Gluconeogenesis

Synthesis of "new glucose" from common metabolites

•Humans use ~160 g of glucose per day

•75% is used by the brain

•Body fluids contain only 20 g of glucose

•Glycogen stores yield 180-200 g of glucose

•So the body must be able to make its own glucose

• 90% of gluconeogenesis occurs in the liver and kidneys

Gluconeogenesis

Why is gluconeogenesis not just the reverse of glycolysis?

• The reverse of glycolysis is• 2 Pyruvate + 2ATP + 2 NADH + 2H+ + 2H2O glucose +2ADP +2Pi + 2 NAD + (G = +74 kJ/mol)• This is thermodynamically unfavorable, so energetically unfavorable

steps in the reverse glyolysis reaction are replaced and energy is added in the form of GTP and ATP to give:

• The actual equation for gluconeogenesis• 2Pyruvate + 4ATP +2GTP+ 2NADH + 2H+ + 6H2O glucose +4ADP +2GDP +6Pi + 2 NAD + (G = -38 kJ/mol)

• Notice the extra ATPs and GTPs drive the process

Glycolysis <-> gluconeogenesis

Gluconeogenesis is not the reversal of glycolysis !!!

Glycolysis: in the cytosol

Gluconeogenesis: major part in cytosol-> 1st step in mitochondria -> shuttle

Reverse reaction of glycolysis thermodynamically not favorable !!!

Biotin: prosthetic group -> carrier for CO2

4

Glycolysis vs Gluconeogenesis

• Glycolosis• Glucose (6C) to 2 pyruvates (3

C)• Creates energy 2ATP• Reduces 2 NAD+ to 2 NADH• Active when energy in cell low• 10 steps from glucose to pyruv

ate • Pyruvate to AcCoA before Kre

bs

• Gluconeogenesis• 2 pyruvates (3C) to Glucose (6

C)• Consumes energy 4ATP+2GT

P• Oxidizes 2NADH to 2 NAD+• Active when energy in cell high• 11 steps from pyruvate to gluc

ose• AcCoA isn’t used in gluconeog

enesis

Gluconeogenesis uses 7 of the 10 enzymatic reactions of glycolysis but in the reverse direction. The 3 not used are the ones requiring ATP in glycolysis.

Synthesis of glucose from non-carbohydrate precursors:

-> gluconeogenesis

• Brain and blood cells depend on glucose -> 160g/day (mainly for the brain)

• Glucose in the blood: 20g

• Starvation > 1day other metabolites for energy!

-> Gluconeogenesis pathway:

• Takes place in liver (and kidneys)

• Important to maintain blood glucose level

• Major precursors: glycerol, amino acids, lactic acid

• Specific enzymes in addition to glycolysis

(for the irreversible steps in glycosis) 7

Synthesis of glucose from non-carbohydrate precursors: -> gluconeogenesis

Pyruvate (end product of glycolysis) -> under aerobic conditions -> shuttle into Mitochondria -> converted into acetyl-CoA -> citric acid cycle

Gluconeogenesis -> start with pyruvate in mitochondria

1st Step: convertion to oxaloacetate

-> malate/oxaloacetate shuttleglycolysis

8

Synthesis of glucose from non-carbohydrate precursors: -> gluconeogenesis

Triacylglycerols (Lipids) taken up by diet

-> brocken down to fatty acids and glycerol

cannot be converted to glucose

glucose

9

Substrates for gluconeogenesis:

Not substrates for gluconeogenesis:

PyruvateLactateGlycerolTCA cycle intermediatesMost amino acids

Acetyl-CoAFatty acidsLysineLeucine

Regulatory enzymes of gluconeogenesis

• Pyruvate carboxylase• PEP carboxykinase (PEPCK)• Fructose-1,6-bisphosphatase• Glucose-6-phosphatase

The pyruvate carboxylase reaction.

First Reaction of Gluconeogenesis - recall that pyruvate is the final product of gl

ycolysis.

Oxaloacetate is the starting material for gluconeogenesis

Regulation of Gluconeogenesis

Glucose-6-phosphatase is subject to substrate level control.

- at higher G6P concentrations reaction rate increases

- recall, this happens in the liver. Other tissues do not hydrolyze their G6P, thereby trapping it in the cells.

Glycolysis and gluconeogenesis are reciprocally regulated.

- regulatory molecules that inhibit gluconeogenesis often activate glycolysis, and vise versa.

