BIOCHEMISTRY: TCA CYCLE, GLUCONEOGENESIS, ENZYME KINETICS

Gluconeogenesis

Gluconeogenesis Definition: The formation of glucose from noncarbohydrates, such as protein or fat.

· Gluconeogenesis Overview

· Some tissues require a constant supply of glucose to remain viable. These include:

· CNS

· RBCs

· Exercising muscle

· Sources of glucose

· Carbohydrate rich meal

· Provides source of glucose up to 3-4 hrs postprandial

· Liver stores glycogen

· Glycogenolysis supplies glucose for up to 10-18 after eating

· Gluconeogenesis (During prolonged fasting)

· Glucose synthesized from substrates, including:

· Lactate, pyruvate

· Glycerol from triglycerides

· α-ketoacids from glucogenic AA’s

· Glucose synthesized by gluconeogenic tissues

· First 12 hours of fasting

· Liver sources 90%

· Kidneys source 10%

· After first 12 hours of fasting

· Kidneys source 40%

· Substrates for gluconeogenesis

· All intermediates of glycolysis & TCA cycle

· Glycerol

· Liberated from triacylglycerols stored in adipose tissue by hydrolysis

· Lactate

· Released into blood by exercising muscle and RBCs which lack mitochondria

· Cori cycle converts lactate back to glucose

· Blood glucose converted to lactate by cells

· Lactate imported into hepatocyte and converted to glucose

· Glucose released by hepatocyte into circulation

· Glucogenic AA’s

· From catalysis of tissue proteins

· Primary source of glucose during a fast

· α-Keto acids derived from deamination of glucogenic AA’s

· Enter TCA cycle to form oxaloacetate or enter as α-Ketoglutarate

· Note:

· No net synthesis of glucose from Acetyl-CoA

· Reaction catalyzed by pyruvate dehydrogenase irreversible

· Reactions unique to gluconeogenesis:

· Gluconeogenesis is not a simple reversal of glycolysis

· Three reactions of glycolysis are irreversible

· These three reactions are bypassed by four unique reactions of gluconeogenesis for production of glucose

· Unique reactions #1 & #2 bypass pyruvate kinase

· Unique reaction #3 bypasses PFK-1

· Unique reaction #4 bypasses hexokinase

· #1) Carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase

· Biotin is coenzyme

· Reaction occurs in mitochondria of liver and kidney

· Oxaloacetate transported to cytosol

· Gluconeogenic enzymes located in cytoplasm

· Oxaloacetate not permeable to inner mitochondrial membrane

· Reduced to malate by mitochondrial malate dehydrogenase

· Malate transported to cytoplasm

· Converted to oxaloacetate by cytoplasmic malate dehydrogenase

· Allosteric regulation

· Pyruvate carboxylase

· Activated by acetyl-CoA

· Deactivated by low levels of acetyl-CoA

· Pyruvate dehydrogenase is inhibited by acetyl-CoA

· When PDH active, acetyl-CoA used for TCA cycle energy production

· #2) Oxaloacetate converted to PEP by PEP carboxykinase (PEPCK)

· Driven by GTP hydrolysis

· Occurs in cytoplasm

· #3) Dephosphorylation of fructose 1,6-bisphosphate to Fructose 6-phosphate by fructose 1,6-bisphosphatase

· Glycolysic reactions are reversed until reaction reaches fructose 1,6-bisphosphate

· This reaction is the primary regulatory step in gluconeogenesis

· Allosteric regulation of fructose 1,6-bisphosphatase

· Regulated by intracellular energy levels

· Activated by ATP

· Inhibited by AMP (energy poor)

· Regulated by sugar levels of the blood

· When sugar levels increase and the hormone glucagon decreases, intracellular levels of Fructose 2,6-bisphosphate increase

· Levels of fructose 2,6-bisphosphate reflect blood glucose levels

· Levels of dependent on blood glucagon

· Fructose 1,6-bisphosphatase allosterically inhibited by fructose 2,6-bisphosphate

· PFK-1 activated by fructose 2,6-bisphosphate

· Allows for reciprocal control and prevents a futile cycle

· #4) Dephosphorylation of Glucose 6-P to glucose by glucose 6-phosphatase

· Dephosphorylation required for cell export, can’t export phosphorylated glucose

· Reaction produces glucose for export

· Activity of enzyme primarily found in ER of liver and kidney cells

· Glucose 6-phosphatase activity in ER only in gluconeogenic cells

· Hepatocytes and kidney release glucose into blood

· Note: Muscle lacks glucose 6-phosphatase

· Cannot supple blood with glucose by gluconeogenesis

· Glucose 6-P from glycogen can’t be dephosphorylated to yield glucose