The sweet side of catabolism: carbohydrates as cellular fuels Russian National Research Medical University Maxim A. Abakumov Moscow, 2014

Carbohydrates metabolism Usually comes as polysaccharides Two main polysacharides are glicogen and starch Polysacharides can not be used in native form Breakdown into monosacharides and transport from gut to blood stream and periheral tissues are needed

Digestion of carbohydrates Digestion – enzyme driven breakdown of large polysacharide molecules into monosacharides Usually takes plase in gastrointestinal tract Glucose polymers Starch, glycogen Disaccharides MaltoseSucroseLactose Digestion by amylase Monosaccharides 2xGlucose Glucose+Fructose Glucose+Galactose MaltaseSucraseLactase

Absorption of carbohydrates Process of monosacharides transport from gut to blood stream or lymph Involves special transporting proteins located on membrane of intestine cells

Digestion Composition of carbohydrates in your diet: ~ 70% starch (polysaccharide) ~ 20% sucrose (disaccharide) ~ 6% lactose (disaccharide) ~ 2% maltose (disaccharide) Polysacharides digestion occurs in mouth and small intestine This process is driven by salivary and pancreatic amylases

Digestion

Absorption Transmembrane transporter proteins are involved First, monosacharides are transported into cell from intestine Second, monosacharides are released into blood stream

Absorption

Glucose metabolism Glucose decomposition for energy release (ATP synthesis) called glycolysis Glucose synthesis with energy consume (ATP hydrolysis) calles gluconeogenesis Glycolysis can be diveded into: a) aerobic b) anaerobic Aerobic products are CO 2 and H 2 O Anaerobic product is lactate For both intermediate is pyruvate

Glucose metabolism in cell Glucose Pyruvate AcetylCoa CO 2 + H 2 O + ATP TCA Aerobic Glycolisis ETC+OP Lactate Anaerobic Glycolisis TCA

Glycolisis 1. Glucokinase 2. Phosphogluco isomerase 3. Phosphofructo kinase-1 4. Aldolase 5. Triosophosphate isomerase GlucoseGlucose-6-PFructose-6-P Fructose-1,6-diP ATPADP ATPADP Glyceraldehyde phosphate isomerase 7. Phosphoglycerate kinase 8. Phosphoglycerate mutase 9. Enolase 10. Pyruvate kinase 1,3-bisphospho glycerate 3-phospho glycerate 2-phospho glycerate ATPADP H2OH2O ATP NAD + NADH Phosphoenol pyruvate Pyruvate

Sequence of reactions + Pyruvate Glucose Glucose metabolism in cell Anaerobic Glycolisis CoA 2x + CO 2 Aerobic Glycolisis TCA, ETC, OP Lactate

Glucose phosphorylation First step in glucose metabolism – phoshorylation of OH-group at 6th carbon atom Phosporylated glucose (glucose-6-phospate) is charged and cannot be transported out of the cell Glucose-6-P goes to metabolism Catalyzed by two types of enzyme (isozymes)

Glucose phosphorylation Hexokinase Glucokinase Low K m value High affinity to glucose Located in most tissue cells Three isoforms (I, II, III) High K m value Low affinity to glucose Located mostly in liver cells Actually IV isoform of hexokinase

Glucose F PET tracer Indicates glucose cosumption by cells Phosphorylates after transport in cell OH-group at 2nd carbon atom is substituted by 18 F Further metabolism is blocked Cells with more active metabolism increase glucose consumption, glucose F level and consequently signal on PET scanner

Phase I Coversion of glucose (6 carbon) to dihydroaceton phosphate and gliceraldehyde- 3-phosphate (2x3 carbon) 2 ATP are required (will be regenerated later) 1st and 3rd reaction are irreversible

Phase I. Preparatory phase. 1. Glucokinase 2. Phosphogluco isomerase 3. Phosphofructo kinase-1 4. Aldolase 5. Triosophosphate isomerase GlucoseGlucose-6-PFructose-6-P Fructose-1,6-diP ATPADP ATPADP Dyhydroxy acetone phosphate D-glyceraldehyde -3-phosphate

Glucose to glucose-6-P 1st ATP is hydrolysed Total ATP count: -1 ATP Total NADH count: 0 NADH

Glucose-6-P to fructose-6-P Total ATP count: -1 ATP Total NADH count: 0 NADH Phosphohexose isomerase

Fructose-6-P to fructose-1,6-diP 2nd ATP is hydrolysed Total ATP count: -2 ATP Total NADH count: 0 NADH Phosphofructokinase-1 ATP ADP Fructose-6-P Fructose-1,6-diP

Fructose-2,6-diP to gliceraldehyde-3- phosphate and dihidroxyacetone-phosphate Total ATP count: -2 ATP Total NADH count: 0 NADH Aldolase Fructose-2,6-diP Dyhydroxyacetone phosphate D-glyceraldehyde- 3-phosphate

Phase II. Payoff phase Coversion of dihydroaceton phosphate and gliceraldehyde-3-phosphate (2x3 carbon) to pyruvate (2x3 carbon) 4 ATP are restored Last reaction is irreversible

