1. Chapter 6
Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway
A basket of fresh bread. Carbohydrates such as these provide a significant portion of human caloric intake

2. Outline What is gluconeogenesis, and how does it operate?
How is gluconeogenesis regulated?
How are glycogen and starch catabolized in animals?
How is glycogen synthesized?
How is glycogen metabolism controlled?
Can glucose provide electrons for biosynthesis?
What is PPP?

3. What Is Gluconeogenesis, and How Does It Operate?
Definition: the biosynthesis of glucose from simpler molecules, primarily pyruvate and its precursors.
The liver is the major location for gluconeogenesis
The gluconeogenesis pathway is similar to the reverse of glycolysis but differs at critical sites.
Glucose is needed to maintain glucose levels range in blood.
Some tissue for example brain, erythrocytes,and muscles use glucose at a rapid rate and sometimes require glucose in addition to dietary glucose.
The brain uses mostly glucose and erythrocytes can use only glucose as a source of energy.

4. The Gluconeogenesis Pathway
pyruvate carboxylase 11
BYPASS 3
2. Phosphoenolpyruvate carboxykinase
3. enolase 10
BYPASS 2
4. phosphoglyceromutase 9
5. phosphoglycerate kinase 8
6. Glyceraldehyde-3- phosphate-
dehydrogenase 7
7. triose phosphate isomerase 6
8. aldolase 5
9. Fructose-1,6-bisphosphatase 4
10. phosphogluco-isomerase 3
11. Glucose-6-phosphatase 2 1
BYPASS 1
(3C)
lactate, alanine, other amino acids

5. Gluconeogenesis Pyruate is the major precursor for gluconeogenesis.
Cost: The production of glucose is energy expensive.
Input: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH
Output: glucose + 4 ADP + 2 GDP + 2 NAD++ 6 Pi
6 high energy bonds used per glucose synthesized
Pyruate is the major precursor for gluconeogenesis.
Lactate is the primary source for pyruvate.
In muscle, lactate is produced in great quantities during exercise and this extra lactate cannot be further oxidized in muscle.
So, Lactate is released from the muscles to the blood and travels to the liver for conversion to pyruvate and, ultimately to glucose.

6. Gluconeogenesis
In glycolysis, there are three irreversible kinase reactions at control points involving: hexokinase, phosphofructokinase, and pyruvate kinase.
In gluconeogenesis, these reactions must be forced the opposite way.
The control points are the same for gluconeogenesis and for glycolysis.
Four unique enzymes are used to replace or bypass these irreversible steps.
The rest of the steps use the same enzymes as glycolysis.

7. Gluconeogenesis Gluconeogenesis retains seven steps of glycolysis:
Steps 2 and 4-9
Three steps are replaced or bypassed.
1. Pyruvate carboxylase and PEP carboxykinase replace the pyruvate kinase reaction of glycolysis.
2. Fructose-1,6-bisphosphatase replaces the phosphofructokinase reaction of glycolysis.
3. Glucose-6-phosphatase replaces the hexokinase reaction of glycolysis.
The new reactions provide for a spontaneous pathway (DG negative in the direction of sugar synthesis), and they provide new mechanisms of regulation.

8. pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi
Bypass No. 1) Pyruvate to oxaloacetate & then PEP
The enzyme Pyruvate Carboxylase is located inside mitochondria. Only this enzyme of the gluconeogenesis pathway is mitochondrial.
pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi
Carboxylations reaction involve CO2 and almost always use the vitamin biotin (Vit B) as a cofactor.
For gluconeogenesis, oxaloacetate must leave the mitochondria because all the rest of the gluconeogenesis enzymes are in the cytosol.
mitochondrial membranes are nearly impermeable to oxaloacetate.

