Gluconeogenesis - The Pathway and Regulation

Gluconeogenesis: Defined as biosynthesis of glucose from non-carbohydrate precursors
-Gluconeogenesis: an intro
-Thermodynamic Barriers (Each barrier detail explanation)
- Energetics of gluconeogenesis
-Substrates of gluconeogenesis (each substrate and pathway explained)
-Regulation of Gluconeogenesis, hormonal and transcriptional regulation

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

2. Gluconeogenesis: an intro
•Definedasbiosynthesisofglucosefromnon-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 pyruvateor as oxaloacetateand DHAP
•Whenfasting,mostofthebody’sglucoseneedsmustbe
metbygluconeogenesis
•Occursmainlyinliverandtosomeextentinkidney
•Responsiblefor64%oftotalglucoseproductionoverthe
first22hoursofthefastandaccountsforalmostallthe
glucoseproductionby46hours

3. The pathway
2-Phosphoglycerate
3-Phosphoglycerate
ATP
ADP
1,3-Bisphosphoglycerate
PiNAD+
NADH+ H+
Glyceraldehyde3-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. Pyruvateto Phosphoenolpyruvate
•Endergonic& requires free energy input.
•This is accomplished by first converting the pyruvateto
oxaloacetate, a “high-energy” intermediate
•ExergonicdecarboxylationOAA provides the free energy
necessary for PEP synthesis.
•CO
2is added to pyruvateby pyruvatecarboxylase
enzyme
•CO
2 that was added to pyruvateto 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. Pyruvateto PEP
PYRUVATE CARBOXYLASE
•A tetramericprotein of
identical 130-kD subunits
•Has a biotin prosthetic
group.
•Biotin functions as a CO2
carrier by acquiring a
carboxyl substituent at its
ureidogroup
•Biotin is covalently bound
to the enzyme by an amide
linkage of Lys residue to
form abiocytin

7. Pyruvateto PEP
PHOSPHOENOLPYRUVATE CARBOXYKINASE
•OAA is converted to PEP by PEPCK.
•Mg
2+
-dependent reaction requires
GTP as the phosphorylgroup 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 +CO
2
Pyruvateto 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-
bisphosphateto fructose 6-phosphate and Pi.
•Fructose 1,6-bisphosphatase (FBPase-1) Mg 2+ dependent
enzyme catalyzes this exergonichydrolysis.
•It is present in liver, kidney, and skeletal muscle, but is
probably absent from heart and smooth muscle.
•it is an allostericenzyme 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 +H
2O

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 compartmentalisationcan only be seen in glucose
producing cells like hepatocytes, renal cells and epithelial cells of
small intestine
•An associated Ca
2+
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 +H
2O

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-Ca
2+
binding protein
The glucose transporter in the endoplasmic reticulum membrane
is like those found in the plasma membrane.
Glucose 6-P to Glucose

13. Energeticsof 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 glucogenicamino acids, lactate, glycerol, and
propionate.
Entry of glucogenicamino acids
•Amino acids that are degraded to pyruvate, α-ketoglutarate, succinylCoA,
fumarate, or oxaloacetateare termed glucogenicamino acids.
•The net synthesis of glucose from these amino acids is feasible because
these citric acid cycle intermediates and pyruvatecan 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 transaminationor deamination, glucogenicamino acids yield either
pyruvateor intermediates of the citric acid cycle

15. Entry of glucogenicamino 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,
pyruvateis reduced to lactate by lactate
dehydrogenase(LDH).
•Lactate is readily converted into pyruvateby 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 acidsthat occur in ruminant
lipids, as well as the oxidation of
isoleucineand the side-chain of
cholesterol, and is a (relatively
minor) substrate for
gluconeogenesis.
Entry of Propionate

18. •The hydrolysis of triacylglycerolsin fat cells yields glycerol and
fatty acids.
•Glycerol may enter either thegluconeogenicor the glycolytic
pathway at DHAP
•In the fasting state glycerol released from lipolysisof adipose
tissue triacylglycerolis 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
–Allostericregulation
–Hormonal Regulation
–Transcriptional Regulation
Regulation of gluconeogenesis

21. Allostericregulation
•Phosphofructokinase-1 (PFK-1)
–Enzyme has several regulatory sites at which allostericactivators or
inhibitors bind
–ATP inhibits PFK-1 by binding to an allostericsite and lowering the
affinity of the enzyme for fructose 6-phosphate
–ADP and AMP act allostericallyto 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. Allostericregulation
•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 allostericsite on PFK-1 increases that its affinity for
substrate F 6-P, & reduces its affinity for the allostericinhibitors 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 phosphorylationof 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
ionof
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, FBTaseand Glucose 6 –
phosphatase
Transcriptional Regulation

27. •FOXO1
•Forkheadbox other -1
•Stimulates synthesis of
gluconeogenicenzymes
•Suppresses the synthesis of
enzymes of glycolysis, pentose
phosphate pathway,
triacylglycerolsynthesis
•Insulin phosphorelateFOXO1
there by inhibiting the
gluconeogenesis. Glucogon
prevents this phosphorylation
and FOXO1 remains active in
the nucleus
Transcriptional Regulation
FOXO
1
FOXO
1P
P
Binds with
activators
Insulin
binds
Inactive

28. •ChREBP
•A TF. Carbohydrate response element binding protein
•Dephosphorelatedby phosphoproteinphosphatase 2A.
Xylulose5-phosphate an intermediate in pentose posphate
pathway activates PP2A
•ActicatedChREBPjoins with Mlxa parterprotein and binds
with ChoRE
•Turns on:
–Pyruvatekinase
–Fatty acid synthasecomplex
–Acetyl –CoAcarboxylase
Transcriptional Regulation

29. •SREBP-1c
•A TF. A member of family of sterol response element binding
proteins.
•Turns on:
–Pyruvatekinase
–HexokinaceIV
–Lipoprotein lipase
–Acetyl-CoAcarboxylase
–Fatty acid synthasecomplex
•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 BiochemistyA clinical Approach, Second
Edition, Lippincott Williams and Wilkins

31. Thank you