Tutorial: Glucose Metabolism in the -Cell

Richard BertramDepartment of Mathematics

AndPrograms 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 -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

-cell Signaling

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

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.

Glycolysis

Energy is Invested at the Beginning of Glycolysis

(G6P)

(F6P)

(FBP)

Two ATP moleculesare used to make onemolecule of FBP

(PFK)

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:

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

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.

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

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

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 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

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.

Anaplerosis Involving Pyruvate

Pyruvatepyruvatecarboxylase

Anaplerosis Involving Amino Acids

Glutamate Glutamine

HistidineProlineArginine

+Leucine

GDH

Anaplerosis Involving Amino Acids

ValineIsoleucineMethionine

Anaplerosis Involving Amino Acids

PhenylalanineTyrosine

Anaplerosis Involving Amino Acids

Aspartate

Asparagine

Cataplerosis of Malate

Malate

Oxaloacetate

Phosphoenolpyruvate (PEP)

Cataplerosis of Citrate

Acetyl-CoA

Oxaloacetate

Malonyl CoA

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 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

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 notexist.

Elements of Oxidative Phosphorylation

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

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

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 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

Cytosolic ATP Can Be Measured in Single -cells

ATP measured using adenovirally driven expression ofrecombinant firefly luciferase.

Ainscow and Rutter, Diabetes, 51:S162, 2002

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+

Mitochondrial Ca2+ Concentration Not Measured Yet in Islets

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

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)

O2 Can Also Be Measured in Islets

Measured using an oxygen electrode

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

Thank You!!