Gluconeogenesis

Question 1

Blood glucose level

Answer 1

1. Blood glucose level normally ranges from 4-8 mM (translating into approximately 70-150 mg/dl or 3.5-7.5 grams in 5 liters)
2. Fasting of vigorous exercise can reduce amount of blood glucose.
3. Gluconeogenesis is the synthesis of “new” glucose.
4. Gluconeogenesis and glycogenolysis are essential for the maintenance of blood sugar during fasting and also during vigorous exericse.
5. Glucose is essential for:
   
   1. Brain
   2. Erythocytes
   3. Adipose tissue (as a source of glycerol-glyceride)
   4. Skeletal muscle opering anaerobically
   5. Mammary gland (precursor of lactose)
   6. etc.

Question 2

Cori Cycle

Answer 2

1. Muscles generate lactate from using glucose as energy source when they are working vigorously
2. Lactate enters the bloodstream and transported to Liver
3. Liver converts lactate into glucose via gluconeogenesis, where glucose enters the bloodstream
4. “Newly” generated glucose is taken up by muscles to be used as energy source once again
5. Repeat of the cycle

Question 3

Interconversion of pyruvate and lactate

Answer 3

1. Glycolysis generates NADH from NAD+
2. NADH is oxidized to NAD+ in the mitochondria (oxidative phosphorylation)
3. Inthe absence of oxygen, the NAD+ is regenerated by the formation of lactate. This reaction is catalyzed by lactate dehydrogenase.

Question 4

Sources of carbon for gluconeogenesis

Answer 4

1. Glycerol released from adipose lipolysis; glycerol from triglycerides can be used for gluconeogenesis.
2. Muscle lactate
   
   1. Muscle does not engage in gluconeognesis as it is missing the essential enzyme glucose 6-phosphatase and instead liver & kidneys do.
   2. The muscle lactate is transported to the liver, in which gluconeogenesis can occur.
3. Amino acids derived from muscle proteolysis (glucogenic amino acids) when a person is really in starvation. e.g. Glucose-Alanine cycle

Question 5

Glucose-Alanine cycle

Answer 5

1. Glucose is used in muscle working vigorously or in starvation and is converted to pyruvate.
2. At a low glucose level, proteins of the muscle yield glucogenic amino acid such as Alanine, which can also be derived from pyruvate.
3. Alanine is transported to liver via bloodstream and is converted back to pyruvate and subsequently into glucose (gluconeogenesis)
4. Glucose reenters the bloodstream and transported back to muscle

Question 6

Location of gluconeogenesis

Answer 6

1. Gluconeognesis primarily occurs in the liver and to a limited extent in the kidney, etc.
2. Gluconeogenesis enzymes are located primarily in cytosol
3. Glucose 6-phosphatase is bound to smooth endoplasmic reticulum; since smooth ER is involved in exocytosis, glucose needs to be here at the end of gluconeogenesis to be released from liver cells and sent into the blood stream.
4. Pyruvate carboxylase (irreversible carboxylation of pyruvate into oxaloacetate (OAA)) is located in mitochondrial matrix

Question 7

Overview of gluconeogenesis

Answer 7

1. 7 of the 10 glycolytic enzymes (involved in glycolysis) are also involved in gluconeogenesis.
2. There are, however, three irreversible reactions (due to the large negative change in free energy) that are bypassed by gluconeogenesis.
3. Energy input is necessary for carrying out gluconeogenesis (Endergonic)
4. 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H₂O → Glucose + 4 ADP + 2 GDP + 6 P_i + 2 NAD⁺ + 2 H⁺

Question 8

Pyruvate → Oxaloacetate →Phosphoenolpyruvate (PEP)

Answer 8

• The reaction takes place within Mitochondrion matrix
• A mechanism to overcome the irreversible conversion of PEP to pyruvate during glycolysis; Endergonic
• Buildup of Acetyl-CoA signals brain is starving of glucose. Buildup of Acetyl-CoA leads to build up of pyruvate, which then go through the gluconeogenesis

