Gluconeogenesis is the process by which glucose is synthesized from non-carbohydrate precursors in the liver and kidneys. During periods of fasting, hepatic stores are depleted and glucose must be formed from substrates like lactate, pyruvate, glycerol, and amino acids. The key steps involve carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase, transport of oxaloacetate to the cytoplasm, its decarboxylation to phosphoenolpyruvate by PEP carboxykinase, and the reversal of glycolytic reactions to generate glucose-6-phosphate and free glucose. Gluconeogenesis is regulated by hormones like glucagon and the

1. Gluconeogenesis
 Some tissues, such as brain, RBCs, kidney
medulla, testes, embrionic tissues and exercising
muscle require a continuous supply of glucose for
metabolic energy.
– The human brain requires over 120 gm of glucose per
day.
 Mammalian cells make glucose from simpler
precursors.
 Liver glycogen can meet these needs for only 10 to
18 hours without dietary carbohydrate.

2. During a prolonged fast,
 Hepatic liver stores are depleted, glc is formed from
other molecules, such as
• Lactate
• Pyruvate
• Glycerol
• Alpha keto acids
 The formation of glc from nonhexose precursors is
called gluconeogenesis (formation of new sugar).

7. Pyruvate precursors
 The direct Glc reserves are sufficient to meet Glc needs
for about a day!
 Gluconeogenic pathway makes Glc from pyruvate
precursors.
 Triacyl glycerol  Glycerol + Fatty acids
 Glycerol is a precursor of glc, glycerol enters glycolytic
pathway as dihydroxyacetone phosphate.

9. Gluconeogenesis is NOT a reversal of glycolysis
 Several reactions MUST differ because of the
irreversible steps.
• HK (hexokinase)
• PFK (phosphofructokinase)
• PK (pyruvate kinase)

14. Let’s make Glc from pyruvate
1. Carboxylation of pyruvate
Pyruvate + CO2 + ATP + H2O  OA +ADP + Pi + 2H
» Enzyme: Pyruvate carboxylase
OA + GTP  PEP+ GDP + CO2
» Enzyme: PEP-carboxykinase

16. Domain structure of pyruvate carboxylase
ATP grasp:
• Activates bicarbonate ions and transfers CO2 to the
biotin domain.
• From there, CO2 is transferred to pyruvate.

17. Carboxylation of pyruvate
 Pyruvate carboxylase contains BIOTIN, which is
covalently bound to the enzyme through lysine
• Enzyme + CO2 + ATP-----> Carboxybiotin-enzyme
+ADP +Pi
• Carboxybiotin-enzyme + pyruvate------->OA +
Enzyme
– BIOTIN carries CO2...

18. Biotin is covalently attached group
 Biotin serves as a carrier for activated CO2.
 -amino group and carboxylate group of biotin are linked.
 CO2 is found mainly as HCO3 in our system.
 When Acetyl CoA is high, then biotin is carboxylated.
 The activated carboxyl group is transferred from
carboxybiotin to pyruvate to form oxaloacetate.

22. 2. Transport of OA to the cytoplasm
• Pyuvate carboxylase is a mitochondrial enzyme,
whereas the other enzymes in gluconeogenesis are
cytoplasmic.
• OA should be transported to the cytoplasm.
• How?
– It is reduced to MALATE first and then transferred to
the cytoplasm.
– In the cytoplasm, it is reoxidized to OA.

24. 3. Decarboxylation of cytoplasmic OA
 OA is decarboxylated and P-lated by PEP carboxykinase
in the cytosol (PEP is made then!)
 The overall reaction catalyzed by the combined action of
pyruvate carboxylase and PEP carboxykinase provides a
pathway from Pyruvate  PEP.
 Therefore, once PEP is formed, it enters the reversed
reactions of glycolysis until it reaches F-1,6 Bisphosphate!

27. 4. Dephosphorylation of F-1,6BP
Fructose 1,6-bisphosphate + H2O  F-6-P + Pi
» Enzyme: Fructose1,6-bisphosphatase
 This enzyme plays an important role in regulation.
 It is inhibited by F 2,6 BisP, an allosteric modifier
whose concentration is influenced by the levels of
circulating glucagon.
 This enzyme is found in liver and kidney.

28. 5. Generation of free Glc
Dephospharylation of Glc 6-P
Glc 6-P + H2O  D-Glc + Pi
» Enzyme: Glc 6-phosphatase
 It is found in liver and kidney but not in muscle and brain.
 Thus, muscle and brain cannot make Glc by
gluconeogenesis
 Type I glycogen storage disease results from an inherited
deficiency of glc 6-phosphatase.

