1

An analysis of metabolic fluxesin contracting human muscle

Gregory J. Crowther

Dept. of Physiology & Biophysics

University of Washington (Seattle)

ATP

2

Energy metabolism in muscle

H+ & Lactate

Glucose

ATP

ATP supplyATP demand

(contractile cost)

(oxidative phosphorylation)

(glycolysis)

3

Questions

Why study cellular metabolism in muscle?

How can metabolic fluxes be quantified using phosphorus NMR spectroscopy?

What turns glycolysis on and off?

How is muscle metabolism affected by type 1 diabetes mellitus?

Intro:

Methods:

Results:

4

Definitions

NMR nuclear magnetic resonance

PCr phosphocreatine (an ATP buffer)

HP hexose phosphates (substrates of glycolysis)

“metabolites” Pi, ADP, AMP (products of ATP hydrolysis)

5

Introduction:

Why study cellular metabolism in muscle?

6

GAPDH

glycogen

GP

PFK

PGK

PK

lactate

PGI

PGM

ALD

PGM

ENO

LDH

Why study cellular metabolism?

Cellular metabolism . . .

• is central to the existence of all cells.

• has important whole-organ and whole-body consequences.

• can be harnessed to spawn advances in medicine and industry.

7

X

GAPDH

glycogen

GP

PFK

PGK

PK

lactate

PGI

PGM

ALD

PGM

ENO

LDH

Why study cellular metabolism?

Cellular metabolism . . .

• is central to the existence of all cells.

• has important whole-organ and whole-body consequences.

• can be harnessed to spawn advances in medicine and industry.

8

X

GAPDH

glycogen

GP

PFK

PGK

PK

lactate

PGI

PGM

ALD

PGM

ENO

LDH

Why study cellular metabolism?

GAPDH

PFK

PGK

PK

ethanol

PGI

HK

ALD

PGM

ENO

PDC

glucose

ADH

Cellular metabolism . . .

• is central to the existence of all cells.

• has important whole-organ and whole-body consequences.

• can be harnessed to spawn advances in medicine and industry.

9

Why study cellular metabolism?

We don’t know . . .

• What are the pathway flux rates in vivo?

• How are these rates up- and downregulated?

GAPDH

glycogen

GP

PFK

PGK

PK

lactate

PGI

PGM

ALD

PGM

ENO

LDH

10

Why study cellular metabolism in muscle?

Advantages of muscle . . .

• huge range of fluxes

• ~40% of human body mass

• critical to physical performance and well-being

• can study muscles specialized for different tasks McArdle et al., Essentials of

exercise physiology, 1994

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Methods:

How can metabolic fluxes be quantified using phosphorus NMR spectroscopy?

12

The basic idea

Force

[PCr]

Time

13

Simultaneous collection of NMR and force data

NMR coil

force transducer

• in vivo

• noninvasive

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NMR reveals metabolic changes

10

20

30[PCr](mM)

250 300 350 400 450

Time (s)

• good time resolution

Am

plitu

de

-10-50510

Chemical shift (ppm)

HP

Pi PCr

ATPpH

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[ATP] remains constant during exercise and recovery

[PCr]

Time

[ATP]

[Pi]

ischemia

exercise

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PCr keeps [ATP] constant

ADP + PCr + H+ Cr + ATP

ATP ADP + Pi + H+

PCr + H+ Cr + Pi

Therefore changes in [PCr] reflect changes in ATP production/consumption.

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Quantifying contractile cost

ATP consumption at the start of ischemic exercise is not “contaminated” by glycolytic or oxidative ATP production.

10

15

20

25

30

35

[PCr](mM)

0 50 100

Time (s)

y = -0.244x + 32.508

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Quantifying oxidative phosphorylation

15

25

35

[PCr] (mM)

0 50 100 150 200

Recovery time (s)

tau

rate constant kPCr = 1/tau

rapid recovery --> high kPCr --> high oxidative capacity

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Quantifying glycolysis

Recall:

Under ischemic conditions,

glycolytic H+ production = (pH)*() + ()*(PCr)

is the muscle buffer capacity

(Conley et al., Am J Physiol 273: C306, 1997)

PCr + H+ Cr + Pi

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Results:

What turns glycolysis on and off?

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Potential controllers of glycolysis

Metabolites

Pi, ADP, and AMP are substrates and allosteric activators of glycolytic enzymes

Calcium

glycolysis stimulation frequency (Conley et al., Am J Physiol 273: C306, 1997)

Hexose phosphates

substrates for glycolysis

+

+

++

+

GAPDH

glycogen

GP

PFK

PGK

PK

lactate

PGI

PGM

ALD

PGM

ENO

LDH

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Turning off glycolysis after exercise

0

1

2

3

4

[HP](mM)

0 20 40 60

ischemic exercise

ischemic rest

Post-exercise time (s)

0

5

10

15

20

25

30

[Pi]

(mM)

calcium important

Post-exercise time

Gly

coly

tic r

ate

calcium unimportant

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Evidence of post-exercise glycolysis

• pH falls due to continued lactic acid production

• [PCr] rises due to continued glycolytic ATP production

0

1

2

PCr(mM)

0 15 30 45 60

Post-exercise time (s)

-0.15

-0.1

-0.05

0

pH

0 15 30 45 60

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Agreement of two estimatesof post-exercise glycolysis

0

1

2

3

4

Glycolytic H+ production (mM)

0 15 30 45 60

Post-exercise time (s)

"pH method""PCr method"

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Time course of post-exercise glycolysis

The cessation of glycolysis must reflect the decline of a muscle activation-related factor such as calcium.