Phase II. Payoff phase ,3-bisphospho glycerate 3-phospho glycerate 2-phospho glycerate ATPADP H2OH2O ATP NAD + NADH 6. Glyceraldehyde phosphate isomerase 7. Phosphoglycerate kinase 8. Phosphoglycerate mutase 9. Enolase 10. Pyruvate kinase Phosphoenol pyruvate Pyruvate

Gliceraldehyde-3-phosphate to 1,3- bisphosphoglycerate 2x Total ATP count: -2 ATP Total NADH count: 2 NADH NAD + NADH PiH+H+ Glyceraldehyde-3-phosphate dehydrogenase D-glyceraldehyde- 3-phosphate 1,3-bisphosphoglycerate

1,3-bisphosphoglycerate to 3- phosphoglycerate 2x Total ATP count: 0 ATP Total NADH count: 2 NADH 2 ATP are synthesized 1.3-bisphosphoglycerate ADP ATP Phosphoglycerate kinase 3-Phosphoglycerate

3-phosphoglycerate to 2- phosphoglycerate 2x Total ATP count: 0 ATP Total NADH count: 2 NADH 3-Phosphoglycerate Phosphoglycerate mutase 2-Phosphoglycerate

2-phosphoglycerate to phosphoenolpyruvate 2x Total ATP count: 0 ATP Total NADH count: 2 NADH 2-Phosphoglycerate H2OH2O Phosphoenolpyruvate Enolase

Phosphoenolpyruvate to pyruvate 2x Total ATP count: 2 ATP Total NADH count: 2 NADH 2 ATP are synthesized Phosphoenolpyruvate ADP ATP Pyruvate kinase Pyruvate

Glucose→Pyruvate Total energy output 2 ATP are consumed 4 ATP are synthesized Total 2 ATP from 1 glucose 2 NADH are synthesized All ATP is synthesized without O 2 (substrate-level phosphorylation) Anaerobic glycolysis

Glucose→AcCoA→CO 2 + H 2 O Total energy output Total 2 ATP + 2 NADH from anaerobic glycolisis. 2 NADH from PDH 6 NADH+ 2 FADH 2 from TCA 2 GTP from TCA Total 10 NADH+4 ATP + 2FADH 2 = 32 ATP All ATP is synthesized with O 2 (oxidative phosphorylation) Aerobic glycolysis

Sequence of reactions + Pyruvate Glucose Glucose metabolism in cell Anaerobic Glycolisis CoA 2x + CO 2 Aerobic Glycolisis TCA, ETC, OP Lactate 32 ATP2 ATP

Glycolysis regulation Glucose Glucose-6-P Fructose-6-P Fructose-1,6-diP Phosphoenol pyruvate Pyruvate Hexokinase Glucose 6- phosphatase Fructose-1,6- bisphosphatase Phosphofructo kinase1 (PFK1) Pyruvate carboxylase Pyruvate kinase AMP ATP Citrate ATP Acetyl-CoA AMP Acetyl-CoA Inhibition Activation

Glycolysis regulation 3 enzymes catalyzing irreversible steps are regulated: 1) Hexokinase 2) Phosphofructokinase-1 3) Pyruvate kinase Feedback or hormonal control

Hexokinase regulation Feedback mechanism

PFK1 and PFK2. Distinguish them. Only kinase activity Phosporylates F-6-P Produces F-1,6-BP for further glycolysis Insulin activated Glucagon inhibited Both kinase and phospatase activity Regulates F-6-P and F-2,6-BP amount F-2,6-BP doesn’t go to glycolisis PFK1PFK2

PFK1regulation. Feedback mechanism.

Pyruvate kinase regulation Feedback mechanism

Hormonal control Insulin and glucagon are two main hormones controlling glucose methabolism Insulin – fed state hormone Insuline provides glycolysis, glicogen and fatty acid synthesis Glucagon – fasting state hormone Glucagon provides gluconeogenesis, glicogen and fatty acids decomposition

Hormonal control over PFK1 and pyruvate kinase PFK2 FBPase-2 PFK2 FBPase-2 ATP ADP ATPADP H2OH2O PiPi Active P H2OH2OPiPi Protein kinase-1 Protein phosphatase-1 Glucagon Insulin Activation of gluconeogenesisActivation of glycolysis Fructose-1-P Fructose-2,6-diP Fructose-1-P

Sequence of reactions + Pyruvate Glucose Aerobic and anaerobic glycolysis ATP production Anaerobic Glycolisis CoA 2x + CO 2 Aerobic Glycolisis TCA, ETC, OP Lactate 32 ATP2 ATP

In mammals Anaerobic (lactic acid fermentation Aerobic OxidationAnaerobic (alcoholic fermentation) LactatePyruvateEthanol Pyruvate fate

Pyruvate dehydrogenase NAD + NADH Pyruvate to AcCoA PDH

PDH regulation

Pyruvate to lactate

Pyruvate to oxaloacetate Pyruvate kinase reaction is irreversible In cytosol glucose and oxaloacetate can not be synthesized from pyruvate Oxaloacetate is TCA intermediate If unsufficient can be synthesized from pyruvate in mytochondria Catalyzed by pyruvatecarboxylase

Pyruvate carboxylase Aspartate (transamination) Citrate (TCA cycle) Phosphoenolpyruvate (gluconeogenesis)