Pyruvate is converted to oxaloacetate before being changed to Phosphoenolpyruvate

1. Pyruvate carboxylase catalyses the ATP-driven formation of oxaloacetate from pyruvate and CO2

2. PEP carboxykinase (PEPCK) concerts oxaloacetate to PEP that uses GTP as a phosphorylating agent.

Biotin is an essential cofactor in most carboxylation reactions.

It is an essential vitamin in the human diet, but deficiencies are rare.

Pyruvate carboxylase requires biotin as a cofactor

Biotin is an essential nutrient

There is hardly any deficiencies for biotin because it is abundant and bacteria in the large intestine also make it.

However, deficiencies have been seen and are nearly always linked to the consumption of raw eggs.

Raw eggs contain Avidin, a protein that binds biotin with a Kd = 10-15 (that is one tight binding reaction!)

It is thought that Avidin protects eggs from bacterial invasion by binding bioitin and killing bacteria.

Acetyl-CoA regulates pyruvate carboxylase

Increases in oxaloacetate concentrations increase the activity of the Krebs cycle and acetyl-CoA is a allosteric activator of the carboxylase.

However when ATP and NADH concentrations are high and the Krebs cycle is inhibited, oxaloacetate goes to glucose.

Oxaloacetate cannot be transported directly across the mitochondrial membrane so it is converted to malate, then transported, then oxidized back to oxaloacetate.

Pyruvate is converted to oxaloacetate in the mitochondria

Transport between the mitochondria and the cytosol

Generation of oxaloacetate occurs in the mitochondria only, but, gluconeogenesis occurs in the cytosol.

Oxaloacetate are produced through pyruvate carboxylase, requires exiting the mitochondrion. Moreover, the inner mitochondrial membrane is not permeable to this compound.

Therefore, oxaloacetate is converted to malate inside the mitochondrion through mitochondrial malate dehydrogenase, the malate is transported by the mitochondrial membrane through a special transport protein and then the malate is converted back to oxaloacetate in the cytoplasm through a cytosolic malate dehydrogenase.

The PEP carboxykinase reaction.

Nucleotide diphosphate kinases

– Both glycolysis and Oxidative phosphorylation produce ATP with its high energy phoshoanhydride bonds: How does GTP get made from GDP?

– Directly from a single step in the Krebs cycle AND from the following reaction

– GDP + ATP → GTP + ADP– This is carried out in the cell by an enzyme called– Nucleotide diphosphate kinase which carries out the g

eneral reaction– NDP + ATP → NTP + ADP (where N is T, G, or C)

Regulatory enzymes of gluconeogenesis

• Pyruvate carboxylase• PEP carboxykinase (PEPCK)• Fructose-1,6-bisphosphatase• Glucose-6-phosphatase

Last step of gluconeogenesis

Free glucose is important control point -> pathway ends mostly with glucose-6-P -> finished just if glucose is needed (in blood) -> advantage of stopping at glucose-6-P -> trapped in the cell (cannot shuttle outside)

Last step of gluconeogenesis: in ER lumen -> glucose shuttled back to cytosol -> leaves cell

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Regulatory enzymes of gluconeogenesis

• Pyruvate carboxylase• PEP carboxykinase (PEPCK)• Fructose-1,6-bisphosphatase• Glucose-6-phosphatase

-enzyme unique to liver and kidney allowing them to supply glucose to other tissues. Found in ER (endoplasmic reticulum).

Glucose-6-phosphatase

Glucose-6-phosphate cannot exit the cell, and so, in the liver, glucose 6-phosphate is transported into the endoplasmic reticulum, where it is converted into glucose by glucose 6-phosphatase.

Fructose-2,6-bisphosphate

•A potent allosteric regulatory molecule.•- activates phosphofructokinase.•- inhibits fructose-1,6-bisphosphatase.•- its synthesis and degradation are catalyzed by the same bifunctional enzyme.

Fructose-2,6-bisphosphate

• F2,6BP activates phosphofructokinase (PFK1) - the enzyme in glycolysis that converts fructose-6-phosphate to fructose-1,6-bisphosphate.

• F2,6BP also inhibits fructose-1,6-bisphosphatase (F1,6BPase) - the enzyme in gluconeogenesis.

• F1,6BPase is ten times more sensitive to F2,6BP than AMP.

• F2,6BP stimulates glycolysis

• F2,6BP inhibits gluconeogenesis

Fructose-2,6-bisphosphate activates glycolysis and inhibits gluconeogenesis, so its level is very important.

