Gluconeogenesis: Objectives

1.  There are many questions, including:

  1. How can glucose be synthesized from lactate (i.e. gluconeogenesis)?
  2. Lactate is converted to Pyruvate, followed by a series of reverse glycolytic steps to get to Glucose. An endorgonic, regulated process.
  3. Where (in which organs) does gluconeogenesis take place?
  4. Gluconeogenesis occurs mainly in the liver (90%) and also in the kidneys
  5. What parts of the cell participate in gluconeogenesis?
  6. In the mitochondria and the cytoplasm
  7. Name the most common precursors for gluconeogenesis.
  8. Lactate from exercising muscle and red blood cells
  9. The breakdown of proteins, which gives rise to amino acids (which can be utilized to make new glucose)
  10. Glycerol (use only part of the pathway)
  11. Fructose (use only part of the pathway)
  12. Glyceraldehyde-3-phosphate
  13. Alanine
  14. Oxaloacetate is needed because
  15. it is the precursor for PEP
  16. Helps to bypass pyruvate kinase
  17. Glycolytic molecules such as F6P and G3P
  18. Where do these precursors for gluconeogenesis come from?
  19. Lactate – comes from the exercising muscles and the breakdown of glucose in the RBCs (anaerobic glycolysis)
  20. Glycerol – comes from triglycerides

2.  Name the three irreversible steps in glycolysis. Name the enzymes.

  1. GlucoseàGlucose-6-Phosphate (Hexokinase/Glucokinase)
  2. Fructose-6-PhosphateàFructose-1,6-Bisphosphate (PFK)
  3. Phosphoenol PyruvateàPyruvate (Pyruvate Kinase)

3.  How are these steps by-passed in gluconeogenesis? Name the enzymes.

  1. Glucose-6-Phosphate à Glucose
  2. Cleave the phosphate group
  3. Enzyme: “Glucose-6-Phosphatase”
  4. Fructose-1,6-BisphosphateàFructose-6-Phosphate
  5. Cleave the phosphate group
  6. Enzyme: “Fructose-1,6-Bisphosphatase”
  7. The REGULATORY enzyme!

  1. Pyruvate à Phosphoenol Pyruvate
  2. Pyruvate à Oxaloacetate
  3. Add CO2 + ATP
  4. (Generates ADP)
  5. Enzyme: “Pyruvate Carboxylase” and coenzyme: Biotin
  6. In the mitochondria
  7. Oxaloacetate + NADHà Malate; Malate + NAD+àOxaloacetate
  8. Requires the Malate Shuttle to go to the cytoplasm
  9. Turned into Malate, given to cytoplasm, returns to oxaloacetate
  10. Enzyme: Malate Dehydrogenase
  11. Oxaloacetate à PEP
  12. Add GTP
  13. Loses CO2
  14. Enzyme: “PEPCarboxykinase” (PEPCK)
  15. In the cytoplasm

4.  Several questions…

  1. What is the role of pyruvate carboxylase in gluconeogenesis and in anaplerosis?
  2. Pyruvate Carboxylase adds CO2 to pyruvate to yield Oxaloacetate in the mitochondria
  3. In anaplerosis: regenerates a catalytic intermediate for TCA cycle
  4. In kidney/liver: Needs to continue regeneration to continue to move towards formation of new glucose
  5. How is it regulated?
  6. (+) regulated by Acetyl CoA.
  7. Write the sequence of steps involved in the conversion of pyruvate to phosphoenol pyruvate.
  8. Three Steps:
  9. PyruvateàOxaloacetic Acid
  10. this reaction uses “biotin” (to carboxylate) and ATP
  11. Pyruvate Carboxylase
  12. In mitochondria
  13. Oxaloacetate + NADHàMalate + NAD+à Oxaloacetate
  14. Used for transport from Mitochondrial matrix to cytoplasm
  15. Enzyme: Malate Dehydrogenase
  16. OxaloacetateàPEP
  17. Uses GTP and liberates CO2
  18. PEPCK
  19. In cytoplasm

5.  Explain why Acetyl CoA cannot serve as a precursor for gluconeogenesis?

  1. Acetyl CoA cannot be used to make new glucose because TCA cycle oxidizes Acetyl CoA completely; no way to make acetyl CoA from pyruvate (irreversible reaction of acetyl coA + CO2 into pyruvate)
  2. Thus cannot use even numbered fatty acids because they produce acetyl CoA.

6.  What key role does Acetyl CoA play in gluconeogenesis?

  1. Acetyl Co A is a positive modulator of pyruvate carboxylase
  2. Without Acetyl CoA, the pyruvate carboxylase would not function!