0

0.05

0.1

0.15

Glycolytic rate

(mM H+/s)

0 20 40 60

Post-exercise time (s)

ischemic exercise

ischemic rest

calcium unimportant

calcium important

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Turning on glycolysis at the start of exercise

What mechanism is responsible for this delayed onset?

0123456

lactate (mM)

6.95

7

7.05

7.1

7.15

pH

Time

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Testing the importance of metabolites

2 bouts of exercise

Will glycolysis begin sooner in Bout 2 (when metabolites are high) than in Bout 1 (when metabolites are low)?

10

20

[P i](mM)

0

75

150

[ADP](M)

0 50 100

Time (s)

10

20

30

[PCr](mM)

ischemic exercise

ischemic rest

Bout 1 Bout 2

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Glycolysis begins earlier in Bout 2 than in Bout 1

0

2

4

6

8

Gly

coly

tic

H+

pro

duct

ion (

mM

)

5 15 25 35 5 15 25 35

Time (s)

5 15 25 35

Bout 1(B1 = 12 s) (B1 = 24 s)

* * *

Bout 2Bout 2

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The onset of glycolysis coincides with elevated metabolites

aerobic rest onset of flux

[Pi] 3.4-3.8 16.2-20.6

[ADP] 0.013-0.014 0.080-0.121

[AMP] 2x10-5-3x10-5 0.0008-0.0012

(All concentrations in mM.)

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Summary of glycolysis data

To initiate and sustain high rates of glycolysis, both elevated metabolite levels and a muscle activation-related factor such as calcium are needed.

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Results (continued):

How is muscle metabolism affected by type 1 diabetes mellitus?

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Little is known about muscle metabolism in people with type 1 diabetes

Rat models suggest that diabetes-induced changes may be completely reversed by insulin treatment (Ianuzzo et al., J Appl Physiol 52: 1471, 1982; Noble & Ianuzzo, Am J Physiol 249: E360, 1985).

We asked whether careful treatment of type 1 diabetes with insulin restores human muscle metabolism to normal.

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Subjects

10 men with type 1 diabetes and 10 male age- and activity-matched control subjects

All diabetic subjects used insulin injections to keep blood glucose levels under good clinical control:

• glycosylated hemoglobin (HbA1c) levels 7%

• no glucose in the urine

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No significant difference in force

10

30

50

70% of max

force

0 50 100

Time (s)

CONTROL

DIABETIC

6.5

6.7

6.9

7.1

pH

0 50 100

Time (s)

10

30

50

70% of max

force

CONTROL

DIABETIC

*

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Glycolysis occurs “early and often” in diabetic subjects

Earlier onset Higher peak rate

0

1

2

3

4

5

Gly

coly

tic

H+

pro

duct

ion (

mM

)

0 5 10 15 20 25 30

Time (s)

CONTROL DIABETIC

*

*

0

0.1

0.2

0.3

0.4

0.5

Peak

gly

coly

tic

rate

(mM

H+

/s)

CONTROL DIABETIC

*

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Oxidative recovery rateis slower in diabetic subjects

10

15

20

25

30

[PC

r] (

mM

)

0 25 50 75

Recovery time (s)

CONTROL

DIABETIC

0

0.005

0.01

0.015

0.02

0.025

0.03

kPC

r (1

/s)

CONTROL DIABETIC

*

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Contractile cost: difference not significant

0

0.25

0.5

0.75

1

1.25

Contractile cost(mM PCr/s)

CONTROL DIABETIC

P = 0.12

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Summary of diabetes data

Elimination of symptoms of type 1 diabetes does not normalize muscle properties.

The observed abnormalities in glycolytic and oxidative fluxes suggest that diabetes causes a shift in the metabolic profile of the muscle.

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Future work

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Acknowledgments

Advisers

Kevin Conley

Marty Kushmerick

UW Departments

Physiology & Biophysics

Radiology

Funding

National Institutes of Health

National Science Foundation

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Acknowledgments(continued)

Coworkers

Cathy Amara

Iris Asllani

Outi Hyyti

Melissa Lambeth

Donghoon Lee

Dave Marcinek

Ken Marro

Daryl Monear

Brad Moon

Eric Shankland

Rudy Stuppard

Nina VØllestad

Coauthors

Mike Carey

Rod Gronka

Sharon Jubrias

Will Kemper

Jerry Milstein

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This talk, like glycolysis after exercise, is over.