Gluconeogenesis is the process by which glucose is synthesized from non-carbohydrate precursors in the liver and kidneys. It occurs mainly during periods of fasting and involves converting substrates like lactate, glycerol, and certain amino acids into glucose. The pathway overcomes three thermodynamic barriers of glycolysis through smaller successive steps. Regulation occurs through allosteric control of enzymes, hormonal control of fructose 2,6-bisphosphate levels, and transcriptional control of key genes like PEPCK and FOXO1. Together these mechanisms help direct carbon fluxes towards gluconeogenesis or glycolysis based on energy demands.

1. Gluconeogenesis
The pathway and regulation
Arun.V.
M.Sc. BMB
14368005

2. Gluconeogenesis: an intro
• Defined as biosynthesis of glucose from non-carbohydrate
precursors.
• The major non-carbohydrate precursors are lactate, amino
acids, glycerol and the carbon skeletons of most amino
acids
• Non-carbohydrate precursors of glucose are first converted
into pyruvate or as oxaloacetate and DHAP
• When fasting, most of the body’s glucose needs must be
met by gluconeogenesis
• Occurs mainly in liver and to some extent in kidney
• Responsible for 64% of total glucose production over the
first 22 hours of the fast and accounts for almost all the
glucose production by 46 hours

3. The pathway
2-Phosphoglycerate
3-Phosphoglycerate
ATP
ADP
1,3-Bisphosphoglycerate
PiNAD+
NADH+ H+
Glyceraldehyde 3- P
Glycerol
DHAP
Glycerol 3- P

4. Thermodynamic Barriers
• Gluconeogenesis is not reversal of glycolysis.
• There are three major thermodynamic barriers for the
pathway which are three irreversible steps in glycolysis
• These three major barriers are bypassed by successive
smaller steps with relatively lesser ΔG.
Glucose + ATP Glucose 6 – P + ADP
ΔG = -8.0 kcal/ mol (-33 kJ /mol)
Fructose 6 – P+ ATP Fructose 1, 6 – P + ADP
ΔG = -5.3 kcal/ mol (-22 kJ /mol)
Phosphoenolpyruvate+ ADP Pyruvate + ATP
ΔG = -4.0 kcal/ mol (-17 kJ /mol)

5. Pyruvate to Phosphoenolpyruvate
• Endergonic & requires free energy input.
• This is accomplished by first converting the pyruvate to
oxaloacetate, a “high-energy” intermediate
• Exergonic decarboxylation OAA provides the free energy
necessary for PEP synthesis.
• CO2 is added to pyruvate by pyruvate carboxylase
enzyme
• CO2 that was added to pyruvate to form OAA is released
in the reaction catalyzed by phosphoenolpyruvate
carboxykinase (PEPCK) to form PEP
• GTP provides a source of energy & phosphate group of
PEP.

6. Pyruvate to PEP
PYRUVATE CARBOXYLASE
•A tetrameric protein of
identical 130-kD subunits
•Has a biotin prosthetic
group.
•Biotin functions as a CO2
carrier by acquiring a
carboxyl substituent at its
ureido group
•Biotin is covalently bound
to the enzyme by an amide
linkage of Lys residue to
form a biocytin

7. Pyruvate to PEP
PHOSPHOENOLPYRUVATE CARBOXYKINASE
•OAA is converted to PEP by PEPCK.
•Mg2+-dependent reaction requires
GTP as the phosphoryl group donor
•Reaction is reversible under
intracellular conditions

8. • Δ G‘o = 0.9 kJ/mol (Vs -17 kJ/mol of glycolysis) for this
reaction which make the reaction quite reversible
• But actually the Δ G under cellular condition is strongly
negative due to very lesser concentration of PEP favoring
a forward way of reaction (-25 kJ/mol)
• Thus the reaction is strongly irreversible
PEP + ADP + GDP +Pi + CO2
Pyruvate to PEP (overall reaction)
Pyruvate + ATP + GTP +HCO-
3

9. Alternative pathways
•There are two pathways for
PEP synthesis.
• one route involve
movement of reduction
equivalents to the cytoplasm,
which provides balance
between NADH produced and
consumed in the cytosol.
•Another route is prominent
when lactate is a source
which yields NADH for
gluconeogenesis. Hence
conversion to Malate is
unnecessary.