9. How does oxaloacetate transported from Mitochondria to cytosol??
1. At first Mitochindrial enzyme malate dehydrogenase convert Oxaloacetate to malate. This is reversible reaction.
2. Then malate is permeable to mitochnodrial membrane and goes out.
3. Now again the cytosolic malate dehydrogenase convert Malate to oxaloacetate.
Now this Oxaloacetate is converted to the phosphoenolpyruvate by enzyme enzyme PEP carboxykinase.
(Other steps are simple reaction)

10. REGULATION OF GLUCONEOGENESIS
Gluconeogenesis serves as an alternative source of glucose when supplies are low and is largely controlled by diet.
High carbohydrate in meal reduce gluconeogenesis and fasting increases.
key enzymes targeted.
Gluconeogenesis and glycolysis are controlled in reciprocal fashion.
Glucose-6-phosphatase is under substrate-level control, not allosteric control.
The fate of pyruvate depends on acetyl-CoA.
Fructose-1,6-bisphosphatase is inhibited by AMP, activated by citrate - the reverse of glycolysis.
Fructose-2,6-bisphosphate is an allosteric inhibitor of fructose-1,6-bisphosphatase.

11. REGULATION OF
GLUCONEOGENESIS
The principal regulatory mechanisms in glycolysis and gluconeogenesis. Activators are indicated by plus signs and inhibitors by minus signs.

12. Summary of Gluconeogenesis
REGULATION OF GLUCONEOGENESIS
Glucagon: hormone released when glucose levels are low. This is a signal to elevate blood glucose levels which increases intracellular levels of cAMP in liver.
cAMP activates protein kinase A that stimulate gluconeogenesis and glycogenolysis.
Summary of Gluconeogenesis
purpose- alternative source of glucose rather than dietary carbohydrates or glycogen breakdown.
primary precursors are pyruvate, lactate, glycerol, part of fatty acids and certain amino acids.
Three essentially irreversible steps of glycolysis are bypassed.
Regulated via pyruvate carboxylase, fructose 1,6 bisphosphatase, and 2-phosphofructokinase.

13. Lactate Formed in Muscles is Recycled to Glucose in the Liver
How your liver helps you during exercise:
Recall that vigorous exercise can lead to a buildup of lactate and NADH, due to oxygen shortage and the need for more glycolysis.
NADH can be reoxidized during the reduction of pyruvate to lactate.
Lactate is then returned to the liver, where it can be reoxidized to pyruvate by liver LDH.
Liver provides glucose to muscle for exercise and then reprocesses lactate into new glucose.
This is referred to as the Cori cycle.

14. Lactate Formed in Muscles is Recycled to Glucose in the Liver
The Cori cycle.

15. GLYCOGEN METABOLISM
Glycogen: These are highly branched polymer of glucose. Chains have glycosidic links α 14.
Branches are linked α 16.
Glucose stored in polymeric form as glycogen mostly in the liver and
skeletal muscle.
Glucose can be rapidly delivered to the blood stream when needed upon degradation of glycogen. (This process is called as glycogenolysis).
Enough glucose and energy triggers synthesis of glycogen. This process is called as glycogenesis.

16. Glycogenesis: GLYCOGEN BIOSYNTHESIS
Glucose is transported into the liver cell by a specific glucose transporter and immediately phosphorylated.
Most of the glucose in a cell is in the form of glucose-6-phosphate.
There are four steps in Glycogen Biosynthesis
1. Conversion of glucose-6-phosphate to glucose-1-phosphate
2. Synthesis of Uridine Diphosphoglucose
3. Glycogen synthesis
4. Branching

17. α-D-glucose-6- α-D- glucose-1- phosphate phosphate
Step1- Conversion of glucose-6-phosphate to glucose-1-phosphate by Enzyme phosphoglucomutase
α-D-glucose α-D- glucose-1-
phosphate phosphate
Reversible reaction allows G1P conversion to G6P in glycogenolysis.
 Mechanism involves phosphorylated enzyme intermediate and glucose-1,6 bisphosphate bound intermediate similar to phosphoglycerate mutase
phosphoglucomutase

18. Step 2- Synthesis of Uridine Diphosphoglucose
With help of enzyme UDP-glucose pyrophosphorylase.
UDP-glucose pyrophosphorylase
α-D- glucose-1- phosphate
Uridine diphosphate glucose
UDP-glucose is one of the sugar nucleotides. Discovered by Luis Leloir in the 1950s, they are “activated forms of sugar”.

