Lec5 Gluconeogenesis Biochemistry 2024/2025 PDF

Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Gluconeogenesis Formation of glucose from non-carbohydrate sources Gluconeogenesis is a metabolic pathwa...

Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Gluconeogenesis Formation of glucose from non-carbohydrate sources Gluconeogenesis is a metabolic pathway that leads to the synthesis of glucose from pyruvate and other non-carbohydrate precursors, even in non-photosynthetic organisms. It occurs in all microorganisms, fungi, plants and animals, and the reactions are essentially the same, leading to the synthesis of one glucose molecule from two pyruvate molecules. Therefore, it is in essence glycolysis in reverse, which instead goes from glucose to pyruvate, and shares seven enzymes with it. Some tissue, such as the Brain, R.B.C, kidney medulla, lens and cornea of the eye, tests and exercising muscle, require a continuous supply of glucose as a metabolic fuel.  liver glycogen can only meet these needs for 10-18hr, in the absence of the dietary intake of carbohydrate. 1 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism  During prolonged fast, hepatic glycogen stores are depleted and glucose is formed from compounds that are not carbohydrate by the gluconeogenesis.  Gluconeogenesis, occurs mainly in the cytoplasm and mitochondria of Liver ~90% and to a small degree in the kidney ~10%. During fasting, as in between meals or overnight, the blood glucose levels are maintained within the normal range due to hepatic glycogenolysis, and to the release of fatty acids from adipose tissue and Ketone bodies by the liver. Fatty acids and Ketone bodies are preferably used by skeletal muscle, thus sparing glucose for cells and tissues that depend on it, primarily red blood cells and neurons. However, after about 18 hours of fasting or during intense and prolonged exercise, glycogen stores are depleted and may become insufficient. At that point, if no carbohydrates are ingested, gluconeogenesis becomes important. Importance of gluconeogenesis And, the importance of gluconeogenesis is further emphasized by the fact that if the blood glucose levels fall below 2 m mol/L, unconsciousness occurs. The excretion of pyruvate would lead to the loss of the ability to produce ATP through aerobic respiration, i.e., more than 10 molecules of ATP for each molecule of pyruvate oxidized. 1-Maintenance of blood glucose during starvation, fasting and prolonged exercise. 2- Removal of lactic acid. 3- Removal of glycerol produced by lipolysis. The major precursors (substrates) for gluconeogenesis are: - lactate, amino acids and glycerol are the major precursors for gluconeogenesis in humans.: - 1. Lactate (Lactic acid): - is oxidized by NAD+ in a reaction catalyzed by lactate dehydrogenase (LDH), to form pyruvate. which can be converted to glucose, sources of lactate include R.B.C, and exercising muscle (in vigorous skeletal muscle activity, large amount of lactic acid produced) pass to liver through blood stream converted into pyruvate and lastly to glucose (by Cori cycle). 2 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism 2. Glucogenic amino acids: - (which form pyruvate as TCA cycle intermediates). Proteins are considered as one of the main sources of blood glucose especially after 18 hr. due to depletion of liver glycogen. The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism. Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. Dietary & muscle proteins Amino acids From degradation of muscle protein. α-keto acid: (α-keto acid are derived from the metabolism of glycogenic amino acids by deamination and converted into α-keto acid such as pyruvate oxaloacetate, and α-keto glutarate). This substance can enter the citric cycle, and form oxaloacetate, a direct precursor of phosphoenol pyruvate. Intermediates in citric acid cycle can be used for gluconeogenesis through oxaloacetate 3. Glycerol (which forms dihydroxy acetone phosphate). Glycerol, derived from hydrolysis of triacylglycerol in fat cells, is also a significant input to gluconeogenesis. glycerol Triglycerole Glycerol reacts with ATP to form glycerol -3-phosphate, which oxidized to Dihydroxy acetone phosphate. And converted to glucose. 