7.  Several questions:

  1. What is the role of biotin in gluconeogenesis?
  2. Biotin is the coenzyme for carboxylation reactions. It is the carrier of active CO2 in carboxylations. (p.13 of notes)
  3. ATP in presence of pyruvate carboxylase +biotin
  4. P from ATP is given to biotin’s imidizole ring: “active biotin complex”
  5. Allows the enzyme to add the CO2 to the enzyme and produce oxaloacetate
  6. Recognize the structure of biotin
  7. It has an imidozole ring fused to a Thiophene ring with a fatty acid. The carboxylate group will bind with lysine (CONH) residue in enzyme. (it looks like two houses that are united at the floor. The top house has nitrogen and the bottom house has a sulfur roof. The fatty acid is attached to the bottom house).
  8. What is the nature of the biotin-enzyme linkage?
  9. It is an amide bond between the biotin and the specific enzyme
  10. What kind of reaction is biotin known to participate in?
  11. Carboxylations- adding CO2 (the carboxylate group will bind with lysine residues in enzyme)
  12. Thiamine – decarboxylation reactions (in PPP and glycolosis)
  13. Give the names of 3 such reactions.
  14. Pyruvate à Oxaloacetate (pyruvate carboxylase)
  15. Propionyl CoA + CO2àSuccinyl CoA (few reactions involved, Key enzyme: Propionyl CoA Carboxylase)
  16. Malonyl CoAàAcetyl Co A (Acetyl-CoA Carboxylase)

8.  State the steps involved in the transport of oxaloacetate from mitochondria to cytoplasm. What role does malate play in this process?

  1. Oxaloacetate is transported to the cytoplasm from the mitochondrial matrix by way of the malate shuttle. OxAc is first converted to malate by malate Dehydrogenase.
  2. Malate then crosses the membrane and is converted back to OxAc by the same enzyme.

9.  Describe why the glycerol-moiety of triglycerides and not the even-numbered fatty acids can be converted to glucose. Name the first glycolytic intermediate formed from glycerol. (see pg. 16)

  1. The essence of this question is based in the number of carbons. Glycerol comes from the breakdown of triglycerides. Glycerol is converted to glycerol-3-phosphate, which then is reacted with NAD+ + glycerol-3-phosphate dehydrogenase to produce: dihydroxyacetone phosphate (DHAP).

10.  Explain why muscle and brain cannot synthesize glucose from lactate.

  1. Muscle and Brain cannot synthesize glucose from lactate because they lack Glucose-6-Phosphatase. This is good because then all the glucose can only be used for energy rather than be exported (like the liver and kidney).

11.  How and where are (a) lactate and (b) alanine (produced by active skeletal muscle) converted to glucose?

  1. Lactate (from muscles) and alanine are converted in the liver.
  2. Lactate is converted to Pyruvate…eventually, make new glucose.
  3. Alanine is converted to Pyruvate…eventually make new glucose.

12.  Why does adipose tissue triglyceride biosynthesis depend on the availability of glucose?

  1. Adipose Tissue lacks Glycerol Kinase so the glycerol that is liberated in the break down of fatty acids cannot be phosphorylated.
  2. Cannot make DHAP
  3. Must have GLUCOSE if want to make it in adipose tissue

13.  Explain how amino acids can serve as precursors for the synthesis of glucose.

  1. Amino Acids can be converted to glycolytic intermediates of the TCA and can then eventually become Oxaloacetate.
  2. When remove the alpha amine group, can be transformed into TCA intermediates.
  3. There are different AAs which can form glutamic acid, furmerate, pyruvate, Propinyl-CoA (go to succinyl-CoA)
  4. Eventually, produce oxaloacetate, which can be made into malate, and can be shuttled out of mitochondria to form new glucose.

14.  State the primary and secondary control steps in the synthesis of glucose from pyruvate. (pg. 16)

  1. Acetyl CoA positively modulates pyruvate carboxylase. KEY ENZYME!
  2. Secondary Control is regulated at the fructose-1,6-bisphosphotase. Glucagon inhibits this enzyme. F16BP by inhibiting F26Bisphophate formation (F26BP stimulates PFK). Look at Pg221 of Unit 2 for more information. ATP and citrate stimulates phosphatase.
  3. Never have glycolysis and gluconeogenesis occurring at the same time!

15.  How much ATP, GTP, and NADH are required to synthesize 1 molecule of glucose from pyruvate in liver?

  1. Need 2 molecules of pyruvate to produce 1 molecule of glucose
  2. 4 ATP, 2 GTP and 2 NADH – highly endorgonic process!
  3. ATP at 2 spots
  4. PyruvateàOxaloacetate
  5. 3-phosphoglycerateà1,3 Bisphosphoglycerate
  6. GTP from OxaloacetateàPEP
  7. NADH from 1,3 BisphosphoglycerateàGlyceraldehyde-3-P

16.  How does ethanol inhibit gluconeogenesis?

  1. Ethanol inhibits the conversion of Lactate to Pyruvate by having a strong hold on the NADH population (depleting NAD+ stores!). Inhibits Lactic Acid Dehydrogenase. Causes Lactic Acidosis, hypoglycermia, possible coma.