Biochemistry: Gluconeogenesis PDF

1A BIOCHEMISTRY CARBOHYDRATES: GLUCONEOGENESIS DR. JANDOC  Gluconeogenesis...

1A BIOCHEMISTRY CARBOHYDRATES: GLUCONEOGENESIS DR. JANDOC  Gluconeogenesis – synthesis of carbohydrates from OUTLINE noncarbohydrate precursors I. OVERVIEW OF GLUCONEOGENESIS  Location: II. SUBSTRATES  Normal conditions: Liver (85-95%) of the glucose A. Gluconeogenic Precursors that is made B. Ketogenic Compounds : Kidneys(10%) III. GLUCONEOGENIC PATHWAY  *Liver glycogen- essential postprandial source of A. Pyruvate Carboxylation glucose B. OAA Transport to the Cytosol  Starvation/Metabolic Acidosis: Kidneys (may C. Cytosolic OAA Decarboxylation contribute up to 50% of glucose formed) D. Conversion of PEP to F1,6-BP  Other tissues: Small Intestinal Epithelial Cells E. Fructose 1,6-Biphosphate Dephosphorylation (5%) F. Conversion of F6P to G6P  Precursors: G. Glucose 6-Phosphate Dephosphorylation  Lactate IV. REGULATION OF GLUCONEOGENESIS  Pyruvate A. Hormonal  Alanine B. Non-Hormonal  Glycerol (from backbone of TAGs) V. CLINICAL DEFECTS ASSOCIATED WITH GLUCONEOGENESIS  Alpha keto-acids (amino acid catabolism) A. Multiple Carboxylase Deficiency  Some amino acids B. Von Gierke’s Disease  Tissues Requiring Continuous Glucose Supply  Brain  Lens and cornea of eye  RBCs I. OVERVIEW OF GLUCONEOGENESIS  Testes  Renal Medulla  Exercising muscle  Functions:  Generates glucose from intermediates of glycolysis, fatty acid or TCA cycle  Maintains adequate blood sugar – during starvation or periods of low carbohydrate intake  Metabolic acidosis- allows excretion of protons by the kidneys  Allows use of dietary protein in carbohydrate pathways  Allows use of lactate during extended exercise II. SUBTRATES FOR GLUCONEOGENESIS  Most important:  Glycerol  Lactate  Alpha keto-acids (from glucogenic amino acid deamination) A. GLUCONEOGENIC PRECURSORS 1. Glycerol  triacylglycerol hydrolysis glycerol liver phosphorylated by glycerol kinase  glycerol phosphate oxidized by glycerol phosphate dehydrogenase dihydroxyacetone phosphate Trans 1 | ABACCO, ALDERITE, ASISTIN, BALANZA, BAYAS, BIANG 1 of 11  CARBOHYDRATES GLUCONEOGENESIS  adipocytes lack glycerol kinase – cannot phosphorylate glycerol 3. Alpha-Keto Acids  derived from glucogenic amino acid metabolism - OAA 2. Lactate - Alpha-ketoglutarate  Released into the blood by  Enter TCA cycle – OAA (direct precursor of PEP) - cells lacking mitochondria 4. Alanine - exercising muscle in Cori cycle  Pyruvate from glycolysis – transamination reaction –  a. Anaerobic Metabolism and Cori Cycle alanine i. Function: - shuttles lactate from muscle to liver – allow muscle to function anaerobically ii. Location: - muscle - liver iii. Products - 2 net ATP, 2 pyruvate per glucose 5. Amino Acids molecule  Converted pyruvate, TCA cycle intermediate – metabolized - Lactate converted to glucose to OAA  b. Lactate to Pyruvate Conversion - occurs in the liver - reverse reaction occurs in the muscles - catalyzed LDH - gluconeogenic tissues – LDH runs the reaction to pyruvate formation - direction of reaction depends on: + i. NAD /NADH Ratio ii. Lactate/Pyruvate Ratio iii. LDH Isoenzyme present S1T1 2 of 11  CARBOHYDRATES GLUCONEOGENESIS B. KETOGENIC COMPOUNDS - irreversible pyruvate dehydrogenase reaction (pyruvate b. Circumvented by 4 Alternate Reactions that Energetically acetyl CoA) acetyl CoA and compounds Favour Glucose Synthesis Catalyzed by that give rise to acetyl CoA (acetoacetate, ketogenic amino 1. Glucose 6-phosphate acids - lysine, leucine) cannot give rise 2. Fructose 1,6-biphosphate to net synthesis of glucose ketone bodies (ketogenic) 3. PEP Carboxykinase 4. Pyruvate Carboxylase III. GLUCONEOGENIC PATHWAY A. PYRUVATE CARBOXYLATION  Essentially the reversal of glycolytic pathway  7 of the glycolytic reactions are reversible – used in - pyruvate carboxylation by pyruvate carboxylase (found in glucose synthesis from pyruvate to lactate hepatocyte and renal cell mitochondria but in muscle cells) to a. 3 Irreversible Reactions of the Glycolytic Pathway oxaloacetate (OAA) converted to PEP by PEP- 1. Glucose Phosphorylation by glucokinase/hexokinase carboxykinase 2. Conversion of Fructose 