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
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:
High
Glucose
ATP formed
through
metabolism
K(ATP)
channels
close
b-cell
depolarizes
Insulin
secreted
b-cell Signaling
Kahn et al., Nature, 444:840, 2006
Three Steps Involved in Glucose Metabolism
Glucose
Glycolysis
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.
ATP
Pyruvate, NADH
Citric Acid
Cycle
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).
NADH, FADH2
Oxidative
Phosphorylation
ATP
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.
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
(GPDH)
Two ATP molecules
produced for each of
two glyceraldehyde-3phosphate molecules,
total of 4 ATP generated.
Net ATP: 4  2  2
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?
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
With moderate glucokinase
activity
With high glucokinase
activity
Bertram et al., Biophys. J., 87:3074, 2004
d F 6P
 0.3( J GK  J PFK )
dt
d FBP
1
 J PFK  J GPDH
dt
2
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
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
0.1 ratio
5 min
Nunemaker et al., Biophys. J., 91:2082, 2006
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 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
Pyruvate
pyruvate
carboxylase
Anaplerosis Involving Amino Acids
Leucine
+
GDH
Glutamate
Histidine
Proline
Arginine
Glutamine
Anaplerosis Involving Amino Acids
Valine
Isoleucine
Methionine
Anaplerosis Involving Amino Acids
Phenylalanine
Tyrosine
Anaplerosis Involving Amino Acids
Aspartate
Asparagine
Cataplerosis of Malate
Phosphoenolpyruvate
(PEP)
Oxaloacetate
Malate
Cataplerosis of Citrate
Malonyl CoA
Acetyl-CoA
Oxaloacetate
Fatty Acids
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
Some of the coenzyme NADH is made during glycolysis. How does
this get into the mitochondria where it can power oxidative
phosphorylation?
3
2
6
1
OAA=oxaloacetate
MDH=malate dehydrogenase
Asp=aspartate
Glu=glutamate
7
5
4
4
Oxidative Phosphorylation
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.
Elements of Oxidative Phosphorylation
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
NADH flux from citric acid cycle increases NADH concentration.
NADH is oxidized when it supplies electrons to the electron
transport chain, decreasing NADH concentration.
d NADH m
 J DH  J o
dt
JH,res
Mitochondrial inner
membrane
Jo
JDH
NADH Concentration Can Be Measured in
Islets
NADH autofluorescence is measured
Bertram et al., Biophys. J., 92:1544, 2007
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.
d ADPm
 J ANT  J F 1F 0
dt
JANT
ADPm  ATPm  Atot
JF1F0
H+
ATP
ATP ADP
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 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.
d Cam
 f m ( J uni  J NaCa )
dt
Ca2+
JNaCa
Juni
Ca2+
Mitochondrial Ca2+ Concentration Not
Measured Yet in Islets
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.
d m
 ( J H ,res  J H , ATP  J ANT  J H ,leak  J NaCa  2 J uni ) / Cm
dt
(Negative terms represent positive charge entering across the inner
membrane)
JH,res
JH,ATP
JANT
JH,leak
JNaCa
Juni
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
O2 Can Also Be Measured in Islets
Measured using an oxygen electrode
Kennedy et al., Diabetes, 51:S152, 2002
Thank You!!