GluconeogenesisThe pathway and regulation

Arun.V.M.Sc. BMB14368005

Gluconeogenesis: an intro

• Defined as biosynthesis of glucose from non-carbohydrateprecursors.

• 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 bemet 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 theglucose production by 46 hours

The pathway

2-Phosphoglycerate

3-Phosphoglycerate

ATPADP

1,3-Bisphosphoglycerate

PiNAD+

NADH+ H+

Glyceraldehyde 3- P

Glycerol

DHAP

Glycerol 3- P

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 + ADPΔG = -8.0 kcal/ mol (-33 kJ /mol)

Fructose 6 – P+ ATP Fructose 1, 6 – P + ADPΔG = -5.3 kcal/ mol (-22 kJ /mol)

Phosphoenolpyruvate+ ADP Pyruvate + ATPΔG = -4.0 kcal/ mol (-17 kJ /mol)

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 phosphoenolpyruvatecarboxykinase (PEPCK) to form PEP

• GTP provides a source of energy & phosphate group of PEP.

Pyruvate to PEPPYRUVATE 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

Pyruvate to PEPPHOSPHOENOLPYRUVATE CARBOXYKINASE

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

• 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

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.

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

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

T1- transports glucose 6-phosphate into the lumen of the ERT2- transport Pi to the cytosolT 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

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.

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

Entry of glucogenic amino acids

• 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

• 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

• The hydrolysis of triacylglycerols in fat cells yields glycerol and fatty acids.

• Glycerol may enter either the gluconeogenic or the glycolyticpathway 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.

Glycolysis and gluconeogenesis

• Need of regulation

• There are three major types of regulation– Allosteric regulation

– Hormonal Regulation

– Transcriptional Regulation

Regulation of gluconeogenesis

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.

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

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.

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, bifunctionalProtein, which is regulated by glucagon and Insulin

Glucagon [cAMP] increases Protein

Kinase APhosphorelat

ion of enzyme

FBPase-2 Activity

Stimulate Glyconeogenesis

Hormonal Regulation

• 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

• 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. Glucogonprevents this phosphorylationand FOXO1 remains active in the nucleus

Transcriptional Regulation

FOXO1

FOXO1 P

PBinds with activators

Insulin binds

Inactive

• ChREBP• A TF. Carbohydrate response element binding protein

• Dephosphorelated by phosphoprotein phosphatase 2A. Xylulose 5-phosphate an intermediate in pentose posphatepathway 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

• 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

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

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