THE UNIVERSITY OF ZAMBIA

SCHOOL OF MEDICINE

DEPARTMENT OF PHYSIOLOGICAL SCIENCES

BIOCHEMISTRY

Mr Mwale Nicholas K.

Email: mwalenicholask@unza.zm/ mwalenicholask@yahoo.com

GLUCONEOGENESIS

OBJECTIVES

Gluconeogenesis

Precursors of gluconeogenesis

Regulation of gluconeogenesis

RESOURCES

All images and notes are taken from the following unless otherwise stated;

Nelson, D. L & Cox, M. M; Lehninger Principles of Biochemistry Fifth Edition (2008). W.H. Freeman & Co. Pp 551-558, 583-590.

Introduction

o Sugar- almost a universal energy source

o Some tissues solely exist on glucose

o Major fuel source for brain, renal medulla

(kidney), embryonic tissues, testes, red blood

cells and nervous system.

o Brain alone requires 120g.

o More than half of all glucose is stored as

glycogen in muscles and liver.

Introduction cont…. • Blood glucose and glycogen stores are easily

depleted after periods of fasting or vigorous exercise.

• During these periods, organisms (plants, animals fungi and microorganisms) need to synthesize glucose from NON CARBOHYDRATE sources.

• Gluconeogenesis= new formation of sugar.

GLUCONEOGENESIS

• Important gluconeogenic precursors include; Lactate Pyruvate Glycerol Certain amino acids

• Generally three and four carbon compounds serve as precursors.

• Gluconeogenesis in mammals occurs mainly in the liver (to lesser extent in kidney and intestinal wall cells).

GLUCONEOGENESIS cont… • New glucose produced goes via blood to supply

tissues

• Lactate from skeletal muscle goes via blood to liver.

• In liver this get converted back to glucose and in muscle will be stored as glycogen.

• This cycle is called the Cori Cycle.

Cori Cycle Active muscles use up glycogen for energy. Lactate is generated during anaerobic respiration. Lactate liver (generates glucose). Glucose stored as glycogen in muscle.

Gluconeogenesis cont….

• Occurs in opposite direction to glycolysis (but steps not completely identical).

• 7 of 10 reactions use the same enzymes but 3 reactions are irreversible (high negative free energy change) in glycolysis and use unique enzymes during the gluconeogenetic pathway; – 1. Conversion of phosphoenolpyruvate to pyruvate

– 2. Conversion of Fructose-6-phosphate to

fructose 1,6-phosphate.

– 3. Conversion of Glucose to Glucose-6-phosphate

Opposing pathways of glycolysis and gluconeogenesis

GLYCOLYSIS Vs GLUCONEOGENESIS CONT’D.

Gluconeogenesis cont… • In the gluconeogenesis pathway, the 3

irreversible reactions of glycolysis require enzymes and input of energy.

• These enzymes ensure that the reactions are

exergonic and thus irreversible.

• Both glycolysis and gluconeogenesis occur in the cytosol and are regulated by the irreversible enzymatic stages.

Conversion of pyruvate to PEP • Pyruvate from cytosol is transported to

mitochondria.

• Pyruvate can be obtained from alanine by transamination.

• When pyruvate or alanine are the glucogenic precursor, the following reactions are predominant;

Pyruvate oxaloacetate phosphoenolpyruvate

Enzymes: pyruvate carboxylase and PEP carboxykinase

Alanine

Conversion of pyruvate to PEP cont…

• First regulatory reaction of pyruvate to oxaloacetate requires Pyruvate carboxylase (with biotin prosthetic group).

• Acetyl CoA produced during fatty acid metabolism is a positive effector.

• Oxaloacetate produced must be transported to cytosol. • Done by reducing to malate by mitochondrial malate DHase then it passes through the malate transporter and cytosolic malate dehydrogenase reconverts malate to oxaloacetate.

Conversion of pyruvate to PEP cont….

• Two high energy phosphate based molecules are required to convert pyruvate to PEP.

• Each yields ~50kJ/mol.

• During glycolysis 1 PEP to

pyruvate only uses 1 ATP.

• Carboxylation of pyruvate serves

to activate the pyruvate molecule

in prep for subsequent reactions.

• If lactate is the pyruvate

precursor a slightly different

path is followed.

Conversion of Fructose 1,6 BP to fructose 6 P

• In glycolysis this is the second irreversible reaction catalyzed by phosphofructokinase-1.

• To reverse this during gluconeogenesis, the Mg²⁺ dependent fructose 1,6-bisphophatase is used here.