• In the liver, the most important coordinating modulator is fructose 2,6-bisphophate (F2,6BP)

• It is formed from F6P by the enzyme phosphofructokinase-2 (PFK-2)

• It is broken down by the same enzyme, but at a different catalytic site in the enzyme – it’s a bifunctional protein

-It is called fructose 2,6-bisphosphatase (FBPase-2) for this activity

- Balance of PFK-2 to FBPase-2 activity controlled by

-Glucagon

-Insulin

Reciprocal regulation of glycolysis & gluconeogenesis

• Pathways not active at same time

• Regulated by products of reaction and precursors (allostery)

• Regulated by hormones: glucagon & insulin, through F-2,6-BP

• Regulated at the transcriptional level of genes

In the liver: aim is to maintain blood glucose level

glucagoninsulin

transcription

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Balance between glycolysis and gluconeogenesis in the liver -> sensitive to blood glucose concentration

-

Regulated by a bifunctional enzyme: PFK2/FBPase2-> formed by PFK2-> hydrolysed (dephosphorylated) by FBPase2

Phosphofructokinase 2 Fructose bisphophatase 2

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6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

P

High glucagon

Increased phosphorylation

Phosphorylation of the enzyme results in the inactivation of the phosphofructokinase-2 activity and activation of the fructose-2,6-bisphosphatase activity. This results in a down regulation of glycolysis and increased gluconeogenesis.

Low glucose

Balance between glycolysis and gluconeogenesis in the liver -> sensitive to blood glucose concentration

Low blood-glucose level -> glucagon-> low level of F-2,6-BP

High blood-glucose level -> insulin-> high level of F-2,6-BP

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• Fructose-2,6-bisphosphate, the most potent allosteric regulator of the glycolysis and gluconeogenesis pathways

F2,6 BP

ATPADP

Pi

F2,6 BPPFK-1

PFK-2

INHIBITS

F2,6 BP

STIMULATES

Regulators of gluconeogenic enzyme activity

Enzyme Allosteric Allosteric Enzyme Protein Inhibitors Activators Phosphorylation Synthesis

PFK ATP, citrate AMP, F2-6P

FBPase AMP, F2-6P

PK Alanine F1-6P Inactivates

Pyr. Carb. AcetylCoA

PEPCK Glucogon

PFK-2 Citrate AMP, F6P, Pi Inactivates

FBPase-2 F6P Glycerol-3-P Activates

Synthesis of other saccharides through gluconeogenesis

42

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Regulation of Gluconeogenesis

• Fate of pyruvate

•Go on to citric acid cycle – requires conversion to Acetyl Co-A by the pyruvate dehydrogenase complex

•Gluconeogenesis – first step is conversion to oxaloacetate by pyruvate carboxylase

• Acetyl Co-A accumulation

• inhibits pyruvate dehydrogenase

• activates pyruvate carboxylase

Coordinated regulation of PFK-1 and FBPase-1 (1) Phosphofructokinase-1 (PFK-1) (glycolysis)(2) Fructose 1,6-bisphosphatase FBPase-1 (gluconeogenesis)

• Modulating one enzyme in a substrate cycle will alter the flux through the two opposing pathways

• Two coordinating modulators•AMP•Fructose 2,6-bisphosphate

• Inhibiting PFK-1 stimulates gluconeogenesis

• Inhibiting the phosphatase stimulates glycolysis

• AMP concentration coordinates regulation• stimulates glycolysis• Inhibits gluconeogenesis

Pathway Integration during a sprint

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Cori Cycle• Muscular activity leads to the release of epinephrine by t

he adrenal medulla. • During muscle contractions, ATP is constantly being use

d to supply energy and more ATP is produced.• At first glycolysis produces pyruvic acid which is then con

verted into acetyl CoA and is metabolized in the citric acid cycle to make ATP.

• If muscular activity continues, the availability of oxygen becomes the limiting factor and the cells soon exhaust their supplies of oxygen.

• However, glycolysis continues even under anaerobic conditions even though the citric acid cycle works only under aerobic conditions.

Cori Cycle (cont.)

• The final limiting factor in continued muscular activity is the build up of lactic acid. The lactic acid eventually produces muscular pain which force discontinuation of activity.

• The lactic acid is sent in the blood to the liver which can convert it back to pyruvic acid and then to glucose through gluconeogenesis.

• The glucose can enter the blood and be carried to muscles

• If by this time the muscles have ceased activity, the glucose can be used to rebuild supplies of glycogen through glycogenesis.

Cori Cycle (cont.)