3 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism ketogenic compounds: - Acetyl-CoA and compounds that give rise to Acetyl-CoA (Aceto acetate and Ketogenic amino acids, cannot give rise to a net synthesis of glucose. This is due to the irreversible nature of the pyruvate dehydrogenase reaction (enzyme which converts pyruvate to Acetyl-CoA). These compounds give rise instead to ketone bodies and therefore termed (Ketogenic). Pyruvate dehydrogenase Pyruvate ▬▬▬▬▬▬▬▬► acetyl CoA + CO2 NAD+ NADH+H+ Even-chain Fatty acid: - do not provide carbon for gluconeogenesis (do not produce any net glucose). β-oxidation of fatty acid provides ATP that drives gluconeogenesis. Odd-Chain Fatty acid: -The 3 carbon at the ω-end of Odd-chain fatty acid is converted to propionate. Propionate enters the TCA cycle as succinyl -CoA which forms malate, an intermediate in glucose formation. Reactions of Gluconeogenesis:- Gluconeogenesis involves several enzymatic steps that do not occur in glycolysis; thus, glucose is not generated by simple reversal of glycolysis. Seven of the reaction of glycolysis is used in the synthesis of glucose from lactate as pyruvate. However, three of reaction are irreversible and must be circumvented. By four alternate reactions that energetically favors the synthesis of glucose. These reactions, unique to gluconeogenesis 4 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Irreversible glycolytic steps bypassed Glycolysis Gluconeogenesis  Hexokinase (or Glucokinase) By (Glucose-6-phosphatase)  Phosphofructokinase By (Fructose 1,6-bisphosphatase)  Pyruvate Kinase. By (Pyruvate Carboxylase) and (Phosphoenolpyruvate carboxykinase) 5 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism First bypass reaction Carboxylation Of Pyruvate: -The first (road block) to overcome the synthesis of glucose from pyruvate is irreversible conversion of pyruvate to phosphoenol pyruvate (PEP). IN THREE STEPS: - step_1: - pyruvate (produces from lactate, alanine and other amino acids), is first converted to oxaloacetate (OAA) by the action of pyruvate carboxylase (this enzyme is found in the mitochondria of LIVER and kidney cells, but not of muscles). this enzyme requires biotin ATP. In Mitochondria Pyruvate carboxylase (PC) exists in the mitochondria of liver and kidney but absent in muscle ATP, biotin, Mn++ and CO2 are required. step_2: - Oxaloacetate cannot directly cross the mitochondrial membrane, therefore, it is converted to malate which can cross the mitochondrial membrane and be reconverted to OAA in the cytosol. (OAA, formed in the mitochondrial, must enter the cytosol, where the other enzymes of gluconeogenesis are located). Transport of Oxaloacetate into cytosol as Malate 6 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Step_3: - Decarboxylation of cytosolic OAA, by phosphoenol pyruvate carboxykinase. to form PEP, the reaction requires GTP. In cytosol First bypass step is generation of PEP from pyruvate via oxaloacetate In order to cross the mitochondrial membrane, oxaloacetate must: 1. Be reduced to malate 2. Go through the malate shuttle 3. Be reoxidized to oxaloacetate 7 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Second bypass reaction Conversion of fructose 1,6_bisphoshate to fructose-6-phosphate:- The second glycolytic reaction (phosphorylation of fructose 6-phosphate by PFK) is irreversible. Hence, for gluconeogenesis fructose 6-phosphate must be generated from fructose 1,6-bisphosphate by a different enzyme which is Fructose 1,6-bisphosphatase. Fructose 1,6-bisphosphatase presents in liver and kidney. This reaction is also irreversible. Phosphofructokinase (In Glycolysis): Fructose-6-P + ATP  fructose-1,6-bisP + ADP Fructose-1,6-bisphosphatase (In Gluconeogenesis): fructose-1,6-bisP + H2O  fructose-6-P + Pi Third bypass reaction Conversion of Glucose 6_Phosphate to Glucose: - Because the hexokinase reaction is irreversible, the final reaction of gluconeogenesis is catalyzed by Glucose 6-phosphatase. Glucose 6-phosphate, release inorganic phosphate (Pi) which produces free glucose that enters the blood, by the action of G -6-phosphatase.  