Gluconeogenesis.pdf

GLUCONEOGENESIS SHARI R. BABU [email protected] DEPT. OF PHYSIOLOGICAL SCIENCES SCHOOL OF MEDICINE, UNZA Shari Babu  OVERVIEW OF GLUCONEOGENESIS Some tissues, such as the brain, red blood cells, kidney medull...

GLUCONEOGENESIS SHARI R. BABU [email protected] DEPT. OF PHYSIOLOGICAL SCIENCES SCHOOL OF MEDICINE, UNZA Shari Babu  OVERVIEW OF GLUCONEOGENESIS Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for only 10–18 hours in the absence of dietary intake of carbohydrate. The brain uses almost 70% of the total glucose produced by liver during normal fasting. During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is formed from non-carbohydrate precursors. Gluconeogenic precursors include intermediates of glycolysis and the tricarboxylic acid (TCA) cycle, as well as glycerol, lactate, and the a-keto acids obtained from the transamination of glucogenic amino acids. Shari Babu ALANINE LACTATE OXALOACETATE Aspartate, α- Ketoglutarate, PYRUVATE Fumarate, Succinyl-CoA GLUCONEOGENESIS GLUCOGENIC AMINO ACIDS DIHYDROXYACETONE PHOSPHATE GLYCEROL FATTY ACID TRIACYLGLYCEROL Shari Babu  The formation of glucose does not occur by a simple reversal of glycolysis. Instead, glucose is synthesized by a special pathway that requires both mitochondrial and cytosolic enzymes. During an overnight fast, approximately 90% of gluconeogenesis occurs in the liver, with the kidneys providing 10% of the newly synthesized glucose molecules. However, during prolonged fasting, the kidneys become major glucose- producing organs, contributing an estimated 40% of the total glucose production. Shari Babu REACTIONS UNIQUE TO GLUCONEOGENESIS Seven out of the ten glycolytic reactions are reversible and use the same enzymes in the synthesis of glucose from pyruvate via gluconeogenesis. However, three of the glycolytic reactions are irreversible and must be circumvented by four alternate reactions that energetically favors the synthesis of glucose. The three irreversible reactions that needs to be circumvent are the glycolytic reactions catalyzed by hexokinase, phosphofructokinase-1 and pyruvate kinase. Shari Babu  Pink arrows Blue arrows indicate indicate glycolytic the gluconeogenic pathway pathway Shari Babu FORMATION OF PHOSPHOENOLPYRUVATE (PEP) FROM PYRUVATE Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two reactions. 1. Carboxylation of pyruvate: Pyruvate Carboxylase catalyzes an ATP- requiring reaction in which the vitamin biotin is the coenzyme: Pyruvate + HCO3- + ATP  Oxaloacetate + ADP + Pi Pyruvate carboxylase is allosterically activated by acetyl CoA and inhibited by ADP. Shari Babu Transport of oxaloacetate (OAA) to the cytosol OAA must be converted to PEP for gluconeogenesis to continue. The enzyme that catalyzes this conversion is found in both the mitochondria and the cytosol in humans. The PEP that is generated in the mitochondria is transported to the cytosol by a specific transporter, whereas for PEP to form in the cytosol it requires the transport of OAA from the mitochondria to the cytosol. OAA is unable to directly cross the inner mitochondrial membrane; it must first be reduced to malate by mitochondrial malate dehydrogenase. Malate can be transported from the mitochondria to the cytosol, where it is reoxidized to oxaloacetate by cytosolic malate dehydrogenase as NAD is + reduced. Shari Babu CYTOSOL MITOCHONDRIAL MATRIX NAD+ NADH MALATE OXALOACETATE Malate Dehydrogenase Malate Transporter 2. Decarboxylation of cytosolic oxaloacetate: Oxaloacetate is decarboxylated and phosphorylated to PEP in the cytosol by PEP-carboxykinase. The reaction is driven by hydrolysis of GTP. Oxaloacetate + GTP  Phosphoenolpyruvate + GDP + CO2 Then, PEP is acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate. PEPCK is inhibited by ADP. Shari Babu DEPHOSPHORYLATION OF FRUCTOSE 1,6-BISPHOSPHATE Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase bypasses the irreversible phosphofructokinase-1 reaction. Fructose 1, 6-bisphosphate + H2O  Fructose 6-phosphate + Pi This reaction is an important regulatory site of gluconeogenesis. Fructose 1,6-bisphosphatase is inhibited by elevated levels of AMP while high levels of ATP and citrate stimulate gluconeogenesis, an energy-requiring pathway. Fructose 1,6-bisphosphatase, found in liver and kidney, is also inhibited by fructose 2,6-bisphosphate. Shari Babu  SB7 Fructose-2,6-bisphosphate is synthesized & degraded by a bi-functional enzyme (PFK2/F2,6BPase) that includes 2 catalytic domains: Phosphofructokinase-2 (PFK2) domain catalyzes: Fructose-6-phosphate + ATP  Fructose-2,6-bisphosphate + ADP Fructose-2, 6 Bisphosphatase (F2,6BPase) domain catalyzes: Fructose-2,6-bisphosphate + H2O  Fructose-6-phosphate + Pi Shari Babu Slide 12 SB7 Phosphofructokinase 1 (PFK), which catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6- bisphosphate, is a key regulatory step in the glycolysis. When glucose