Gluconeogenesis PDF

Gluconeogenesis Gluconoegenesis is the biosynthesis of new glucose from non-carbohydrate substrates. In the absence of dietary intake of carbohydrate liver glycogen can meet these needs for only 10 to 18 hours During prolonged fast hepatic glycogen stores are depleted and glucose is formed from p...

Gluconeogenesis Gluconoegenesis is the biosynthesis of new glucose from non-carbohydrate substrates. In the absence of dietary intake of carbohydrate liver glycogen can meet these needs for only 10 to 18 hours During prolonged fast hepatic glycogen stores are depleted and glucose is formed from precursors such as lactate, pyruvate, glycerol and keto acids. Approximately 90% of gluconeogenesis occurs in the liver whereas kidneys provide 10 % of newly synthesized glucose molecules. The kidneys thus play a minor role except during prolonged starvation when they become major glucose producing organs. 1 2 Reactions Unique to Gluconeogenesis Seven of the reactions of glycolysis are reversible and are used in the synthesis of glucose from lactate or pyruvate. However three of the reactions are irreversible and must be bypassed by four alternate reactions that energetically favor the synthesis of glucose. 1) Carboxylation of Pyruvate In gluconeogenesis, pyruvate is first carboxylated by pyruvate Carboxylase to oxaloacetate (OAA). where it is converted to Phosphoenolpyruvate (PEP) by the action of PEP carboxykinase Note: pyruvate carboxylase is found in the mitochondria of liver and kidneys, but not in muscle 1. Biotin is a coenzyme of pyruvate carboxylase derived from vitamin B6 covalently bound to the apoenyme through an ε-amino group of lysine forming the active enzyme. 3 2. Allosteric regulation Pyruvate carboxylase is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA may signal one of several metabolic states in which the increased synthesis of oxaloacetate is required. For example, this may occur during starvation where OAA is used for the synthesis of glucose by gluconeogenesis At low levels of acetyl COA, pyruvate carboxylase is largely inactive and pyruvate is primarily oxidized in the TCA cycle B. transport of Oxaloacetate to the Cytosol Oxaloacetate, formed in mitochondria, must enter the cytosol where the other enzymes of gluconeogenesis are located. However, oxaloacetate is unable to cross the inner mitochondrial membrane directly. It must first be reduced to malate which can then be transported from the mitochondria to the cytosol. In the cytosol, Malate is reoxidized to oxaloactate 2) Decarboxylation of Cytosolic Oxaloacetate. Oxaloacetate is decarboxylated and phosphorylated in the cytosol by PEP- carboxykinase. The reaction is driven by hydrolysis of GTP 4 The combined action of pyruvate carboxylase and PEP carboxykinase provides an energetically favorable pathway from pyruvate to PEP. PEP then enters the reversed reactions of glycolysis until it forms fructose 1, 6- bisphosphate. Phosphoenolpyruvate Carboxykinase (PEPCK) Deficiency. PEPCK, an essential marker for gluconeogenesis, catalyzes the conversion of phosphoenolpyruvate to oxaloacetate. There are different isoforms of PEPCK that is, PEPCK1 (cytosolic) and PEPCK2 (mitochondrial). PEPCK1 is regulated by the mitochondrial GTP-dependent pathways, including hormones, substrate supply, and purine nucleotides. Although this enzyme helps in gluconeogenesis, it has an important role in glyceroneogenesis where it helps in the synthesis of glyceride-glycerol from glucose or glycerol in adipose tissue and liver. It plays another role in citric acid cycle and helps in the entry of carbon skeletons to amino acids. PEPCK2 is known for its ability to fix carbon dioxide by converting pyruvate into oxaloacetic acid. Moreover, PEPCK2 is principally involved in gluconeogenesis, providing the cytosolic NADH through its conversion to pyruvate from lactic acid. This enzyme deficiency is an autosomal recessive disorder whose phenotype is not 5 expressed clearly. Lactic acidosis and hypoglycemia are the primary symptoms for PEPCK deficiency. Reye syndrome develops due to inhibition of gluconeogenesis which, in turn, is due to PEPCK enzyme deficiency. The specific symptoms of PEPCK deficiency are associated with lactic acidosis, hypoglycaemia, hepatomegaly, glucagon