1. Pyruvate carboxylase is covalently attached to Biotin
2. CO2 is activated and transferred by py a pyruvate carboxylase to its biotin prosthetic group; this step requires ATP hydrolysis; activated by accumulating Acetyl-CoA (just downstream of pyruvate)
3. The enzyme then transfers the CO2 to pyruvate, generating Oxaloacetate
4. Oxaloacetate cannot cross the mitochondrial membrane (inner and outer) so it is reduced to malate that can, with the help of NADH
5. In the cytosol, malate i reoxidized to oxaloacetate, which is converted to phosphoenolpyruvate by PEP carboxykinase (PEP CK); NADH is regained and GTP hydrolyzed in the process.

• Biotin is an essential nutrient
• Acetyl-CoA inhibits pyruvate dehydrogenase**/PDC (pyruvate → Acetyl-CoA), but activates pyruvate carboxylase (pyruvate → oxaloacetate)
• In some species, PEP CK is located in the mitochondrai, while in others, it is located in the cytosol. In humans, it is located in both the cytosol and the mitochondria, while only the cytolic enzyme participates in gluconeognesis.

Question 9

F 1,6-P to F 6-P

Answer 9

• Fructose 1,6-bisphosphatase or FBP-1 catalyzes the reaction: Fructose 1,6-biphosphate + H2O –> Fructose 6-phosphate + Pi
• This reaction is inhibited by Fructose 2,6-bisphosphate

1. High glucagon/insulin ratio causes elevateed cAMP and increased levesl of active protein kinase A (PKA)
2. Increased PKA activity favors the phosphorylated form of the PFK-2(Phosphofructosekinase-2)/FBP-2 (Fructose (2,6-) Biphosphatase-2) complex.
3. Phosphorylated PFK-2 is inactive, whereas FBP-2 is active; this impedes the formation of fructose 2,6-bisphosphate and thus the glycolysis itself, and instead yields F 6-P
4. Decreased levels of fructose 2,6-bisphophate decreases the inhibition of FBP-1 (Fructose bisphophatase-1), which leads to an increased rate of gluconeognesis.

• Fructose 2,6-bisphosphate can be seen as a second messenger, as its concentration changes in response to hormonal stimulation

Question 10

G 6-P to D-Glucose

Answer 10

1. Glucose 6-phosphatase is an ER enzyme that catalyzes the conversion of glucose 6-phosphate to D-glucose
2. Glucose 6-phosphate is translocated to the ER membrane by glucose 6-phosphate translocase, where G6 phosphatase will take away the phosphate inside the ER yielding D-glucose
3. Glucose 6-phosphatase is NOT found in muscle

Question 11

Energy (Gluconeogenesis) Summary

Answer 11

4 ATP + 2 GDP + 2 NADH invested in gluconeogenesis

Question 12

‘Futile’ cycle

Answer 12

1. When Glucose/Insulin level is high, the active protein kinase A also deactivates pyruvate kinase so that the gluconeogenesis is not counteracted.

Question 13

Activators of gluconeogenesis

Answer 13

1. Glucagon
   
   1. increase of protein kinase A and cAMP
   2. induction of synthesis of key enzymes of gluconeogenesis and repression of glycolytic enzymes
2. Elevated levels of gluconeogenesis precursors
3. Acetyl CoA
   
   1. can be formed by beta oxidation of fatty acids
   2. inhibits pyruvate dehydrogenase (complex) as a way of negative feedback regulation

Question 14

Deactivators of gluconeogenesis

Answer 14

1. Insulin
   
   1. reduction of hepatic cAMP
   2. Induction of glycolytic enzymes and repression of gluconeogenic enzymes
   3. Regulation of enzyme activity by phosphorylation and by allosteric modification is rapid, while regulation of enzyme synthesis can require a number of hours
2. AMP
   
   1. Elevated levels of AMP signal lower level of ATP