6-phosphate to fructose 1,6- biphosphate by PFK 1. Biotin is a Coenzyme 3. Conversion PEP to pyruvate to pyruvate kinase - pyruvate carboxylase contains biotin (covalently bound to apoenzyme though an -amino group of lysine (covalently bound biotin is called biocytin) - high energy phosphate cleavage of ATP drives formation of apoenzyme-biotin-CO2 intermediate (high energy complex) pyruvate carboxylation to OAA a. Occurs in the mitochondria of  Liver cells  Kidney cells b. 2 purposes  Provide substrate for gluconeogenesis  Provide oxaloacetate - replenish TCA cycle intermediates that may become depleted c. Muscle cells S1T1 3 of 11 CARBOHYDRATES GLUCONEOGENESIS  also contain pyruvate carboxylase provide pathway from pyruvate PEP reversed oxaloacetate to replenish the TCA cycle reactions of glycolysis fructose 1,6-biphosphate intermediates that may become depleted  site of reaction – cytosol ++ ++ 2. Mg , Mn  metals  required for enzymatic activity 3. Site of Reaction  mitochondrial matrix 4. Allosteric Regulation  Activated by Acetyl CoA -increased acetyl CoA levels signals metabolic states requiring increased OAA synthesis - starvation OAA glucose through gluconeogenesis - pyruvate carboxylation replenish TCA cycle intermediates that may become depleted  Low Acetyl CoA levels - pyruvate carboxylase is largely inactive pyruvate is primarily oxidized in the TCA cycle B. OAA TRANSPORT TO THE CYTOSOL 1. OAA  formed in the mitochondria  must enter cytosol – other gluconeogenic enzymes are located  unable to cross mitochondrial membranemalate cytosol OAA 2. Conversion of OAA to Malate  Enzyme - Reversible reaction catalyzed by malate dehydrogenase - Mitochondria – mitochondrial malate dehydrogenase catalyzes the reaction as written - Cytosol – cytosolic malate dehydrogenase catalyzes the reverse reaction – regenerate OAA  Malate – also transfer reducing equivalents from the mitochondria to the cytosol C. CYTOSOLIC OAA DECARBOXYLATION  Driven by GTP hydrolysis  OAA – decarboxylated and phosphorylated in the cytosol  Catalyzed by PEP carboxykinase (PEPCK) - Requires Mn++ for activation  combined action of pyruvate carboxylase and PEP carboxykinase provides energetically favored S1T1 4 of 11 CARBOHYDRATES GLUCONEOGENESIS D. CONVERSION OF PEP TO F1,6BP  reverse of those that occur in glycolysis  catalyzed by same enzymes E. FRUCTOSE 1,6-BIPHOSPHATE DEPHOSPHORYLATION  provides energetically favorable pathway for F6P formation  catalyzed by fructose 1,6-biphosphatase (allosteric enzyme) bypasses the irreversible PFK-1 reaction  important regulatory site of gluconeogenesis S1T1 5 of 11 CARBOHYDRATES GLUCONEOGENESIS 1. Regulation by Energy Levels Within Cells  Elevated AMP Levels - signals energy-poor state inhibition of fructose 1,6-biphosphatase  Stimulation of Gluconeogenesis - Citrate 2. Regulation by Fructose 2,6-Biphosphate G. GLUCOSE 6-PHOSPHATE DEPHOSPHORYLATION  Fructose 2,6-Biphosphate  catalyzed by glucose 6-phosphatase (occurs in liver, - Allosteric modifier kidneys, and small intestinal epithelial cells) but not - Concentration – influenced by circulating in muscles glucose 6-phosphate derived from muscle glucagon glycogen cannot be dephosphorylated to yield free glucose) - inhibits fructose 1,6-biphosphatase (occurs bypasses the irreversible glucokinase and hexokinase in the liver and kidneys) reactions provides energetically favourable pathway for 3. Reciprocal Control the formation of free glucose  the allosteric effects are exactly the opposite of those observed with PFK, the regulatory enzyme of glycolysis (example of reciprocal control of opposing metabolic pathways) F. CONVERSION OF F6P TO GLUCOSE 6-PHOSPHATE  reverse of that of glycolytic pathway  catalyzed by the same enzyme ENZYMES REQUIRED  Glucose 6-Phosphate Translocase - transports glucose 6-phosphate across the endoplasmic reticulum membrane  Glucose 6-Phosphatase - found only in gluconeogenic cells a. Enzyme Deficiency - Type Ia glycogen storage disease b. Muscle Cells - lack glucose 6-phosphatase cannot provide blood glucose by gluconeogenesis S1T1 6 of 11  CARBOHYDRATES GLUCONEOGENESIS - glucose 6-phosphate from muscle glycogen cannot be