insensitivity, failure to thrive, Fanconi syndrome, developmental delay, hypotonia, and massive fat deposition in liver and kidneys. Treatment of PEPCK deficiency includes the maintenance therapy similar to FBPase deficiency to treat acute attacks (glucose and bicarbonate infusions). There is no specific treatment other than maintaining normoglycaemia and correcting metabolic disorders. C- Dephosphorylation of fructose 1, 6 bisphosphate Hydrolysis of fructose 1, 6-bisphosphate by fructose 1, 6-bisphosphatase passes the irreversible PFK- 1 reaction and provides energetically favorable pathway for the formation of fructose 6-phosphate. This reaction is an important regulatory site of gluconeogenesis, 1. Regulation by energy levels within the cell Fructose1, 6 bisphatase is inhibited by elevated levels of AMP, which signal an energy poor state in the cell 6  Conversely high levels of ATP and low concentrations of AMP stimulate gluconeogensis Fructose-1,6-bisphosphatase (FBPase) Deficiency. FBPase is an unique enzyme in the gluconeogenetic pathway, regulated via alteration of the active (R) and inactive (T) conformational isomeric states, which catalyzes the magnesium dependent reversible production of fructose- 1,6-bisphosphate from fructose-6- phosphate and inorganic phosphate. The molecular weight of human FBPase is 36.7KDa and consists of four identical subunits of one substrate and one allosteric site. FBPase activity is regulated by fructose-2,6-bisphosphate (binds to substrate site) and adenosine monophosphate (binds to allosteric site). This enzyme is encoded by the FBP1 gene in liver and kidney at 9q22.2 and q22.3 chromosomal site. FBPase deficiency is a metabolic recessive disorder in the liver that is characterized by the life-threatening episodes of hyperventilation, hypoglycemia, apnoea, lactic acidosis, and ketosis. The diagnosis of FBPase enzyme deficiency was determined through spectrophotometric and load tests (radiochemical) in liver, kidney, and jejunum. Calcitriol stimulated FBP1 gene expression is similar to expression of vitamin D receptor. The measurement of FBPase deficiency is observed in leucocytes. Later, similar activity is determined in monocytes where stimulation with calcitriol resulted in four- to six fold enhancement of activity. Further immunoblotting technique revealed the presence of enzymes in monocytes but not in lymphocytes. Moreover, both clinical symptoms and mutation analysis are the common methods for FBPase activity. In addition, activity assessment of liver tissue is generally used for a reliable diagnosis. 7 Glucose (10–12mg/kg/minute, newborns) and bicarbonate (200mmol/24 h) are given to control hypoglycemia and acidosis. Starch and gastric drip are frequently given during treatment but not sucrose, sorbitol, fructose, fat (20–25%), and protein (10%) E. Dephosphorylation of glucose 6.phosphate Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the irreversible hexokinase reaction provides energetically favorable pathway for the formation of free glucose, Glucose 6-phosphatase like pyruvate carboxylase, occurs in liver and kidney, but not in muscle. Thus muscle cannot provide blood glucose from muscle glycogen. Glucose-6-phosphatase (G6Pase) Deficiency G6Pase helps in the formation of glucose-6-phosphate from glucose in the lumen of endoplasmic reticulum (ER). Here in, the enzyme is a part of the multicomponent system, including several integral membrane proteins, G6Pase catalytic subunit (G6PC), a regulatory Ca2+ binding protein, and glucose- 6-phosphatase translocase (G6PT). G6Pase activity is restricted to the various gluconeogenic tissues like liver, kidney, small intestine, and 𝛽-cells of the endocrine pancreas. G6Pase enzyme is encoded by G6PC1, G6PC2, and G6PC3 genes which are responsible for metabolic disorders. G6PC1 is expressed in the liver, kidney, and small intestine, whereas G6PC2 is expressed in the pancreas and G6PC3 is expressed ubiquitously in the human body. G6PC1 and G6PC3 are located on the 17q21 chromosome and G6PC2 is on the 2q31 chromosome. The cytosolicglucose-6-phosphate is transported to ER through SLC37A4 encoded gene. 8 Deficiency of G6Pase activity in liver, kidney, and intestinal mucosa with excessive accumulation of glycogen in these organs