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Tutorial: Glucose Metabolism in the b -Cell. Richard Bertram Department of Mathematics And Programs in Neuroscience and Molecular Biophysics. Metabolites as Signaling Molecules. All cells in the body convert glucose and other fuels to adenosine

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tutorial glucose metabolism in the b cell
Tutorial: Glucose Metabolism in the b-Cell

Richard Bertram

Department of Mathematics

And

Programs in Neuroscience and Molecular Biophysics

metabolites as signaling molecules
Metabolites as Signaling Molecules

All cells in the body convert glucose and other fuels to adenosine

triphosphate (ATP), the primary energy molecule. The ATP powers

many of the energy-requiring chemical reactions that occur in the cell.

However, in b-cells the ATP molecule and several intermediates of

metabolism act also as signaling molecules. They tell the b-cell the

level of blood glucose, so that the cell can adjust its electrical and

Ca2+ activity to secrete the appropriate amount of insulin.

A primary target of the signaling molecule ATP is the ATP-dependent

K+ channel (the K(ATP) channel). This is inactivated by ATP, so:

ATP formed

through

metabolism

K(ATP)

channels

close

High

Glucose

b-cell

depolarizes

Insulin

secreted

b cell signaling
b-cell Signaling

Kahn et al., Nature, 444:840, 2006

three steps involved in glucose metabolism
Three Steps Involved in Glucose Metabolism

Glucose

Anaerobic production of ATP. Occurs in the cytosol.

However, not much ATP is produced by glycolysis, only

two ATP molecules for each glucose molecule metabolized.

Glycolysis

ATP

Pyruvate, NADH

Found in all aerobic organisms, takes place in mitochondria

of eucaryotes. Most of the coenzyme NADH is made here

Through a series of redox reactions (NAD+ is reduced).

Citric Acid

Cycle

NADH, FADH2

Found in eucaryotes, takes place in mitochondria. O2

is consumed by the electron transport chain. Most of the

ATP is produced here, 28 ATP molecules for each

glucose molecule metabolized.

Oxidative

Phosphorylation

ATP

energy is invested at the beginning of glycolysis
Energy is Invested at the Beginning of Glycolysis

Two ATP molecules

are used to make one

molecule of FBP

(G6P)

(F6P)

(PFK)

(FBP)

energy is generated during second step
Energy is Generated During Second Step

(GPDH)

Two ATP molecules

produced for each of

two glyceraldehyde-3-

phosphate molecules,

total of 4 ATP generated.

Net ATP:

glycolysis can be oscillatory
Glycolysis Can Be Oscillatory

Sustained NADH oscillations in yeast,

very simple (single cell) eucaryotes.

Oscillations are in the presence of glucose

and cyanide (which blocks electron

transport, inhibiting oxidative phosphorylation).

Dano et al., Nature, 402:320, 1999

Oscillations in three glycolytic

intermediates in muscle extracts.

Tornheim, JBC, 263:2619, 1988

what is the mechanism for glycolytic oscillations
What is the Mechanism for Glycolytic Oscillations?

In muscle extracts the mechanism is

known to be the allosteric enzyme

Phosphofructokinase (PFK). The

key feature of this enzyme is that

its product FBP feeds back and

stimulates the enzyme.

The muscle form of this enzyme,

PFK-M, dominates the PFK

activity in b-cells.

model glycolytic oscillations
Model Glycolytic Oscillations

With moderate glucokinase

activity

With high glucokinase

activity

Bertram et al., Biophys. J., 87:3074, 2004

JGK is the glucokinase reaction rate

JPFK is the PFK reaction rate

JGPDH is the GPDH reaction rate

glycolytic oscillations occur only for moderate gk rates

0.1 ratio

5 min

Glycolytic Oscillations Occur Only for Moderate GK Rates

15 mM

A model prediction is that it should be possible to turn on the

GOs by simply increasing the glucose concentration. We have

evidence for this from Ca2+ measurements in islets:

8 mM

8 mM

Nunemaker et al., Biophys. J., 91:2082, 2006

coenzymes are produced by the citric acid cycle
Coenzymes are Produced by the Citric Acid Cycle

Acetyl group has 2 carbons

Oxaloacetate has 4 carbons

Citrate has 6 carbons

As the cycle progresses, first one

carbon is lost and then another

Cycle ends where it began, except

that 4 NADH, one FADH2, and

one GTP molecule have been made

The coenzymes NADH and FADH2

are electron carriers that are used to

transfer electrons between molecules.

This transfer is key for powering

oxidative phosphorylation

anaplerosis and cataplerosis
Anaplerosis and Cataplerosis

Anaplerosis is a series of enzymatic reactions in which metabolic

intermediates enter the citric acid cycle from the cytosol.

