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Tutorial: Glucose Metabolism in the -Cell
Richard BertramDepartment of Mathematics
AndPrograms 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 -cells the ATP molecule and several intermediates of metabolism act also as signaling molecules. They tell the -cell thelevel of blood glucose, so that the cell can adjust its electrical andCa2+ activity to secrete the appropriate amount of insulin.
A primary target of the signaling molecule ATP is the ATP-dependentK+ channel (the K(ATP) channel). This is inactivated by ATP, so:
HighGlucose
ATP formedthroughmetabolism
K(ATP) channels close
-celldepolarizes
Insulinsecreted
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-cell Signaling
Kahn et al., Nature, 444:840, 2006
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Three Steps Involved in Glucose Metabolism
Glycolysis
Citric Acid Cycle
Oxidative Phosphorylation
Glucose
ATP
Anaerobic production of ATP. Occurs in the cytosol. However, not much ATP is produced by glycolysis, onlytwo ATP molecules for each glucose molecule metabolized.
ATPPyruvate, NADH
NADH, FADH2
Found in all aerobic organisms, takes place in mitochondriaof eucaryotes. Most of the coenzyme NADH is made hereThrough a series of redox reactions (NAD+ is reduced).
Found in eucaryotes, takes place in mitochondria. O2
is consumed by the electron transport chain. Most of theATP is produced here, 28 ATP molecules for eachglucose molecule metabolized.
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Glycolysis
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Energy is Invested at the Beginning of Glycolysis
(G6P)
(F6P)
(FBP)
Two ATP moleculesare used to make onemolecule of FBP
(PFK)
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Energy is Generated During Second Step
Two ATP moleculesproduced for each oftwo glyceraldehyde-3-phosphate molecules, total of 4 ATP generated.
(GPDH)
224 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 glucoseand cyanide (which blocks electrontransport, inhibiting oxidative phosphorylation).
Dano et al., Nature, 402:320, 1999
Oscillations in three glycolyticintermediates in muscle extracts.
Tornheim, JBC, 263:2619, 1988
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What is the Mechanism for Glycolytic Oscillations?
In muscle extracts the mechanism is known to be the allosteric enzymePhosphofructokinase (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 -cells.
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Model Glycolytic Oscillations
With moderate glucokinase activity
With high glucokinase activity
)(3.06
PFKGK JJdt
PFd
GPDHPFK JJdt
FBPd
2
1
JGK is the glucokinase reaction rateJPFK is the PFK reaction rateJGPDH is the GPDH reaction rate
Bertram et al., Biophys. J., 87:3074, 2004
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Glycolytic Oscillations Occur Only for Moderate GK Rates
A model prediction is that it should be possible to turn on theGOs by simply increasing the glucose concentration. We haveevidence for this from Ca2+ measurements in islets:
8 mM15 mM
8 mM
5 min
0.1 ratio
Nunemaker et al., Biophys. J., 91:2082, 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 onecarbon is lost and then another
Cycle ends where it began, exceptthat 4 NADH, one FADH2, andone GTP molecule have been made
The coenzymes NADH and FADH2
are electron carriers that are used totransfer 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 metabolicintermediates 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 throughputduring periods of exercise.
There is some evidence that anaplerosis is required for a glucose-inducedrise 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
Pyruvatepyruvatecarboxylase
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Anaplerosis Involving Amino Acids
Glutamate Glutamine
HistidineProlineArginine
+Leucine
GDH
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Anaplerosis Involving Amino Acids
ValineIsoleucineMethionine
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Anaplerosis Involving Amino Acids
PhenylalanineTyrosine
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Anaplerosis Involving Amino Acids
Aspartate
Asparagine
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Cataplerosis of Malate
Malate
Oxaloacetate
Phosphoenolpyruvate (PEP)
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Cataplerosis of Citrate
Acetyl-CoA
Oxaloacetate
Malonyl CoA
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 ShuttleSome of the coenzyme NADH is made during glycolysis. How doesthis get into the mitochondria where it can power oxidative phosphorylation?
1
2 3
4
56
7OAA=oxaloacetateMDH=malate dehydrogenaseAsp=aspartateGlu=glutamate
4
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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 notexist.
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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 -cells.
We have recently published a simpler model that uses curve fitting toreduce 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
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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 electrontransport chain, decreasing NADH concentration.
oDHm JJ
dt
NADHd
JDH
Jo
JH,res
Mitochondrial inner membrane
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NADH Concentration Can Be Measured in Islets
NADH autofluorescence is measured
Bertram et al., Biophys. J., 92:1544, 2007
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The ADP/ATP Equations
ADP is phosphorylated to ATP by the F1-F0 ATP-synthase. This is dueto 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.
01FFANTm JJ
dt
ADPd totmm AATPADP
JANT JF1F0
H+ATP
ADPATP
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Cytosolic ATP Can Be Measured in Single -cells
ATP measured using adenovirally driven expression ofrecombinant firefly luciferase.
Ainscow and Rutter, Diabetes, 51:S162, 2002
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The Ca2+ Equation
Calcium enters the mitochondria from the cytosol through calciumuniporters.
Calcium is pumped out of the mitochondria into the cytosol viaNa+/Ca2+ exchangers.
)( NaCaunimm JJf
dt
Cad
Juni
Ca2+
JNaCa
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 productionby the F1F0 ATP synthase. If membrane potential is 0, then noATP will be made.
muniNaCaleakHANTATPHresHm CJJJJJJ
dt
d/)2( ,,,
(Negative terms represent positive charge entering across the inner membrane)
JH,res
JH,ATP
JANT
JH,leak
JNaCa
Juni
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Mitochondrial Inner Membrane Potential Can Be Measured in Islets
Kindmark et al., J. Biol. Chem., 276:34530, 2001
Measured using the fluorescent dye rhodamine 123 (Rh 123)
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O2 Can Also Be Measured in Islets
Measured using an oxygen electrode
Kennedy et al., Diabetes, 51:S152, 2002
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Thank You!!