10. Fructose 1,6-Bis P to Fructose 6-P
• This step is irreversible hydrolysis of fructose 1,6-
bisphosphate to fructose 6-phosphate and Pi.
• Fructose 1,6-bisphosphatase (FBPase-1) Mg 2+ dependent
enzyme catalyzes this exergonic hydrolysis.
• It is present in liver, kidney, and skeletal muscle, but is
probably absent from heart and smooth muscle.
• it is an allosteric enzyme that participates in the regulation of
gluconeogenesis.
• Δ G = - 16.3 kJ/mol (Vs -22 kJ /mol of glycolysis)
Fructose 6-phosphate + PiFructose 1,6-bisphosphate +H2O

11. Glucose 6-P to Glucose
• This final step in the generation of glucose does not take place in
the cytosol.
• Glucose 6-P is transported into the lumen of the endoplasmic
reticulum, where it is hydrolyzed to glucose by glucose 6-
phosphatase, which is bound to the membrane at the luminal
side.
• This compartmentalisation can only be seen in glucose
producing cells like hepatocytes, renal cells and epithelial cells of
small intestine
• An associated Ca2+ binding stabilizing protein is essential for
phosphatase activity.
• Glucose and Pi are then shuttled back to the cytosol by a pair of
transporters.
Glucose+ PiGlucose 6-phosphate +H2O

12. T1- transports glucose 6-phosphate into the lumen of the ER
T2- transport Pi to the cytosol
T 3 – transport glucose to the cytosol.
SP- Ca2+binding protein
The glucose transporter in the endoplasmic reticulum membrane
is like those found in the plasma membrane.
Glucose 6-P to Glucose

13. Energetics of gluconeogenesis
• 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.
• Thus it is not a simple reversal of glycolysis but it is energetically an
expensive affair.

14. Substrates of gluconeogenesis
• The major substrates are the glucogenic amino acids, lactate, glycerol, and
propionate.
Entry of glucogenic amino acids
• Amino acids that are degraded to pyruvate, α-ketoglutarate, succinyl CoA,
fumarate, or oxaloacetate are termed glucogenic amino acids.
• The net synthesis of glucose from these amino acids is feasible because
these citric acid cycle intermediates and pyruvate can be converted into
phosphoenolpyruvate.
• Amino acids are derived from the dietary proteins, tissue proteins or from
the breakdown of skeletal muscle proteins during starvation.
• After transamination or deamination, glucogenic amino acids yield either
pyruvate or intermediates of the citric acid cycle

15. Entry of glucogenic amino acids

16. • In active skeletal muscle the rate of glycolysis
exceeds the rate of oxidative metabolism which leads
to anaerobic glycolysis in skeletal muscle
• During anaerobic glycolysis in skeletal muscle,
pyruvate is reduced to lactate by lactate
dehydrogenase (LDH).
• Lactate is readily converted into pyruvate by the
action of lactate dehydrogenase.
Entry of Lactate

17. • Propionate is a major precursor of
glucose in ruminants.
• It enters gluconeogenesis via the
citric acid cycle.
• In non-ruminants, including
humans, propionate arises from
the Beta -oxidation of odd-chain
fatty acids that occur in ruminant
lipids, as well as the oxidation of
isoleucine and the side-chain of
cholesterol, and is a (relatively
minor) substrate for
gluconeogenesis.
Entry of Propionate

18. • The hydrolysis of triacylglycerols in fat cells yields glycerol and
fatty acids.
• Glycerol may enter either the gluconeogenic or the glycolytic
pathway at DHAP
• In the fasting state glycerol released from lipolysis of adipose
tissue triacylglycerol is used solely as a substrate for
gluconeogenesis in the liver and kidneys.
Entry of Glycerol
Glycerol Kinase is absent in adipose tissue hence the formed glycerol is transported
to liver and used as per the need of the hour.

19. Glycolysis and
gluconeogenesis

20. • Need of regulation
• There are three major types of regulation
– Allosteric regulation
– Hormonal Regulation
– Transcriptional Regulation
Regulation of gluconeogenesis

21. Allosteric regulation
• Phosphofructokinase-1 (PFK-1)
– Enzyme has several regulatory sites at which allosteric activators or
inhibitors bind
– ATP inhibits PFK-1 by binding to an allosteric site and lowering the
affinity of the enzyme for fructose 6-phosphate
– ADP and AMP act allosterically to relieve this inhibition by ATP.
– High citrate concentration increases the inhibitory effect of ATP.
– Thus glycolysis is down regulated when enough ATP is present in cells.