Cataplerosis is the opposite, a process where intermediates leave the

citric acid cycle and enter the cytosol.

In muscle, anaplerosis is important for increasing citric acid throughput

during periods of exercise.

There is some evidence that anaplerosis is required for a glucose-induced

rise in mitochondrial ATP production.

Some amino acids (the building blocks of proteins) enter and leave the

citric acid cycle through anaplerosis and cataplerosis.

anaplerosis involving pyruvate
Anaplerosis Involving Pyruvate

Pyruvate

pyruvate

carboxylase

anaplerosis involving amino acids
Anaplerosis Involving Amino Acids

Leucine

+

Glutamate

Glutamine

GDH

Histidine

Proline

Arginine

anaplerosis involving amino acids1
Anaplerosis Involving Amino Acids

Valine

Isoleucine

Methionine

cataplerosis of malate
Cataplerosis of Malate

Phosphoenolpyruvate

(PEP)

Oxaloacetate

Malate

cataplerosis of citrate
Cataplerosis of Citrate

Malonyl CoA

Acetyl-CoA

Oxaloacetate

Fatty Acids

subway analogy
Subway Analogy
  • Citric Acid Cycle is like a subway system:
  • Acetyl-CoA is like people getting on at station A
  • NADH is like people getting off at station B
  • Intermediates are like the subway cars
  • Anaplerosis is like adding cars to the system
  • Cateplerosis is like removing cars to use for spare parts
the malate aspartate shuttle
The Malate/Aspartate Shuttle

Some of the coenzyme NADH is made during glycolysis. How does

this get into the mitochondria where it can power oxidative

phosphorylation?

3

2

4

5

6

4

1

7

OAA=oxaloacetate

MDH=malate dehydrogenase

Asp=aspartate

Glu=glutamate

last stage of glucose metabolism produces the most atp
Last Stage of Glucose Metabolism Produces the Most ATP

Keeping score of ATP production:

Glycolysis – 2 ATP for each glucose molecule

Citric Acid cycle – No ATP produced

Oxidative Phosphorylation – up to 34 ATP molecules

Without mitochondria (and thus OP), complex life forms could not

exist.

the magnus keizer model
The Magnus-Keizer Model

Published as a series of papers in the late 1990s. Describes oxidative

Phosphorylation in b-cells.

We have recently published a simpler model that uses curve fitting to

reduce the complexity of the flux and reaction functions (Bertram et al.,

J. Theoret. Biol., 243:575, 2006).

Mitochondrial Variables: NADH concentration

ADP or ATP concentration (ADP+ATP=constant)

Calcium concentration

Inner membrane potential

O2 consumption is also calculated

the nadh equation
The NADH Equation

NADH flux from citric acid cycle increases NADH concentration.

NADH is oxidized when it supplies electrons to the electron

transport chain, decreasing NADH concentration.

JH,res

Mitochondrial inner

membrane

Jo

JDH

nadh concentration can be measured in islets
NADH Concentration Can Be Measured in Islets

NADH autofluorescence is measured

Bertram et al., Biophys. J., 92:1544, 2007

the adp atp equations
The ADP/ATP Equations

ADP is phosphorylated to ATP by the F1-F0 ATP-synthase. This is due

to the flux of protons down the concentration gradient from outside

to inside of the mitochondrial inner membrane.

The ATP made in this way is transported out, and ADP transported in,

by the adenine nucleotide transporter.

JANT

JF1F0

ATP

H+

ATP

ADP

cytosolic atp can be measured in single b cells
Cytosolic ATP Can Be Measured in Single b-cells

ATP measured using adenovirally driven expression of

recombinant firefly luciferase.

Ainscow and Rutter, Diabetes, 51:S162, 2002

the ca 2 equation
The Ca2+ Equation

Calcium enters the mitochondria from the cytosol through calcium

uniporters.

Calcium is pumped out of the mitochondria into the cytosol via

Na+/Ca2+ exchangers.

Ca2+

JNaCa

Juni

Ca2+

the inner membrane potential equation
The Inner Membrane Potential Equation

This membrane potential is the driving force for ATP production

by the F1F0 ATP synthase. If membrane potential is 0, then no

ATP will be made.

(Negative terms represent positive charge entering across the inner

membrane)

JH,leak

JH,res

JH,ATP

JNaCa

Juni

JANT

mitochondrial inner membrane potential can be measured in islets
Mitochondrial Inner Membrane Potential Can Be Measured in Islets

Measured using the fluorescent dye rhodamine 123 (Rh 123)

Kindmark et al., J. Biol. Chem., 276:34530, 2001

o 2 can also be measured in islets
O2 Can Also Be Measured in Islets

Measured using an oxygen electrode

Kennedy et al., Diabetes, 51:S152, 2002