29. Freeing Glc
 The final step, freeing Glc, takes place in ER lumen where it
is hydrolyzed to Glc by Glc 6-Phosphatase, a membrane
bound enzyme.
 Calcium binding protein (SP) is necessary for phosphatase
activity.
 Glc and Pi are shuttled back to the cytosol by a pair of
transporters.
 The glucose transporter in the ER membrane is like those
found in the plasma membrane.

31. Gluconeogenesis is energetically costly!
 The stoichiometry of gluconeogenesis is:
2 pruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O 
Glc + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+
 In contrast, the stoichiometry of reversal of glycolysis is:
2 pyruvate + 2 ATP + NADH + 2 H2O 
Glc + 2 ADP + 2 Pi + 2 NAD+ + 2 H+
 The difference is 4 ATP. This is needed to turn energetically
unfavorable process to a favorable one!

32. Gluconeogenesis and glycolysis
are reciprocally regulated
 Both glycolysis and gluconeogenesis are highly exorgonic
under cellular conditions so there is no thermodynamic
barrier.
 But, amounts and activities of the distinctive enzymes of
each pathway are controlled so that both pathways are not
highly active at the same time.

34. Substrate cycles
F-6-P  F 1,6BisP
 A pair of reactions such as the above one is called
“substrate cycle”
 There is also some cycling in irreversible reactions.
 “Imperfection” in metabolism?
 They are sometimes referred as “futile cycles”
• Futile cycles amplify metabolic signals!
 The other potential biological role of substrate cycles is the
generation of heat produced by the hydrolysis of ATP.

38. Lactate and alanine formed by contracting
muscle are used by other organs
 Lactate is a dead end in metabolism.
 Lactate should be converted to pyruvate.
 The plasma membranes of most cells are highly permeable
to lactate and pyruvate; therefore, they easily diffuse to go
to liver!
 Excess lactate enters the liver and is converted to pyruvate
first and then to glucose.
• Thus, the liver restores the level of glucose necessary for active
muscle cells, which derive ATP from the glycolytic conversion of
glucose into lactate. Contracting skeletal muscle supplies lactate to the
liver, which uses it to make glucose.
• These reactions constitute the CORI CYCLE.

39. LDH enzyme
 Lactate  Pyruvate by LDH (lactate dehydrogenase).
 The interconversion of pyruvate and lactate are done by
different subunits of LDH. LDH is a tetramer.
 H  in the heart
 M  in the muscle

41. REGULATION
1. Control point: Pyruvate carboxylase
 Acetyl CoA is a + allosteric modulator for the pyruvate
carboxylase enzyme.
– Glc is made from pyruvate when there is a lot of Acetyl CoA
(more Acetyl CoA than TCA cycle can handle)
– Acetyl CoA inhibits the pyruvate dehydrogenase enzyme but
stimulates the pyruvate carboxylase.
2. Control point: F 1,6 bisphoshatase reaction
3.Control point: F-2,6 bisphosphate
 Hormonal control

48. Hormonal Control
 The special role of the liver is to maintain constant
blood glucose level and requires additional control
mechanisms.
 When blood glucose decreases, glucagon increases and
glucose is released.
 This hormonal regulation in the liver is mediated by
fructose-2,6-bisphosphate, which is a allosteric effector
for PFK-1, and F-1,6-bisphosphate

50. Role of F2,6BP in regulation of Glycolysis
and Gluconeogenesis

51. What is F-2,6-BP?
 It is structurally related to F-1,6-BP.
 It is not an intermediate.
 It is a “regulator”
 F-2,6-BP activates PFK-1 and glycolysis.
 FBPase and PFK-2 are part of the same enzyme!
 An increase in glucagon (during starvation) leads to a
decrease in F-2,6-BP overall which goes to a decrease in
glycolysis, an increase in gluconeogenesis
 A decrease in glucagon (after a carbohydrate rich diet)
leads to an increase in F-2,6-BP and an increase in
glycolysis.
 Therefore, F-2,6-BP acts as an intracellular signal
indicating “glucose abundant”.

52. Pathway integration
 Glycolysis and gluconeogenesis are coordinated in
a tissue specific fashion
 Consider a sprinter
 Skeletal muscle  lactate will build up
 Cardiac muscle  lactate will be converted into
pyruvate
 Liver  gluconeogenesis, a primary function of
the liver, will take place to ensure that enough Glc
is present in the blood for skeletal and cardiac
muscle, as well as for other tissues.