LLaboratoireaboratoire de de BBiologie et iologie et BBiotechnologies iotechnologies MMarinesarines

UMRUMR PPhysiologie et hysiologie et EEcophysiologie des cophysiologie des MMollusques ollusques MMarinsarins

Characterization of glucidic metabolism of Characterization of glucidic metabolism of

pacific oyster, pacific oyster, Crassostrea gigas, Crassostrea gigas, as a way as a way

to study the reserves managementto study the reserves management

A.C. Hanquet Dufour, C. Heude, K. Kellner, H. Bacca and M. MathiA.C. Hanquet Dufour, C. Heude, K. Kellner, H. Bacca and M. Mathieu eu

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INTRODUCTION : INTRODUCTION : The reserves of The reserves of Crassostrea gigasCrassostrea gigas

The reserves are stored in a specialized tissue :The reserves are stored in a specialized tissue :

This tissue is constituted by This tissue is constituted by vesicular cellsvesicular cells..

The vesicular cells are specialized in The vesicular cells are specialized in storage and mobilizationstorage and mobilization of of glycogen.glycogen.

GlycogenGlycogen is the major source of energy supporting general metabolism andis the major source of energy supporting general metabolism andparticularly during reproduction.particularly during reproduction.

GillsGillsMantleMantle

adductor adductor

MuscleMusclepericardicpericardic

CavityCavity

gonadic gonadic

Region Region

digestivedigestive

GlandGland

Labial Labial palpspalps

(4)(4)

EpithelialCell

vesicular cells

Cellular fragments

Cells : Ø 7-25µm (Heude Bethelin, 2000)

Dissociated cells from labial palps

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INTRODUCTION : glucose transport and these regulationsINTRODUCTION : glucose transport and these regulations

GlucoseGlucoseFoodFood

GlucoseGlucose

GlycogenGlycogen

1st stage of regulation1st stage of regulation

on transport of on transport of

glucoseglucose

GlucoseGlucose

2nd stage of regulation2nd stage of regulation

on synthesis or on synthesis or

degradation of degradation of

glycogenglycogen

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I. Characterization of glucidic metabolismI. Characterization of glucidic metabolism

I.1. Parameters of Glucose uptake : global mechanismI.1. Parameters of Glucose uptake : global mechanism

I.2. Systems of glucose transport : Existence of different I.2. Systems of glucose transport : Existence of different

transporters ?transporters ?

II. II. Seasonal variationsSeasonal variations

III. Experimental trophic conditioningIII. Experimental trophic conditioning

Results :Results :

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I. Characterization of glucidic metabolismI. Characterization of glucidic metabolism

I.1. Parameters of glucose I.1. Parameters of glucose

uptake : global mechanismuptake : global mechanism

K+

K+

Na+

Na+

Na+

Na+Do not

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Results : Kinetic of glucose uptake and effect of Results : Kinetic of glucose uptake and effect of extracellularextracellular

concentrationconcentration

15/07/04

0

0,1

0,2

0,3

0,4

0,5

0,6

1 2 3 4 5 6

time of incubation (hours)

glu

cose u

pta

ke (nm

)/10

6 c

ells

0,25 mM

1 mM

5 mM

•• EquilibriumEquilibrium is obtained after is obtained after 3 hours 3 hours for the highestfor the highest concentration of substrate.concentration of substrate.

•• maximalmaximal uptakeuptake is is # # 0,3 nmol/100,3 nmol/1066 cells.cells.

(2 hours and 0,33 nmol/10(2 hours and 0,33 nmol/1066 cells for cells for MytilusMytilus edulisedulis, Lenoir 1989), Lenoir 1989)

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0

0,05

0,1

0,15

0,2

0,25

0,3

0 5 10 15 20

S

V

SaturableSaturable componentcomponent

Results : determination of two components of glucose uptakeResults : determination of two components of glucose uptake

Kinetic of glucose uptake (october 04)

0

0,05

0,1

0,15

0,2

0,25

0,3

0 5 10 15 20

[glucose] mM

glu

cose u

pta

ke

(nm

ole

/h/1

06 c

ells)

Existence of two components : a Existence of two components : a

passivepassive diffusion diffusion andand aa saturablesaturable

component.component.

The Characteristics of The Characteristics of saturablesaturable

component were calculated with component were calculated with

MichaelisMichaelis--MentenMenten transformation. transformation.

(Km = 1,74 (Km = 1,74 mMmM and Vmax = and Vmax =

0,1057 nmoles/h/100,1057 nmoles/h/1066 cells)cells)

Vmax

Vmax/2

Km

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0

0,05

0,1

0,15

0,2

0,25

0,3

0 5 10 15 20

S

V

SaturableSaturable componentcomponent

Results : determination of two components of glucose uptakeResults : determination of two components of glucose uptake

Kinetic of glucose uptake (october 04)

0

0,05

0,1

0,15

0,2

0,25

0,3

0 5 10 15 20

[glucose] mM

glu

cose u

pta

ke

(nm

ole

/h/1

06 c

ells)

Existence of two components : a Existence of two components : a

passivepassive diffusion diffusion andand aa saturablesaturable

component.component.