level is low, glucagon is released into the bloodstream, triggering a cAMP signal cascade. In the liver Protein kinase A inactivates the PFK-2 domain of the bifunctional enzyme via phosphorylation. The F-2,6-BPase domain is then activated which lowers fructose 2,6-bisphosphate (F-2,6-BP) levels. Because F-2,6-BP normally stimulates phosphofructokinase-1(PFK1) and inhibits F-1,6BPase, the decrease in its concentration leads to the inhibition of glycolysis and the stimulation of gluconeogenesis, respectively. Shari Babu, 22/03/2021  GLUCAGON AND/OR EPINEPHRINE cAMP-dependent Protein Kinase (PKA) + Phosphorylation - P PFK-2 PFK-2 F-2,6-BPase F-2,6-BPase P - + Fructose-2, 6-Bisphosphate Fructose-6-Phosphate + Pi Decreased levels of F-2,6-BP means inhibition of F-1,6-BPase is lifted. This increases the rate of Gluconeogenesis.  INSULIN Protein Phosphatase-1 Dephosphorylation + - P P F-2,6-BPase F-2,6-BPase P PFK-2 PFK-2 P - + Fructose-6-Phosphate + ATP Fructose-2, 6- Bisphosphate + ADP Increased levels of F-2,6-BP activates PFK-1. This increases the rate of Glycolysis. In presence of glucagon or PKA action In presence of insulin, epinephrine, cAMP levels glucose will enter cells. are increased. F2,6BPase The levels of fructose-6- cAMP-dependent protein phosphate will increase. kinase (PKA) is activated. PFK2 Insulin decreases the levels of cAMP. PKA activates F2,6BPase domain via phosphorylation. Activate Insulin also activates Inactive protein phosphatase-1 (PP1). Once activated, F2,6BPase will breakdown PP1 dephosphorylates F2,6BP and lift its inhibition and activates PFK2. PP1 action on F1,6BPase. This results in increased PKA inactivates PFK2 via levels of F26BP which phosphorylation preventing activates PFK1. formation of F2,6BP. Also, PP1 dephosphorylates and Gluconeogenesis inactivates F26BPase. stimulated. Glycolysis is stimulated. SB8 DEPHOSPHORYLATION OF GLUCOSE 6-PHOSPHATE Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the irreversible hexokinase reaction. Glucose 6-phosphate + H2O  Glucose + Pi Release of free glucose requires two proteins: glucose 6-phosphate translocase, which transports glucose 6-phosphate across the ER membrane, and the ER enzyme, glucose 6-phosphatase, which removes the phosphate, producing free glucose. Shari Babu Slide 16 SB8 The deficiency of any one of the four unique gluconeogenic enzymes i.e., pyruvate carboxylase, phoshoenolpyruvate carboxykinase, fructose 2,6-bisphosphatase and glucose-6-phosphatase, can lead to hypoglycemia during periods of fasting. Especially overnight fasting when the liver glycogen reserves have been depleted. Shari Babu, 22/03/2021 SB9 HORMONAL REGULATION OF GLUCONEOGENESIS Glucagon lowers the level of fructose 2,6-bisphosphate, resulting in activation of fructose 1,6-bisphosphatase and inhibition of PFK-1, thus favouring gluconeogenesis. Glucagon elevates cAMP level and cAMP-dependent protein kinase activity, which stimulates the conversion of pyruvate kinase to its inactive (phosphorylated) form. This decreases the conversion of PEP to pyruvate, which has the effect of diverting PEP to the synthesis of glucose. Glucagon increases the transcription of PEP-carboxykinase gene, increasing the levels of the enzyme. Insulin increases the activity of phosphofructokinase-1, pyruvate kinase and the levels of F-2,6-BP. Shari Babu Slide 17 SB9 So in the liver and kidneys, glycolysis and gluconeogenesis do not occur simultaneously. But it is possible for gluconeogenesis to occur in the liver while glycolysis occurs in extra-hepatic tissues. Shari Babu, 22/03/2021 CLINICAL SIGNIFICANCE In the absence of glucose-6-phosphatase, gluconeogenesis is impaired, and this results in fasting hypoglycemia. This can occur in Von Gierke’s disease when the enzyme is deficient. Diabetes is either the result of impaired insulin production or decreased insulin sensitivity. Insulin is an inhibitor of gluconeogenesis. In the absence of insulin, gluconeogenesis occurs at a rapid rate, exacerbating hyperglycemia. Alcoholism can induce hypoglycemia. Alcohol is oxidized to acetaldehyde and then to acetate in the liver. These reactions increase the level of NADH. The high levels of NADH favors the formation of lactate from pyruvate. This lowers the level of pyruvate that can enter gluconeogenesis, which causes hypoglycemia. As a result, heavy ethanol consumption can lead to both lactic acidosis and hypoglycemia. SB10 The Cori Cycle Lactate is formed by active skeletal muscle when the rate of glycolysis exceeds the rate of oxidative metabolism. Lactate is readily converted into pyruvate by the action of lactate dehydrogenase. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). Lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle. Shari Babu Slide 19 SB10 During vigorous muscular activity, epinephrine is released and it stimulates hepatic gluconeogenesis. Shari Babu, 22/03/2021 Cori Cycle Shari Babu