19. Glycogen Synthase Catalyzes Formation of α(1→4) Glycosidic
Step 3- Glycogen synthesis by the enzyme glycogen synthase.
 UDP-glucose + (glucose)n UDP+(glucose)n+1
Glycogen Synthase Catalyzes
Formation of α(1→4) Glycosidic
Bonds in Glycogen.
Very large glycogen particle is
built around a single protein,
glycogenin, at the core.
The first glucose is linked to a
tyrosine -OH on the protein.
Sugar units are then added by
the action of glycogen synthase.
Glycogen synthase transfers
glucosyl units from UDP-glucose
to C-4 hydroxyl at a nonreducing
end of a glycogen strand.

20. Step 4- Branching by the enzyme called branching enzyme.
This branching enzyme introduces branching by transferring
a teminal fragment of 6-7 residues to a growing chain at 6-position.
Makes a branch with an α (16) link creating two ends to add glucose.
Branching accelerates the rate of glucose release during degradation.

21. Release of glucose-1-phosphate by the
GLYCOGENOLYSIS (DEGRADATION OF GLYCOGEN)
Release of glucose-1-phosphate by the
enzyme called glycogen phosphorylase. This enzyme always acts at nonreducing end. The 1,4 glycosidic link is cleaved by phosphorylysis.
This process stops at fourth glucose from a
1,6 branch point.
The de-branching enzyme such glucosidase or transferase breaks 1,6 branch and release glucose and glucose-1-phosphate molecules.

22. How Is Glycogen Metabolism Controlled?
Glycogen reserves are the most immediately available large source of metabolic energy for mammals.
Storage and utilization are under dietary and hormonal control.
1. Primary hormones
a).epinephrine (adrenaline,
b).glucagon
c).insulin
“fight-or-flight”)
2. Primary enzyme targets in glycogen metabolism are glycogen phosphorylase and glycogen synthase. The actions of the hormones are indirect.

23. 1. Control by hormones
a). Glucagon - released at low glucose levels
This is a polypeptide hormone produced in α-cells of the islets of Langerhan of the pancreas.
Acts primarily on liver cells because the
receptors of this hormones are found on surface of liver cells.
Stimulates glycogen breakdown & inhibits glycogenesis.
Glucagon also blocks glycolysis & stimulates gluconeogenesis.
b). Epinephrine – Also released at low glucose levels
 Acts primarily on skeletal muscle.
Stimulates glycogen breakdown & inhibits glycogenesis.
Glucagon and epinephrine both stimulate intracellular pathway via increasing levels of cAMP.

24. 1. Control by hormones
c). Insulin: -High levels of glucose induce release of insulin from β-cells of islets of Langerhan in the pancreas.
Insulin is polypeptide hormone and detected by receptors at surface of muscle cells.
Increases glycogenesis in muscle.
Intracellular signal pathway involves complex sequential Phosphorylations and dephosphorylations.
Insulin triggers protein kinases that stimulate insulin synthesis.
Insulin Modulates the Action of Glycogen Synthase in Several Ways

25. The Actions of Insulin on Metabolism
The metabolic effects of insulin are mediated through protein phosphorylation and second messenger modulation.

26. 2. Control by Enzymes SIMPLISTIC SUMMARY
Glycogen metabolism is a highly regulated process which involve the mutual control of glycogen phosphorylase and glycogen synthase.
GP allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine.
GS is stimulated by glucose-6-P. Both enzymes are regulated by covalent modification called phosphorylation.
SIMPLISTIC SUMMARY
Epinephrine and glucagon stimulate glycogenolysis and inhibit glycogenesis via a cAMP and a phosphorylation cascade.  releases of glucose
Glycogenesis is stimulated by insulin in a pathway ending in the dephosphorylation of glycogen
synthase.
Glycogenolysis is also inhibited via dephosphorylation.  takes up glucose

27. Pentose Phosphate Pathway
Cells are provided with a constant supply of NADPH for biosynthesis by the pentose phosphate pathway.
Also called the hexose monophosphate shunt.
This pathway also produces ribose-5-P .
This pathway consists of two oxidative processes followed by five non-oxidative steps.
It operates mostly in the cytosol of liver and adipose cells.
NADPH is used in cytosol for fatty acid and other biosynthesis.

