1. Gluconeo genesis
Carbohydrates provide a significant portion of human caloric intake

2. All cells are dependent on glucose.
Glucose level in blood plasma must be stable.
Brain is especially sensitive to the decrease of glucose level (the daily glucose requirement of the brain in a typical adult human being is about 120 g).
Red blood cells use only glucose as a fuel.
160 g of glucose needed daily by the whole body.
The amount of glucose present in body fluids is about 20 g, and that readily available from glycogen is approximately 190 g.
During period of fasting glycogen in liver is mobilized but it only lasts 12 to 24 hours and this source of glucose may not fulfill metabolic need.
During a longer period of starvation organism must synthesize glucose from smaller noncarbohydrate precursor molecules.

3. • During starvation or intense exercise, glucose must be replaced by gluconeogenesis.
• Major site of gluconeogenesis: Liver
• Secondary site: Kidney cortex.
• Thus gluconeogensis in the liver and kidney helps to maintain the glucose level in the blood so that brain and muscle can extract sufficient glucose to meet their metabolic demands.

4. Gluconeogenesis – synthesis of glucose from noncarbohydrate precursors
Liver and kidney are major sites of glucose synthesis
Main precursors: lactate, pyruvate, glycerol and some amino acids
Under fasting conditions, gluconeogenesis supplies almost all of the body’s glucose
Gluconeogenesis – universal pathway. It present in animals, microorganisms, plants and fungi
Plants synthesize glucose from CO2 using the energy of sun, microorganisms – from acetate and propionate

5. Gluconeogenesis is not a Reversal Glycolysis
In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is converted into glucose.
However, gluconeogenesis is not a reversal of glycolysis.
There are three irreversible reactions in glycolysis catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.
1. Glucose + ATP  glucose-6-phosphate + ADP (hexokinase) G = -8 kcal mol-1
3. Fructose-6-phosphate + ATP  fructose-1,6-biphosphate + ADP (phosphofructokinase) G = -5.3 kcal mol-1
10. Phosphoenolpyruvate + ADP  pyruvate + ATP (pyruvate kinase) G = -4 kcal mol-1
These three reactions must be bypassed in gluconeogenesis

6. Bypassed Reactions in Gluconeogenesis
1. Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.
Pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi + 2H+
Oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2
2. Fructose 6-phosphate is formed from fructose 1,6-bisphosphate Enzyme - fructose 1,6-bisphosphatase.
Fructose 1,6-bisphosphate + H2O  fructose 6-phosphate + Pi
3. Glucose is formed by hydrolysis of glucose 6-phosphate in a reaction catalyzed by glucose 6-phosphatase.
Glucose 6-phosphate + H2O  glucose + Pi

7. Gluconeogenesis
The distinctive reactions are shown in red.

8. Comparison of glycolysis and gluconeogenesis

9. Bypass I: Pyruvate  Phosphoenolpyruvate
The first step in gluconeogenesis is the carboxylation of pyruvate to form oxaloacetate at the expense of a molecule of ATP.
Enzyme pyruvate carboxylase is present only in mitochondria.
Pyruvate is transported into mitochondria from cytoplasm; the part of pyruvate is formed in mitochondria from amino acids.
Essential cofactor of pyruvate carboxylase is biotin, which serves as a carrier of CO2.
Biotin-binding domain of pyruvate carboxylase
Structure of carboxybiotin

10. Pyruvate carboxylase is allosterically activated by acetyl CoA.
Accumulation of acetyl CoA from fatty acid oxidation signals abundant energy, and directs pyruvate to oxaloacetate for gluconeogenesis.
Pyruvate carboxylase reaction
biotin
This reaction takes place in mitochondria matrix.

11. Oxaloacetate is polar molecule and can not pass through the mitochondria membrane into cytoplasm
Therefore it is reduced: oxaloacetate + NADH2  malate + NAD+ Enzyme – malate dehydrogenase
Malate passes through the mitochondria membrane into cytoplasm and again oxidized to oxaloacetate (enzyme malate dehydrogenase): malate + NAD+  oxaloacetate + NADH2
Cytoplasmic oxaloacetate is decarboxylated to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase

12. Phosphoenolpyruvate carboxykinase reaction
Reaction takes place in the cytosol.
In decarboxylation reaction GTP donates a phosphoryl group.
Oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase.
One molecule of ATP and one molecule of GTP were spent to lift pyruvate to the energy level of phosphoenlpyruvate.

13. Mechanism of phosphoenolpyruvate carboxykinase reaction

14. Bypass II: Fructose 1,6-bisphosphate  Fructose 6-phosphate
A metabolically irreversible reaction
The enzyme responsible for this step is fructose 1,6-bisphosphatase
F1,6BPase is allosterically inhibited by AMP and fructose 2,6-bisphosphate (F2,6BP)

15. Bypass III: Glucose 6-phosphate  glucose
In most tissues, gluconeogenesis ends with the formation of glucose 6-phosphate (G-6P).
Glucose 6-phosphate, unlike free glucose, cannot diffuse out of the cell.
The generation of free glucose is controlled in two ways:
enzyme responsible for the conversion of glucose 6-phos-phate into glucose, glucose 6-phosphatase, is regulated;
enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis — liver and to a lesser extent kidney, pancreas, small intestine.