Glucose -6-phosphatase, the enzyme is involved in both gluconeogenesis and glycogenolysis. This enzymes like pyruvate carboxylase, occurs in liver and kidney but not in brain and muscle. Thus, muscle cannot provide blood glucose by gluconeogenesis, also G-6-P derived from muscle glycogen cannot be dephosphorylated to yield free glucose. The glucose produced by gluconeogenesis in the liver, is delivered by the bloodstream to brain and muscle. Hexokinase or Glucokinase (In Glycolysis): Glucose + ATP  glucose-6-phosphate + ADP Glucose-6-Phosphatase (In Gluconeogenesis): Glucose-6-phosphate + H2O  glucose + Pi 8 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism The reaction common to glycolysis and gluconeogenesis: - In gluconeogenesis, the equilibrium of the seven reversible reactions of glycolysis are pushed in favor of glucose synthesis as a result of the essentially irreversible formation of PEP, F.6.P and glucose catalyzed by the gluconeogenic enzymes. Fig: The key reactions of gluconeogenesis from precursors red arrows indicate steps that differ from those of glycolysis Note: that both Glycolysis and Gluconeogenesis are energetically favorable under physiological conditions and therefore both ~ irreversible processes 9 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Summary of Gluconeogenesis Reactions: - 2Pyruvate+4ATP+2GTP+2NADH+2H++6H2O→Glucose+2NAD++4ADP+2GDP+6Pi+6H+ The synthesis of 1 mole of glucose from 2 moles of pyruvate requires the energy equivalent of about 6 moles of ATP. The Cori Cycle Lactate and glucose shuttle between active muscle/RBC and liver 6 ATP GN (glucagon/insulin reg.) 2 Liver gluconeogenesis buffers the blood glucose for use by muscle, RBC’s and brain (120 g/day) RBCs *Note: the brain fully 2 ATP GL oxidizes glucose, so it does not funnel back lactate The Alanine Cycle The liver can also use the amino acid Alanine similarly to Lactate Following transamination to pyruvate, gluconeogenesis allows the liver to convert it to glucose for secretion into the blood 11 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism Regulation of gluconeogenesis: - The moment-to-moment regulation of gluconeogenesis is determined Primarily by: - A. Circulating level of glucagon. B. The availability of gluconeogenesis substrates. A) Glucagon: - The major hormones that regulated blood glucose are insulin and glucagon. The glucagon is pancreatic islet hormone stimulates gluconeogenesis by 2mechanisms 1. Changes in allosteric effectors: - glucagon lowers the level of fructose 2,6-bisphoshate resulting activation of fructose 1,6-bisphosphatase and inhibition of phosphofructokinase (Regulatory enzyme of glycolysis). 2. Covalent modification of enzyme activity: - Glucagon, via an elevation in cAMP level and cAMP-dependent kinase activity, stimulates the conversion of pyruvate kinase (one of the regulatory enzymes in glycolysis) to its inactive (phospho related form) →↓conversion of PEP to pyruvate. 11 Biochemistry Prof. Dr. Amina Al-Obaidi 2024/ 2025 Lec5 - Carbohydrate metabolism B) Substrate availability: - The availability of gluconeogenesis precursors particularly glucogenic amino acid, markedly influences the rate of hepatic glucose synthesis. Decrease level of insulin favor mobilization of amino acids from muscle protein and provide the carbon skeleton for gluconeogenesis. C)Allosteric activation by acetyl-CoA: - Allosteric activation of hepatic pyruvate carboxylase by acetyl-CoA occurs during starvation, as result of excessive lipolysis in adipose tissue, the liver is flooded with fatty acid. The rate of formation of acetyl-CoA by β-oxidation of these F.A, exceeds the capacity of the liver to oxidize it to CO2 and H2O as a result, acetyl-CoA accumulates and lead to activation pyruvate carboxylase. Pyruvate can go “up” or “down” depending upon energy needs Reciprocal regulation by ATP/AMP  AMP inhibits Fructose-1,6-bisphosphatase but activates Phosphofructokinase  ATP inhibits Phosphofructokinase but activate Fructose-1,6- bisphosphatase In high ATP/AMP ratio: stimulate gluconeogenesis In low ATP/AMP ratio: stimulate glycolysis 12