leads to glycogen storage disease (GSD) type 1 (Von Gierke’s disease). The latter is considered as acute metabolic disorder preferably characterized by hypoglycemia. There are two main types of glycogen storage diseases: the first is due to a defect in G6PC, called GSD type 1a, and the second one is due to the defect in G6PT, called GSD type 1b. GSD-1a patients are clinically diagnosed with prompt induced hypoglycemia and hyperlactacidemia in the neonatal period. Protruded abdomen due to pronounced hepatomegaly is the first symptom developed around 3months of age. Moreover, the other biological hallmarks are hyperlipidemia, hyperuricaemia, round doll-like face, developmental delay, and late onset of puberty. The clinical signs are chronic acidosis and hypertriglyceridemia which led to the development of osteopenia and enlarged kidneys. Long term complications may be the hepatocellular adenomas, renal complications, hyperuricaemia, and severe hypertriglyceridemia which may cause risk of pancreatitis and pulmonary hypertension. In GSD-1b patients along with these symptoms, patients are also diagnosed with neutropenia, which is responsible for development of Crohn’s disease. In the recent studies, the antibacterial flagell in antibodies (anti-CBir1) detection in GSD-1b patients is another indication of Crohn’s disease and this antibody level increased during disease state. In GSD-1b patients, splenomegaly is more common along with hepatomegaly, which is rarely found in GSD-1a patients. Previously, liver biopsy was the main diagnosis for the detection of G6Pase disorder. Recent advances in molecular biology involve DNA based diagnostic tests and genes cloning and G6Pase mutation database helps in diagnosis. 9 Moreover, measurement of granulocyte colony-stimulating factor (GCSF) is an important parameter for GSD-1b diagnosis, as G-CSF may increase the number and improve the function of circulating neutrophils, and G-CSF may improve the symptoms of Crohn-like inflammatory bowel disease in individuals with GSD-1b. Corn starch and other carbohydrates are the primary treatment for G6Pase deficiency. It is also necessary to normalize other physiological parameters during disease state of G6Pase deficiency. All opurinol and angiotensin converting enzyme (ACE) inhibitors are used as supplementary drug to lower the uric acid and microalbuminuria. Adjunct therapy during G6Pase deficiency includes lipid lowering drugs and potassium citrate. Liver transplantation in the patient with GSD- 1a can be performed if dietary therapy becomes unresponsive to hepatocellular adenoma and tumors. Bone marrow transplantation can be undertaken for the patients with GSD-1b related myeloid deficiencies. Substrates for Gluconeogenesis Gluconeogenic precursors are molecules that can give rise to a net synthesis of glucose. They include all the intermediates of glycolysis and the citric acid cycle. Glycerol, lactate, and the α-keto acids obtained from the deamination of glucogenic amino acids are the most important gluconeogenic precursors. A. Gluconeogenic Precursors 1. Glycerol is released during hydrolysis of triacylgycerol in adipose tissue and is delivered to the liver. Glycerol is phosphorylated to glycerophosphate an intermediate of glycolysis. 10 2. Lactate is released in the blood by cells, lacking mitochondria such as red blood cells, and exercising skeletal muscle. B. Ketogenic compounds AcetylCoA and compounds that give rise to acetyl CoA (for example acetocetate 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, (pyruvate to acetyl CoA.) These compounds give rise to ketone bodies and are therefore termed Ketogenic. Advantages of Gluconeogenesis 1. Gluconeogenesis meets the requirements of glucose in the body when carbohydrates are not available in sufficient amounts. 2. Regulate Blood glucose level 3. Source of energy for Nervous tissue and Erythrocytes 4. Maintains level of intermediates of TCA cycle 5. Clear the products of metabolism of other tissues(Muscle) Coris Cycle or Lactic Acid Cycle In an actively contracting muscle, only about 8% of the pyruvate is utilized by the citric acid cycle and the remaining is, therefore, reduced to lactate. 