•  At this time the oxygen debt can be made up so that the citric cycle and electron transport chain also begin to function again. In order for most of the lactic acid to be converted to glucose, some must be converted to pyruvic acid and then to acetyl CoA

Liver Cell

The Cori Cycle

Cori Cycle

Sample questions

• There are four enzymes of gluconeogenesis that circumvent the irreversible steps in glycolysis.  When starting with the substrate pyruvate or lactate they are

• A. Hexokinase, phosphofructokinase-1, phosphofructokinase-2 and pyruvate kinase

• B. Pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase

• C. Glycerol kinase, glycerol-3-phosphate dehydrogenase, fructose-2,6-bisphosphatase, and glucose-6-phosphatase

• D. Amino transferase, phosphoenolpyruvate carboxykinase, fructose-2,6-bisphosphatase, and glucose-6-phosphatase

Sample questions

• The enzymes that remove phosphate groups during the process of gluconeogenesis and circumvent two of the three irreversible reactions of glycolysis are

• A. Pyruvate kinase and glycerol kinase• B. Phosphoenolpyruvate carboxykinase and glycerol kin

ase• C. 3-Phosphoglycerate kinase and fructose-1,6-bisphosp

hatase• D. Fructose-1,6-bisphosphatase and glucose-6-phosphat

ase

Glycogen Metabolism

Glycogen Metabolism

• Glycogenolysis is a process by which glycogen is broken down into glucose to provide immediate energy and to maintain blood glucose levels during fasting.

• Glycogenesis is the formation of glycogen from glucose.

• Glycogenolysis occurs primarily in the liver and is stimulated by the hormones glucagon and epinephrine (adrenaline).

• When blood glucose levels fall, as during fasting, there is an increase in glucagon secretion from the pancreas. This increase is accompanied by a decrease in insulin secretion.

• Insulin is aimed at increasing the storage of glucose in the form of glycogen

Regulation of Glycogen Metabolism

• Muscle glycogen is fuel for muscle contraction

• Liver glycogen is mostly converted to glucose for bloodstream transport to other tissues

• Both mobilization and synthesis of glycogen are regulated by hormones

• Insulin, glucagon and epinephrine regulate mammalian glycogen metabolism (hormones)

• Ca2+ and [AMP]/[ATP] (muscle glycogen phosphorylase)

• [glucose] (liver glycogen phosphorylase)

• [glucose 6-phosphate] (glycogen synthase)

• Hormones

•Insulin is produced by -cells of the pancreas-increases glucose transport into muscle, adipose tissue via GLUT 4 transporter-stimulates glycogen synthesis in the liver

• Glucagon Secreted by the cells of the pancreas in response

to low blood glucose (elevated glucagon is associated with the

fasted state)

-Stimulates glycogen degradation to restore blood glucose

to steady-state levels

-Only liver cells are rich in glucagon receptors

• Epinephrine (adrenaline) Released from the adrenal glands

in response to sudden energy requirement (“fight or flight”)

- Stimulates the breakdown of glycogen to G1P (which is

converted to G6P)

-Increased G6P levels increase both the rate of glycolysis in

muscle and glucose release to the bloodstream from the liver

Glucagon/Epinephrine control of glycogen synthesis/degradation

Signal cascade initiated by epinephrine

• 1) Epinephrine binds to a receptor on the muscle cell membrane and stimulates adenyl cyclase in the membrane.

• 2) Adenyl cyclase in the membrane catalyzes the formation of cyclic AMP from ATP.

• 3) The increase of cyclic AMP activates a protein kinase. The binding of cyclic AMP to an enzyme is an allosteric control where the enzyme is "switched on" for activity.

• 4) The protein kinase causes phosphorylations (addition of phosphate) on a series of phosphorylation enzymes which activates them to finally produce glucose-1-phosphate. At the same time that enzymes are being activated for glycogen breakdown, glycogen synthetase enzyme must be inactivated. Glycogenesis must be "switched off" and glycogenolysis "switched on."

• 5) Glucose-6-phosphate is the final result of the initial stimulation by epinephrine or other hormones such as glucagon. If this happened to a muscle cell, then the glycolysis pathway is the next step in the sequence. If this happened to a liver cell stimulated by glucagon, then glucose is produced to enter the blood stream.

• Epinephrine markedly stimulates glycogen breakdown in muscles and, to a lesser extent, in the liver.

• Muscular activity quickly uses stored ATP as the energy source and more ATP must be generated by the breakdown of glycogen.

• At first glycolysis produces pyruvic acid which is then converted into acetyl CoA and is metabolized in the citric acid cycle to make ATP.

• If muscular activity continues, the cells soon exhaust their supplies of oxygen. When this happens, the citric acid cycle is inhibited and causes pyruvic acid to accumulate.

• However, glycolysis continues even under anaerobic conditions even though the citric acid cycle works only under aerobic conditions. Glycogenolysis is stimulated to make more glucose-6-phosphate.