dephosphorylated to yield glucose H. SUMMARY OF THE REACTIONS COMMON TO GLYCOLYSIS AND GLUCONEOGENESIS AND ENERGETICS IV. REGULATION OF GLUCONEOGENESIS A. HORMONAL REGULATION 1. GLUCAGON  stimulate gluconeogenesis by 3 mechanisms a. Changes in Allosteric Effectors - glucagon lowers fructose 2,6-biphosphate level  fructose 1,6-biphosphatase activation  PFK inhibition b. Covalent Modification of Enzyme Activity - glucagon (via elevated cAMP level and cAMP- dependent protein kinase activity) stimulates conversion of pyruvate kinase to its inactive synthesis S1T1 7 of 11  CARBOHYDRATES GLUCONEOGENESIS c. Induction of Enzyme Synthesis - increased enzymatic activity - insulin - decreases gene transcription 2. GLUCAGON, EPINEPHRINE, GLUCOCORTICOIDS - stimulate the synthesis of the enzymes of gluconeogenesis used to bypass the irreversible steps of glycolysis 3. INSULIN - suppresses the synthesis of the enzymes (of gluconeogenesis used to bypass the irreversible steps of glycolysis) - stimulates the synthesis of key glycolytic enzymes (hexokinase, PFK, pyruvate kinase) B. NONHORMONAL REGULATION 1. SUBSTRATE AVAILABILITY  availability of gluconeogenic precursors markedly influences rate of hepatic glucose synthesis  decreased insulin levels amino acid mobilization from muscles provide carbon skeletons for gluconeogenesis 2. ALLOSTERIC ACTIVATION BY ACETYL COA  starvation excessive adipose tissue lipolysis liver is flooded with fatty acids -oxidation excessive acetyl CoA exceeds capacity of liver acetyl CoA accumulation allosteric S1T1 8 of 11 CARBOHYDRATES GLUCONEOGENESIS activation of hepatic pyruvate carboxylase (pyruvate dehydrogenase inhibition) 3. ALLOSTERIC INHIBITION BY AMP  inhibits fructose 1,6-biphosphatase  activate phosphofructokinase - AMP elevation stimulation of pathways that oxidize nutrients to provide energy S1T1 9 of 11  CARBOHYDRATES GLUCONEOGENESIS 2. Effects  developmental retardation  ketoacidosis  hair loss  diffuse erythematous rash 3. Treatment  therapeutic doses of biotin 4. Case  Presentation o A 2 year old male is admitted to the hospital while suffering severe lactic acidosis. He has had a history of poor growth and development, repeated seizures, ataxia, and persistent metabolic acidosis. Urinalysis indicates high levels of the following organic acids: pyruvate, lactate, -hydroxybutyrate, -hydroxypropionate, and - methylcrotonate  Diagnosis o The organic acids present in the child’s urine are all substrates or metabolites of substrates from biotin-dependent carboxylase suggests a form of multiple carboxylase deficiency due to defect in holocarboxylase synthetase or biotinase  Treatment o oral dose of biotin 10 mg / day o metabolite levels returned to normal within 3 days o the more general symptoms, such as ataxia, improved over a period of several days following biotin therapy V. CLINICAL DEFECTS ASSOCIATED with GLUCONEOGENESIS B. VON GIERKE’S DISEASE (TYPE I GLYCOGEN STORAGE DISEASE) A. MULTIPLE CARBOXYLASE DEFICIENCY 1. Causes 1. Causes  genetic defect in G6Pase a. Genetic defect in the enzyme Holocarboxylase Synthetase - responsible for covalently attaching biotin to 2. Effects pyruvate carboxylase and 3 other biotin dependent  Enlarged Liver enzymes deficient activity - due to accumulation of excessive glycogen  3 Other Biotin-Dependent Enzymes (derived from G6P) that cannot be converted to  acetyl CoA carboxylase glucose and released from the liver  propionyl CoA carboxylase  Hypoglycemia  -methylcrotonyl CoA carboxylase - due to inability of the liver to form glucose from b. Biotinase Deficiency G6P no glucose released to the bloodstream - biotinase removes biotin from proteins during  Lactic Acidemia degradation biotin recycling - because glucose cannot be formed from lactate via pyruvate lactic acid accumulation decreased blood pH depletion of alkali reserves S1T1 10 of 11 CARBOHYDRATES GLUCONEOGENESIS  Hyperlipidemia - lipids primarily fatty acids  Hyperuricemia - G6P accumulation increased activity of pentose phosphate pathway increased production of purine nucleotides increased uric acid production REFERENCES th 1. Lippincott’s Illustrated Reviews: Biochemistry, 6 edition S1T1 11 of 11