28. pentose phosphate pathway
The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

29. pentose phosphate pathway
The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

30. The pentose phosphate pathway.

31. 1. The Oxidative Steps of the Pentose Phosphate Pathway
1. Glucose-6-P Dehydrogenase: Irreversible 1st step - highly regulated!
2. Gluconolactonase
3. 6-Phosphogluconate Dehydrogenase: An oxidative decarboxylation
The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway.

32. 2. The Gluconolactonase Reaction
1. The Oxidative Steps of the Pentose Phosphate Pathway
2. The Gluconolactonase Reaction
3. The 6-phosphogluconate dehydrogenase reaction

33. 2. The Nonoxidative Steps of the Pentose Phosphate Pathway
There are Five steps, but only 4 types of reactions...
Phosphopentose isomerase
converts ketose to aldose
Phosphopentose epimerase
epimerizes at C-3
Transketolase ( a TPP-dependent reaction)
transfer of two-carbon units
Transaldolase (uses a Schiff base mechanism)
transfers a three-carbon unit

34. 2. The Nonoxidative Steps of the Pentose Phosphate Pathway
The phosphopentose isomerase reaction converts a ketose to an aldose. The reaction involves an enediol intermediate.
The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate

35. 2. The Nonoxidative Steps of the Pentose Phosphate Pathway
The transketolase reaction of step 6 in the pentose phosphate pathway.
The transaldolase reaction

36. 2. The Nonoxidative Steps of the Pentose Phosphate Pathway
The transketolase reaction of step 8 in the pentose phosphate pathway. This is another two-carbon transfer, and it also requires TPP as a coenzyme.

37. Utilization of Glucose-6-P Depends on the Cell’s Need for ATP, NADPH, and Rib-5-P
Glucose can be a substrate either for glycolysis or for the pentose phosphate pathway
The choice depends on the relative needs of the cell for biosynthesis and for energy from metabolism
ATP can be made if G-6-P is sent to glycolysis
Or, if NADPH or ribose-5-P are needed for biosynthesis, G-6-P can be directed to the pentose phosphate pathway
Depending on these relative needs, the reactions of glycolysis and the pentose phosphate pathway can be combined in four principal ways

38. Four Ways to Combine the Reactions of Glycolysis and Pentose Phosphate
Both Ribose-5-P and NADPH are needed by the cell
In this case, the first four reactions of the pentose phosphate pathway predominate
NADPH is produced and ribose-5-P is the principal product of carbon metabolism
2) More Ribose-5-P than NADPH is needed by the cell
Synthesis of ribose-5-P can be accomplished without making NADPH, by bypassing the oxidative reactions of the pentose phosphate pathway

39. Four Ways to Combine the Reactions of Glycolysis and Pentose Phosphate
3) More NADPH than ribose-5-P is needed by the cell
This can be accomplished if ribose-5-P produced in the pentose phosphate pathway is recycled to produce glycolytic intermediates
4) Both NADPH and ATP are needed by the cell, but ribose-5-P is not
This can be done by recycling ribose-5-P, as in case 3 above, if fructose-6-P and glyceraldehyde-3-P made in this way proceed through glycolysis to produce ATP and pyruvate, and pyruvate continues through the TCA cycle to make more ATP

40. Xylulose-5-Phosphate is a Metabolic Regulator
In addition to its role in the pentose phosphate pathway, xylulose-5-P is also a signaling molecule
When blood glucose rises, glycolysis and the pentose phosphate pathways are activated in the liver
The latter pathway makes xylulose-5-P, which stimulates protein phosphatase 2A (PP2A)
PP2A dephosphorylates PFK-2/F-2,6-Bpase and also carbohydrate-responsive element-binding protein (ChREBP)
Glycolysis and lipid biosynthesis are both activated as a result