11 The lactic acid thus generated should not be allowed to accumulate in the muscle tissues. The muscle cramps, often associated with strenuous muscular exercise are thought to be due to lactate accumulation. This lactate diffuses into the blood. During exercise, blood lactate level increases considerably. Lactate then reaches liver where it is oxidized to pyruvate. It is then taken up through gluconeogenesis pathway and becomes glucose, which can enter into blood and then taken to muscle. This cycle is called cori's cycle, by which the lactate is efficiently reutilized by the body. Significance of the cycle: Muscle cannot form glucose by gluconeogenesis process because glucose 6 phosphatase is absent. Unlike Liver, muscle cannot supply Glucose to other organs inspite of having Glycogen 12  Glycogen metabolism Introduction Glycogen is the major storage form of carbohydrate in animals.It is mainly stored in liver and muscles and is mobilized as glucose whenever body tissues require. Degradation of Glycogen (glycogenolysis) A. Shortening of chains Golycogen phosphorylase cleaves the α-1, 4 glycosidic bonds between the glucose residues at the non reducing ends of the glycogen by simple phosphorolysis. This enzyme contains a molecules of covalently bound pyridoxal phosphate required as a coenzyme, Glycogen phosphorylase is a phosphotransferase that sequentially degrades the glycogen chains at their non reducing ends until four glucose units remain an each chain before a branch point. The resulting structure is called a limit dextrin and phosphorylase cannot degrade it any further. The product of this reaction is Glucose 1 phosphate. The glucose 1 phosphate is then converted to glucose 6 phosphate by phosphoglucomutase. Conversion of glucose 6 phosphate to glucose occurs in the Liver, Kidney and intestines by the action of Glucose 6 phosphatase. This does not occur in the skeletal muscle as it lacks the Enzyme. B. Removal of Branches A debranching enzyme also called Glucantransferase which contains two activities, Glucantransferase and Glucosidase. The transfer activity removes the terminal 3 glucose residues of one branch and attaches them to a free C4 13  end of the second branch. The glucose in α-(1,6) linkage at the branch is removed by the action of Glucosidase as free glucose. C. Lysosomal Degradation of Glycogen A small amount of glycogen is continuously degraded by the lysosomal enzyme α-(1, 4) glycosidase (acid maltase). The purpose of this pathway is unknown. However, a deficiency of this enzyme causes accumulation of glycogen in vacuoles in the cytosol, resulting in a very serious glycogen storage disease called type II (Pomp’s disease) Synthesis of Glygogen (Glycogenesis) The synthesis of glycogen from glucose-6-phosphate involves the following set of reactions. 1. Synthesis of glucose-1-phosphate. Glucose-6-phosphate is reversibly converted to glucose-1-phosphate by phosphoglucomutase, an enzyme that contains a phosphoryl group attached to a reactive serine residue 2. Synthesis of UDP-glucose. Formation of UDP-glucose, is a reversible reaction catalyzed by UDP-glucose pyrophosphorylase 3. Synthesis of glycogen from UDP-glucose. 14  The formation of glycogen from UDP-glucose requires two enzymes: a) glycogen synthase, which catalyzes the transfer of the glucosyl group of UDP-glucose to the nonreducing ends of glycogen b) amylo (1,4 →1,6)-glucosyl transferase (branching enzyme), which creates the (1,6) linkages for branches in the molecule Glycogen synthesis requires a preexisting tetrasaccharide composed of four (1, 4)- linked glucosyl residues. The first of these residues is linked to a specific tyrosine residue in a “primer” protein called glycogenin. Glycogen storage diseases These are a group of genetic diseases that result from a defect in an enzyme required for either glycogen synthesis or degradation. They result in either formation of glycogen that has an abnormal structure or the accumulation of excessive amounts of normal glycogen in specific tissues, A particular enzyme may be defective in a single tissue such as the liver or the defect may be more generalized, affecting muscle, kidney, intestine and myocardium. The severity of the diseases may range from fatal in infancy to mild disorders that are not 15 life threatening some of the more prevalent glycogen storage diseases are the following. 16