Tutorial: Glucose Metabolism in the b-Cell

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Tutorial Glucose Metabolism in the b-Cell
Richard Bertram Department of Mathematics And Prog
rams in Neuroscience and Molecular Biophysics
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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
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b-cell Signaling
Kahn et al., Nature, 444840, 2006
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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
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Glycolysis
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Energy is Invested at the Beginning of Glycolysis
Two ATP molecules are used to make one molecule
of FBP
(G6P)
(F6P)
(PFK)
(FBP)
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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
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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, 402320, 1999
Oscillations in three glycolytic intermediates in
muscle extracts.
Tornheim, JBC, 2632619, 1988
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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.
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Model Glycolytic Oscillations
With moderate glucokinase
activity
With high glucokinase activity
Bertram et al., Biophys. J., 873074, 2004
JGK is the glucokinase reaction rate JPFK is the
PFK reaction rate JGPDH is the GPDH reaction rate
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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., 912082, 2006
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Citric Acid Cycle
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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
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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.
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Anaplerosis Involving Pyruvate
Pyruvate
pyruvate carboxylase
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Anaplerosis Involving Amino Acids
Leucine

Glutamate
Glutamine
GDH
Histidine Proline Arginine
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Anaplerosis Involving Amino Acids
Valine Isoleucine Methionine
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Anaplerosis Involving Amino Acids
Phenylalanine Tyrosine
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Anaplerosis Involving Amino Acids
Aspartate
Asparagine
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Cataplerosis of Malate
Phosphoenolpyruvate (PEP)
Oxaloacetate
Malate
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Cataplerosis of Citrate
Malonyl CoA
Acetyl-CoA
Oxaloacetate
Fatty Acids
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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

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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?
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2
4
5
6
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1
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OAAoxaloacetate MDHmalate dehydrogenase Aspaspa
rtate Gluglutamate
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Oxidative Phosphorylation
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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.
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Elements of Oxidative Phosphorylation
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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., 243575,
2006). Mitochondrial Variables NADH
concentration
ADP or ATP concentration
(ADPATPconstant)
Calcium concentration
Inner membrane potential
O2 consumption is also calculated
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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
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NADH Concentration Can Be Measured in Islets
NADH autofluorescence is measured
Bertram et al., Biophys. J., 921544, 2007
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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
H
ATP
ADP
ATP
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Cytosolic ATP Can Be Measured in Single b-cells
ATP measured using adenovirally driven expression
of recombinant firefly luciferase.
Ainscow and Rutter, Diabetes, 51S162, 2002
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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
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Mitochondrial Ca2 Concentration Not Measured Yet
in Islets
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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
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Mitochondrial Inner Membrane Potential Can Be
Measured in Islets
Measured using the fluorescent dye rhodamine 123
(Rh 123)
Kindmark et al., J. Biol. Chem., 27634530, 2001
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O2 Can Also Be Measured in Islets
Measured using an oxygen electrode
Kennedy et al., Diabetes, 51S152, 2002
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Thank You!!