22. Allosteric regulation
• Fructose 1,6- bisphosphatase-1 (FBPase1)
– Inhibited by AMP, when energy currency ATP is less
– Thus there gluconeogenesis is down regulated because it is a energy
consuming process.
– The opposing effect of PFK-1 and FBPase-1 helps to regulate glycolysis
and gluconeogenesis according to current need of cell

23. Hormonal Regulation
• hormonal regulation of glycolysis and gluconeogenesis is mediated by
fructose 2,6-bisphosphate.
• F2,6-BP binds to allosteric site on PFK-1 increases that its affinity for
substrate F 6-P, & reduces its affinity for the allosteric inhibitors ATP
and citrate.
•PFK-1 is virtually inactive in the absence of F2,6-BP
•F2,6-BP activates PFK-1 and stimulates glycolysis in liver
•F2,6-BP inhibits FBPase-1 slowing gluconeogenesis.

24. Hormonal Regulation
• F2,6-BP formed by phosphorylation of fructose 6-phosphate,
catalyzed by phosphofructokinase- 2 (PFK-2), and is broken down by
fructose 2,6- bisphosphatase (FBPase-2).
PFK-2 and FBPase-2 are
•two distinct enzymatic activities of a single, bifunctional
Protein, which is regulated by glucagon and Insulin
Glucagon [cAMP]
increases Protein
Kinase A
Phosphorelat
ion of
enzyme
FBPase-2
Activity
Stimulate
Glyconeogenesis

25. Hormonal Regulation

26. • CREB
– Glucagon causes cAMP to rise during fasting.
– Epinephrine acts during exercise or stress.
– cAMP activates protein kinase A, which phosphorylates CREB that
stimulate transcription of the PEPCK
– Increased synthesis of mRNA for PEPCK results in increased synthesis
of the enzyme PEPCK
– Cortisol also induces PEPCK.
– Insulin stimulates inactivation of TF of PEPCK, FBTase and Glucose 6 –
phosphatase
Transcriptional Regulation

27. • FOXO1
• Forkhead box other -1
• Stimulates synthesis of
gluconeogenic enzymes
• Suppresses the synthesis of
enzymes of glycolysis, pentose
phosphate pathway,
triacylglycerol synthesis
• Insulin phosphorelate FOXO1
there by inhibiting the
gluconeogenesis. Glucogon
prevents this phosphorylation
and FOXO1 remains active in
the nucleus
Transcriptional Regulation
FOXO
1
FOXO
1 P
P
Binds with
activators
Insulin
binds
Inactive

28. • ChREBP
• A TF. Carbohydrate response element binding protein
• Dephosphorelated by phosphoprotein phosphatase 2A.
Xylulose 5-phosphate an intermediate in pentose posphate
pathway activates PP2A
• Acticated ChREBP joins with Mlx a parter protein and binds
with ChoRE
• Turns on:
– Pyruvate kinase
– Fatty acid synthase complex
– Acetyl –CoA carboxylase
Transcriptional Regulation

29. • SREBP-1c
• A TF. A member of family of sterol response element binding
proteins.
• Turns on:
– Pyruvate kinase
– Hexokinace IV
– Lipoprotein lipase
– Acetyl-CoA carboxylase
– Fatty acid synthase complex
• Turns off:
– Glucose 6- phosphatase
– PEP Carboxykinase
– FBPae-1
Transcriptional Regulation

30. Reference
• Michael M.Cox, David L. Nelson. Principles of
Biochemistry, Fifth Edition. W.H. Freeman and
Company
• Berg.M.J, Tymoczko.l.John, Stryer.L. Biochemistry,
Fifth Edition, W.H. Freeman and Company
• Voet.D., Voet.G.J, Biochemistry, Fourth Edition,
John Wiley and Sons, INC.
• Smith.C., Marks.D.A, Lieberman.M., Mark’s Basic
Medical Biochemisty A clinical Approach, Second
Edition, Lippincott Williams and Wilkins

31. Thank you