Gluconeogenesis 12/06/ :25

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1 Gluconeogenesis 12/06/ :25 Tissues require constant supply of glucose as metabolic fuel Eg: Brain, RBC s Kidney Medulla, Lens+Cornea of Eye, Testes and Exercising Muscle. Liver Glycogen meets needs for 10-18hrs in absence of dietary intake of Carbs. During prolonged fast, Liver glycogen stores depleted. Glucose is formed from non carb precursors o For example: Lactate Pyruvate Glycerol (from triacylglycerols) α-keto acids (from catabolism of glucogenic aa) You can t form glucose by reversing glycolysis as equilibrium of Glycolysis favours pyruvate formation Gluconeogenesis Requires both mitochondrial & cytosolic enzymes. During overnight fast, 90% of gluconeogenesis occurs in liver. 10% in Kidneys.

2 During prolonged fast, Kidneys = major site for glucose at 40% production SUBSTRATES:

3 1. Glycerol o released during hydrolysis of triacylglycerols in Adipose Tissue. o Delivered by blood to liver o Glycerol then phosphorylated by glycerol Kinase to glycerol Phosphate o Glycerol Phosphate then oxidised by Glycerol Phosphate Dehydrogenase to Dihydroxyacetone phosphate ( an intermediate of Glycolysis) o Adipocytes can t phosphorylate glycerol because they lack glycerol kinase. 2. Lactate o released into blood by excercising skeletal muscle or cells that lack mitochondria (RBC s) o Cori Cycle- blood-born glucose is converted by muscle to lactate, which diffuses into blood. Lactate then taken by liver and converted back to glucose. This is then released into circulation 3. AA o AA from hydrolysis of tissue protein = maj source of glucose during a fast. o Metabolism of glucogenic AA generates α-keto acids. Eg. α- KG which enters TCA and forms OAA as direct precursor of PEP (phosphoenolpyruvate) o N.b. Acetyl coa + compounds cant give rise to net synth of glucose- due to irreversible nature of Pyruvate Dehydrogenase (PDH) reaction which converts Pyruvate to Acetyl-CoA. o Instead they produce ketone bodies, hence called ketogenic Reactions unique to Gluconeogensis 7 reactions in glycolysis are reverisible therefore used in synthesis of glucose from lactate or pyruvate. 3 reactions are irreversible and therefore must be got around by 4 alternative reactions- they energetically favour synthesis of glucose. 1. Carboxylation of Pyruvate.

4 o Must bypass irreversible conversion of PEP to pyruvate by Pyruvate Kinase. Pyruvate is carboxlated by Pyruvate Carboxylase to OAA Then converted to PEP by action of PEP carboxyinase a. Biotin (coenzyme) ú pyruvate carboxylase needs BIOTIN bound to ε-amino group of lysine residue in the enzyme ú hydrolysis of ATP drives formation of enzymebiotin-co2 intermediateú then carboxylates pyruvate to form OAA. Occurs in the Liver Mitochondria and Kidney Cells. 2 purposesú provide important substrate for gluconeogenesis ú provide OAA that replenishes TCA intermediates that are depleted. Muscle cells contain pyruvate carboxylase but use OAA for replenishment anaplerotic purpose- don t synthesise glucose o B. Allosteric Regulation: ú Pyruvate carboxylase allosterically activated by acetyl CoA ú High levels of Acetyl CoA in mitochondria signal that inc synth of OAA is required. Occurs during fasting. OAA used for synthesis of glucose by liver and Kidney ú Low levels of Acetyl CoA= pyruvate carboxylase being inactive ú Pyruvate is oxidised by PDH complex to produce acetyl CoA- then used in TCA. 2. Transport of OAA to CYTOSOL o OAA converted to PEP for gluconeogenesis to continue o Enzyme for this is in mitochondria and cytosol o OAA cant travel across inner Mitochondrial membrane

5 Needs to be reduced to Malate by Malate Dehydrogenase (MD). o Malate transported from Mitochondria to cytosol o Reoidised to OAA by cytosolic MD (NAD+ produced) o NADH used in reduction of 1,3, BPG to G3P 3. Decarboxylation of Cytosolic Oxaloacetate. o OAA decarboxylated + phosphorylated to PEP o In CYTOSOL o By PEP Carboxykinase (PEPCK) o Driven by hydrolysis of GTP o This produces energy favourable pathway from Pyruvate to PEP o Then follows reverse glycolysis to Fructose 1,6- Bisphosphate 4. Dephosphorylation of Fructose 1,6-Bisphosphate o Hydrolysis of Fructose 16 Bisphosphate by fructose 1,6 bisphosphatase o Found in liver and kidney o Bypasses irreversible phosphofructokinase-1 (PFK-1) rxn. o Produces Fructose 6, phosphate o IMPORTANT REGULATION OF GLUCONEOGENESIS A. Regulation by energy levels in cell: ú Fructose 1,6-bisphosphatase inhibited by high levels of AMP ú AMP signals energy poor state in cell. ú High levels of ATP and low AMP stimulate gluconeogenesis B. Regulation by Fructose 2,6 Bisphosphate: ú Fructose 1,6 Bisphosphatase is inhibited by Fructose 2,6-Bisphosphate. (ALLOSTERIC EFFECTOR) ú Influenced by insulin:glucagon ratio. ú When Glucagon is high, effector is made ú Phosphatase = active 5. Dephosphorylation of G6P

6 o Hydrolysis of G6P by G6P-phatase o Bypasses irreversible hezokinas/glucokinase reaction o Provides energetically favourable pathway for formation of free glucose o LIVER +KIDNEY = only organs that release free glucose from G6P Requires 2 proteins ú 1. G6P translocase (transports g6p across ER membrane ú 2. G6P-phatase (removes phosphate making free glucose) o ER membrane proteins required for final step of glycogen degradation o CLINICAL: type 1a and Type 1b glycogen storage disease Caused by deficiency in phosphatase and transferase (respectively) Characterised by sever fasting hypoglycaemia Because free glucose is unable to be produced from either gluconeogenesis/glycogenolysis. o Specific Glucose transporters (GLUTS) responsible for moving glucose into cytosol then blood. o G6P translocase moves Pi out of ER as it moves G6P in. 6. Summary Of reactions of Glycolysis and Gluconeogenesis. o Irreverisble reactions of glycolysis o Catalysed by hexokinase/glucokinase, Pfk-1 and PK o Circumvented by o G6P-phatase, Fructose 1,6 Bisphosphatase and Pyruvate Carboxylase/PEP-carboxykinase Per molecule of Glucose: o Gluconeogenesis of pyruvate couples cleavage of 6 high energy Phosphate bonds o Oxidation of 2 NADH