16. Glucose 6-phosphatase reaction
The final step in the generation of glucose does not take place in the cytosol.
G-6-P is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed by glucose 6-phosphatase, which is bound to the membrane.
Glucose 6-phosphatase reaction

17. Glucose and Pi are then shuttled back to the cytosol by transporters.
Ca2+-binding stabilizing protein is essential for phosphatase activity.
Glucose and Pi are then shuttled back to the cytosol by transporters.
Generation of glucose from glucose 6-phosphate. Several proteins play a role in the generation of glucose. T1 transports G-6-P into the lumen of the ER; T2 and T3 transport Pi and glucose respectively back into the cytosol. SP – Ca-binding protein.

18. The Net Reaction of Gluconeogenesis
2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O  Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi + 2H+
G°' = -9 kcal mol-1
Six nucleotide triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are generated in glycolysis in the conversion of glucose into pyruvate.
The extra cost of gluconeogenesis is four high phosphoryl-transfer potential molecules per molecule of glucose synthesized from pyruvate.

19. Subcellular Locations of Gluconeogenic Enzymes
Gluconeogenesis enzymes are cytosolic except:
(1) Glucose 6-phosphatase (endoplasmic reticulum)
(2) Pyruvate carboxylase (mitochondria)
(3) Phosphoenolpyruvate carboxykinase (cytosol and/or mitochondria)

20. Regulation of Gluconeogenesis
Gluconeogenesis and glycolysis are reciprocally regulated - within a cell one pathway is relatively inactive while the other is highly active.
The amounts and activities of the distinctive enzymes of each pathway are controlled.
The rate of glycolysis is determined by the concentration of glucose.
The rate of gluconeogenesis is determined by the concentrations of precursors of glucose.

21. AMP stimulates phospho-fructokinase, whereas ATP and citrate inhibit it. Fructose 1,6-bisphosphatase is inhibited by AMP and activated by citrate.
Fructose 2,6-bisphosphate strongly stimulates phospho-fructokinase 1 and inhibits fructose 1,6-bisphosphatase.
During starvation, gluconeo-genesis predominates because the level of F-2,6-BP is very low.
High levels of ATP and alanine, which signal that the energy charge is high and that building blocks are abundant, inhibit the pyruvate kinase.
Pyruvate carboxylase is activated by acetyl CoA and inhibited by ADP.
ADP inhibits phosphoenol-pyruvate carboxykinase.
Gluconeogenesis is favored when the cell is rich in biosynthetic precursors and ATP.

22. Regulation of the Enzymes Amount by Hormones
Hormones affect gene expression primarily by changing the rate of transcription.
Insulin, which rises subsequent to eating, stimulates the expression of phosphofructokinase and pyruvate kinase.
Glucagon, which rises during starvation, inhibits the expression of these enzymes and stimulates the production of phosphoenolpyruvate carboxykinase and fructose 1,6-bisphosphatase.
Transcriptional control in eukaryotes is much slower than allosteric control; it takes hours or days in contrast with seconds to minutes.

23. Precursors for Gluconeogenesis
Any metabolite that can be converted to pyruvate or oxaloacetate can be a glucose precursor
Major gluconeogenic precursors in mammals:
(1) Lactate
(2) Most amino acids (especially alanine),
(3) Glycerol (from triacylglycerol hydrolysis)

24. Lactate Glycolysis generates large amounts of lactate in active muscle
Red blood cells steadily produce lactate
Lactate produced by active skeletal muscle and erythrocytes is a source of energy for other organs
The plasma membranes of some cells, particularly cells in cardiac muscle, contain carriers that make them highly permeable to lactate and pyruvate.

25. Lactate
Lactate and pyruvate diffuse out of active skeletal muscle into the blood and then into these permeable cells.
Once inside these well-oxygenated cells, lactate can be reverted back to pyruvate and metabolized through the citric acid cycle and oxidative phosphorylation to generate ATP.
The use of lactate in place of glucose by these cells makes more circulating glucose available to the active muscle cells.
Excess lactate enters the liver.

26. The Cori Cycle
Liver lactate dehydrogenase converts lactate to pyruvate (a substrate for gluconeogensis)
Glucose produced by liver is delivered to peripheral tissues via the bloodstream
Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize glucose.
These reactions constitute the Cori cycle

27. Amino Acids
Carbon skeletons of most amino acids are catabolized to pyruvate or citric acid cycle intermediates
The glucose-alanine cycle: (1) Transamination of pyruvate yields alanine which travels to the liver (2) Transamination of alanine in the liver yields pyruvate for gluconeogenesis (3) Glucose is released to the bloodstream

28. Glucose- alanine cycle:
Glycogen
gluconeogenesis
transamination

29. Gluconeogensis from Glycerol

30. Glycerol (which can be generated by hydrolysis of triacylglycerols (fat) to yield free FAs + glycerol) is an excellent substrate for gluconeogenesis.