lipoic acid, FAD, NAD+ a CoA replenish intermediates to TCA source of acetyl CoA for TCA and FA replenish intermediates to gluconeo- synthesis genesis irreversible, mitochondria irreversible reaction localized in mitochondria  Clinical applications 1. Inborn deficiency of the enzymes a) pyruvate kinase (95%) b) phosphoglucose isomerase (4%) - different expression in the cells and tissues - pyruvate kinase – Leu, Ery - triosophosphate isomerase – Ery, Leu, muscle, CNS Symptoms: hemolytic anemia Therapy: no therapy or folic acid 2. Lactic acidosis -anaerobic glycolysis → energy production during inadequate oxygen suply (MI, pulmonary embolism, uncontrolled bleeding ) - lactate in the blood → information about oxygen debt - diagnosis of the shock status - monitoring of the patients therapy and patients recovery after cerebral stroke and myocardial infarction, or intense training 2. Gluconeogenesis New formation of Glc from non-sacharidic compounds LIVER, kidneys, intestine!!!  Gluconeogenesis- activation Glu deficiency in the food, strenous physical activity, starvation → decrease of Glu concentration in the blood (lower than 4,5-5 mmol/l) → need for continuous supply of Glu → brain, Ery, kidney medulla, lens,cornea, testes, exercising muscle Tissues response: 1. Degradation of the glycogen stores - glycogen in the liver: 10 – 18 hrs 2. new Glu formation from non-saccharide precursors- gluconeogenesis: - 1. lactate- (Er, muscles), pyruvate, 2. glycerol (TAG-adipose) - 3. alfa-oxoacid mostly Ala (metabolism of AA)-muscles - 4. propionyl CoA from odd chain FA via succinyl CoA - gluconeogenesis localized: 90 % in the liver (presence of enzymes) 10 % in the kidney (more important during prolonged starving) 10% Additional intestinal from Glutamine Precursors 1. Lactate (anaerobic glycolysis, RBC, muscle) 2. aminoacids (muscle proteins, or glutamin) 3. glycerol (adipose)  Characterization of gluconeogenesis 1. Reversible reactions of gluconeogenesis -catalyzed by the same enzymes as reversible reactions of glycolysis 2. enzymes which catalyze irreversible reactions of glycolysis (phosphokinases) – in gluconeogenesis are replaced by the enzymes with opposite effect (phosphatases) 3.Glucose 6 phosphatase has special importance → allows Glu transport from the hetatocytes/kidney into the blood stream to increase blood Glu level 4. Rate of gluconeogenesis is increased by inhibition of glycolytic enzymes – allosterically: (ATP +), (Fru 2,6-) 5. hormonally: glucagon low Glu in blood- (stimulatory) insulin (high Glu level) (inhibitory) Metabolic pathway: - gluconeogenesis isn't reverse of glycolysis - three reactions are irreversible: 1. conversion of phosphoenolpyruvate to Pyr enzyme: pyruvate kinase i) - pyruvate → carboxylation → oxaloacetic acid (OAA) enzyme: pyruvate carboxylase (MIT – liver, kidney medulla, no in muscles) coenzyme: biotin; activation – acetyl-CoA ii) - oxalacetic acid → phosphoenolpyruvate enzyme: phosphoenolpyruvate carboxykinase (cytosol) oxaloacetate → transport from MIT into cytosol → malate → reoxidation to oxalacetate 2. dephosphorylation of Fru-1,6-bis-P enzyme: fructose 1,6-bisphosphatase - regulatory step: a) cell energetic status ↓ AMP+; ↑ATP+ ↑ AMP- b) regulation by Fru-2,6-bisphosphate - allosteric inhibition of fructose 1,6-bisphosphatase 3. dephosphorylation of Glu-6-P enzyme: glucose 6-phosphatase All enzymes are exclusively localized: Liver (90%), kidney medulla (10%)+ Intestinal mucosa (10% !!!)  Glu 6-phosphatase Glucose-6-P Glucose Fructose-6-P Phosphofructokinase 1 Fru 1,6-bisphosphatase Fructose-1,6-bis-P Glyceraldehyde-3-P Dihydroxyacetone-P 1,3-bis-phosphoglycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate Phosphoenolpyruvate CO2 Pyruvate carboxylase (MIT) carboxykinase (cytosol) Oxalacetate Gluconeogenesis in enterocytes when glycemia is lower from glutamine!!! Summary reaction: 2Pyr + 4ATP + 2GTP + 2NADH + 2H+ + 6H2O → → Glu + 2NAD+ + 4ADP + 2GDP + 6Pi + 6H+ Substrates for gluconeogenesis: 1. lactate- immediate substrate Cori cycle – Glu (liver) → blood → exercising muscle, RBC → lactate (blood) → liver (gluconeogenesis) 2. Amino acids and α-ketoacids from muscle proteins send as ALANINE - Glu-Ala cycle:- starvation- glucagon i) Glucogenic AA – Ala, Ser, Gly, Cys, Thr (pyruvate); Asp (oxalacetate); Glu (alfa-ketoglutarate) ii) pyruvate, oxalacetate, alfa-ketoglutarate 3. glycerol- prolonged starvation – degradation of adipose in conn. tiss. - product of lipolysis (TAG) in the adipose tissue by Hormone sens. lipase - transport into the liver- cortisol - phosphorylation → glycerol-P (oxidation) → dihydroxyacetone-P Regulation of gluconeogenesis: simultaneous inhibition of enzymes of glycolysis and activation of gluconeogenesis! 