• When the cells become anaerobic, glycolysis continues if pyruvic acid is converted to lactic acid.

Liver Cell

Signal cascade

Insulin lowers blood glucose levels

High Blood Glucose:

Glycogen Storage

• Glycogen is a D-glucose polymer

• (14) linkages

• (16) linked branches every 8-14 residues

Glycogen storage diseases

GlucoseGlycogenGlucose

Glycogen Breakdown

Glycogen Synthesis

Glycogen Synthesis

R

Glycogen Breakdown or Glycogenolysis

• Three steps– Glycogen phosphorylase

Glycogen + Pi <-> glycogen + G1P(n residues) (n-1 residues)

– Glycogen debranching– Phosphofructomutase

GlycogenBreakdown

GlucoseGlycogenGlucose

Glycogen Breakdown

Glycogen Synthesis

Glycogen Breakdown

R

Glycogen Phosphorylase

Requires Pyridoxal-5’-phosphate

PLP

Glycogen Debranching Enzyme

Phosphofructomutase

PKA is activated by cyclic AMP, which is produced by a G-protein in response to epinephrine/glucagon.

Phosphorylase kinase is activated upon phosphorylation by protein kinase A (PKA).

Glycogen phosphorylase is activated upon phosphorylation by phosphorylase kinase.

Reciprocal Regulation of GlycogenPhosphorylase and Glycogen Synthase

• Glycogen phosphorylase (GP) and glycogen synthase (GS) control glycogen metabolism in liver and muscle cells

• GP and GS are reciprocally regulated both covalently and allosterically (when one is active the other is inactive)

• Covalent regulation by phosphorylation (-P) and dephosphorylation (-OH)

COVALENT MODIFICATION (Hormonal control)

Active form “a” Inactive form “b” Glycogen phosphorylase -P -OHGlycogen synthase -OH -P

Allosteric regulation of GP and GS GP a (active form) - inhibited by Glucose

GP (muscle)- stimulated by Ca2+ and high [AMP]

GS b (inactive form) - activated by Glucose 6-Phosphate

Control of glycogen phosphorylase

phosphorylase b (inactive)

phosphorylase a (active)

phosphorylation

glycogen breakdown

• Hormones initiate enzyme cascades

•Catalyst activates a catalyst activates a catalyst, etc.

• When blood glucose is low: epinephrine and glucagon activate protein kinase A

• Glycogenolysis is increased (more blood glucose)

• Glycogen synthesis is decreased

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Regulation of blood glucose level in the liver

Fig 15-12Glycogen

BreakdownStep 1.

Glycogen Phosphorylase

Fig 15-12Glycogen

Breakdown

Phospho- glucomutase

GlycogenBreakdown

Debranching enzyme

Glycogen Synthesis

UDP-glucose Pyrophorylase

Glycogen Synthase

Sample questions• The most important control step in gluconeogenesis is fructose-1,6-

bisphosphatase.  All of the following statements are true EXCEPT

• A. Fructose-1,6-bisphosphatase converts fructose-2,6-bisphosphate to fructose-6-phosphate

• B. During times when insulin is high, fructose-1,6-bisphosphatase is inhibited by fructose-2,6-bisphosphate

• C. During a fast or exercise when glucagon and/or epinephrine are high, fructose-1,6-bisphosphatase is active because of the absence of fructose-2,6-bisphosphate

• D. Glycolysis or gluconeogenesis cannot be active at the same time.  If they were is would be a futile cycle

Sample questions• In the liver, glucagon will activate• A. Glycolysis and glycogen synthesis• B. Gluconeogenesis and glycogenolysis• C. Gluconeogenesis and glycogen synthase• D. Gluconeogenesis and glycogen synthesis

• Which of the following statements about hormonal levels during different states is true?

• A. During the time you are eating a high carbohydrate mixed meal, the insulin to glucagon ratio will decrease

• B. When passing from the fed to fasting state, insulin and glucagon usually decrease

• C. When playing basketball, epinephrine is usually low and insulin is high• D. After running for 20 miles, epinephrine and glucagon are high and insulin

is low

Sample questions

• All of the following will result in activation of glycogen phosphorylase in skeletal muscle EXCEPT

• A. Increased concentrations of AMP from contraction of muscle

• B. Increased epinephrine and cAMP• C. Increased cytosolic [Ca++]• D. Increased protein phosphatase• E. Increased activity of glycogen phosphorylase kinase

Experiment for next week

• Separation of Ferrihemoglobin and Potassium Ferricyanide by Gel Filtration Chromatography

• Changed to Construction of Serum Protein Standard Curve using Folin's Phenol Method