• Hydrolysis of the phosphate group at C1 is promoted by this enzyme.

Conversion of Glucose-6 P to glucose

• This is the reversal of the hexokinase reaction involving the dephosphorylation of glucose-6-phosphate.

• This is energetically unfavorable -requires transfer of Phosphate to ADP forming ATP.

• To overcome this the enzyme glucose-6-phosphatase catalyzes the HYDROLYSIS of the phosphate group.

• Enzyme found in liver and renal cells- glucose made transported to brain and muscle via blood.

Overview of gluconeogenesis • Gluconeogenesis is expensive but highly necessary

for maintenance of blood sugar.

• Overall energy expenditure is;

• Irreversible reactions commit the substrate into a singular metabolic pathway.

• This also serves to regulate the pathway.

Precursors of gluconeogenesis • 4,5,6 carbon molecules from the citric acid cycle

can be oxidized to oxaloacetate.

Precursors of gluconeogenesis cont…

• Carbon atoms of many amino acids can be catabolized to pyruvate or intermediates of the citric acid cycle (which are then oxidized to oxaloacetate).

Precursors of gluconeogenesis cont..

• Acetyl CoA from fatty acid catabolism in contrast does not serve as a precursor for gluconeogenesis.

• Amino acids, glycerol and other compounds that can be catabolized into components for gluconeogenesis are called glucogenic compounds.

• Glycolysis and gluconeogenesis are regulated such that when glycolysis is high gluconeogenesis is low.

• Regulation occurs by allosteric and covalent modification.

REGULATION OF GLUCONEOGENESIS • Both glycolysis (sugar breakdown) and

gluconeogenesis (making sugar from non carb sources) occur in cell cytosol.

• Means these two processes are regulated in a coordinated manner so when glycolysis is favored, gluconeogenesis is hindered.

• The three irreversible reactions serve as a regulatory point.

• Other substrates and by products such as ATP, AMP, reaction intermediates have an effect on the enzymes.

• Hormones also regulate enzyme activity.

Regulation of gluconeogenesis cont.. • Phosphofructokinase-1 which catalyzes the

reaction below is inactive when fructose-2,6-bisphosphate is absent.

• This occurs even at physiological concentrations of other allosteric effectors such as AMP.

• (AMP signals low cell energy, ATP signals the opposite)

Effects of F-2,6-BP on glycolysis and gluconeogenesis

OTHER EFFECTORS INVOLVED IN GLYCOLYSIS AND GLUCONEOGENESIS

Regulation of gluconeogenesis cont..

• The phosphorylated sugar, F-2,6-BP (shown below) is in turn regulated by a bifunctional protein.

• PFK-2= Phosphofructokinase 2

• FBPase = F-2,6 bisphosphatase

PFK-2 PFK-2 FBPase-2

Catalyzes break down of F-2,6-BP

Catalyzes formation of F-2,6-BP

Regulation of Gluconeogenesis cont… • Activity of this bifunctional protein depends

on whether it is phosphorylated or not.

• Phosphorylation of this protein enhances the activity of FBPase- (breaks down F-2,6-BP-inactivates PFK-1-glycolysis stops and gluconeogenesis is promoted).

• Glucagon hormone-signals low glucose levels • Glucagon activates formation of cAMP • cAMP activates cAMP dependent protein kinases • These kinases phosphorylate the bifunctional

protein.

ACTIVATION/INACTIVATION OF ENZYMES BY MODIFICATION OF RESIDUES

Effects of hormones insulin and glucagon on glycolysis and gluconeogenesis

Regulation of gluconeogenesis cont..

• cAMP dependent protein kinases also phosphorylate liver pyruvate kinases (different isozyme in muscle) caused by glucagon.

• This slows use of glucose for energy and diverts it to brain and other organs.

• In muscle, epinephrine (adrenalin) enhances cAMP.

• Kinases enhanced by this activate glycogen breakdown (producing glucose for fuel).

Regulation of gluconeogenesis cont… • Acetyl CoA allosterically enhances the

activity of pyruvate carboxylase- promoting conversion of glucogenic substrates to glucose (gluconeogenesis).

• Acetyl CoA also inhibits the enzyme that converts pyruvate to acetylCoA (pyruvate dehydrogenase complex) thus slowing down the electron transport chain.

• Overall high cell energy (NADH, ATP etc.) reduces glycolysis and favors gluconeogenesis.

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Gluconeogenesis and glycolysis are reciprocally controlled