1. Pyruvate → PEP Fosfoenolpyruvatecarboxykinase - Induction (glukagon, adrenalin, cortisol) - inhibition (insulin) 2. Fructose-1,6-P → Fructose-6-P Fructose-1,6-bisphosphatase – inhibition (fructose-2,6-P) 3. Glucose-6-P → Glucose Glucose -6-phosfatase – induction by fasting  Regulation of gluconeogenesis Glucagon – stimulation of gluconeogenesis a) decreases level of Fru-1,6-bisphosphate which leads to → activation of fructose 1,6-bisphosphatase (gluconeogenesis) → inhibition of phosphofructokinase 1 (glycolysis) b) covalent modification of the enzymatic activity - ↑ cAMP → active protein kinase A → inactive pyruvate kinase (P) => phosphoenolpyruvate accumulation Cortisol-stimulation of muscle proteolysis- sarcopenia +cachexia Cortisol stimulated of adipose TAG hydrolysis- cachexia Substrate availability ↓ insulin → mobilization of proteins → glucogenic AA Allosteric activation by acetyl-CoA ↑ acetyl-CoA → stimulation of pyruvate carboxylase during prolonged starvation (liver)  Cori cycle Cori´s husbands-Charles Univ Prague = glucose – lactate cycle - metabolic transfer of intermediates of carbohydrate metabolism (Glu and lactate) between muscle (degradation) and liver (synthesis) - Lactate produced from the muscle pool of Glu ( glycogen or Glu from blood) → lactate production in the erytrocytes (anaerobic glycolysis product) - lactate released to the blood – into liver → Glu production by gluconeogenesis - transport of Glu – for muscle(brain..) metabolism  Glucose – alanine cycle - anaerobic glycolysis → lactate and alanine, later muscle proteolysis (cortisol) - blood: alanine is transfered to the liver - liver: a/ ammonia is released → converted to urea- excretion via kidneys b/ pyruvate → gluconeogenesis → glucose - less productive process than the Cori cycle - Side product – urea - removal of the urea is energy-dependent → total ATP production is lower!!!  Glucagon (↓) Insulin (↑) receptor Cell membrane Cell membrane Adenylate cyclase Activation of several enzymes ATP cAMP 2. Reduced protein kinase A activity prefer dephosphorylated 1. Elevated rate of insulin / PFK-2 / FBP-2 complex glucagon causes decrease Active protein kinase A (↓) of cAMP and decrease active protein kinase A levels FBP-2 = fructose bisphosphatase 2 PFK-2 = phosphofructokinase 2 Fructose-6-phosphate ATP ADP P P ATP PFK-2 FBP-2 PFK-2 FBP-2 active inactive inactive active Phosphofructokinase 2 Pi ADP + Fructose 2,6- Fructose-1,6-bis-P bisphosphate (↑) 3. Dephosphorylated PFK-2 is 4. Increase fructose-2,6- active and FBP-2 is inactive, this bisphosphate concentration prefer production of fructose-2,6- activate PFK 1, which faces to bisphosphate increased glycolysis  Glucagon (↑) Insulin (↓) receptor Cell membrane Cell membrane Adenylate cyclase ATP cAMP + PPi 2. Increased protein kinase A activity prefer phosphorylated 1. Low rate of insulin / form of PFK-2 / FBP-2 complex glucagon decreases cAMP Active protein kinase A (↑) and increases volume of active protein kinase A; Pyruvate kinase inactive FBP-2 = fructose bisphosphatase 2 Pi PFK-2 = phosphofructokinase 2 Fructose-6-phosphate ATP ADP P Pi PPFK-2 PFK-2 FBP-2 FBP-2 active inactive Fructose bisphosphatase 1 inactive active Pi H2O - Fructose-2,6-bis-P (↓) Fructose-1,6- bis-P 3. Phosphorylated PFK-2 is inactive 4. Decreased fructose-2,6-bis-P and FBP-2 is active, what defend concentration decelerates PFK 1 fructose-2,6-bis-P production activation, what lead to increased gluconeogenesis  Summarization Glycolysis: high Glc in the blood , high In Production of ATP for all tissues also in less oxygen Role of glycolysis in different tissues- production of energy and other substrates Lactic acidosis- low oxygen – low tissue perfusion, low Er Regulation (3 key enzymes- regulated by AMP, Fru2,6P) Gluconeogenesis: low Glc, high Glucagon Activated during fasting, physical activity, high protein diet Precursors: lactate, glycerol, Amino acids 3 key regulatory enzymes: pyruvate → PEP fructose-1,6-P→ fructose-6-P glucose-6-P → glucose Regulation: glucagon- Fru2,6 bisphosphatase, acces of substrates and increase of AcCoA