The Characteristics of The Characteristics of saturablesaturable

component were calculated with component were calculated with

MichaelisMichaelis--MentenMenten transformation. transformation.

(Km = 1,74 (Km = 1,74 mMmM and Vmax = and Vmax =

0,1057 nmoles/h/100,1057 nmoles/h/1066 cells)cells)

Vmax

Vmax/2

Km

Passive DiffusionPassive Diffusion

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I. Characterization of glucidic metabolismI. Characterization of glucidic metabolism

I.2. Systems of glucose transport : I.2. Systems of glucose transport :

Existence of different transporters ?Existence of different transporters ?

K+

K+

Na+

Na+

Na+

Na+Do not

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inhibitors of glucose uptakeinhibitors of glucose uptake

Cytochalasin BCytochalasin B : an effective inhibitor of : an effective inhibitor of saturablesaturable diffusion (GLUT) (Keller et al., 1989)diffusion (GLUT) (Keller et al., 1989)

PhloridzinPhloridzin : a specific inhibitor of Na+: a specific inhibitor of Na+--glucose transport (SGLT) (glucose transport (SGLT) (HedigerHediger et al., 1987)et al., 1987)

Results : Different systems of glucose transport ?Results : Different systems of glucose transport ?

0

20

40

60

80

100

120

140

sans

inhibiteur

0,005mM 0,1mM 1mM 10mM

[phloridzin] mM

D-g

luco

se u

pta

ke (

%)

�� PhloridzinPhloridzin 1mM ~ 60%1mM ~ 60%

0

20

40

60

80

100

120

140

sans

inhibiteur

0,1 µM 1 µM 10 µM 100 µM 770 µM

[cytochalasin B] µM

D-g

luco

se u

pta

ke (

%)

�� CytochalasinCytochalasin B 10µM ~ 40 %B 10µM ~ 40 %Do not

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2. 2. SaturableSaturable

Diffusion Diffusion

(GLUT)(GLUT)

Conclusion : presence of different systems of glucose Conclusion : presence of different systems of glucose

transport ?transport ?

1. Passive 1. Passive

DiffusionDiffusion

GlucoseGlucose

GLUT transporterGLUT transporter

sodiumsodium--potassium potassium PumpPump

SGLT transporterSGLT transporter

K+

K+

3. Na+/glucose 3. Na+/glucose

transport (SGLT)transport (SGLT)Na+

Na+

Na+

Na+

SGLT SequenceSGLT Sequence

available available

(AY551089, (AY551089, HuvetHuvet

et al.,et al., 2004) 2004)

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II. Seasonal Variations II. Seasonal Variations

Evolution of uptake parameters Evolution of uptake parameters

during the seasonsduring the seasons

K+

K+

Na+

Na+

Na+

Na+Do not

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0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0 2 4 6 8 10[glucose] mM

glu

co

se

up

take

(nm

ol/h

/106

ce

lls)

February

April

Results : seasonal variations of the capacity of glucose Results : seasonal variations of the capacity of glucose

transport transport

Difference on the capacity for glucose transport in February andDifference on the capacity for glucose transport in February and April. April.

InterpretationInterpretation :: The system of glucose transport was different on the two sampliThe system of glucose transport was different on the two sampling ng

dates. (variability of the number of glucose transporters ? Modidates. (variability of the number of glucose transporters ? Modification on the affinity ?)fication on the affinity ?)

Variation of the capacity of glucose transport at Variation of the capacity of glucose transport at

different periods of year different periods of year

Similar Similar

DiffusionDiffusion

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0

0,05

0,1

0,15

0,2

0,25

0,3

0 2 4 6 8 10 12 14 16 18 20

S

V

AprilApril

0

0,025

0,05

0,075

0,1

0 5 10 15 20

S

V

FebruaryFebruary

Results : variations of Results : variations of saturablesaturable componentcomponent

0,050,05

0,050,05

DiffusionDiffusion

((nMnM/h/10/h/1066

cellscells))

0,1170,1170,950,95AprilApril

0,0330,0331,781,78FebuaryFebuary

Vmax Vmax ((nmolnmol//h/10//h/1066 cellscells))

Km Km (mM)(mM)

KineticKinetic ofofDD--Glucose Glucose

uptakeuptake

The lower the value of Km,The lower the value of Km,

the higher the the higher the ligandligand affinity to affinity to

substratesubstrate

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0

0,05

0,1

0,15

0,2

0,25

February April

glu

co

se

up

tak

e (

nm

ol/1

06 c

ells

)

standard

cytochalasin B (10µM)

phloridzin (1mM)

Results : comparisons between the constants of Results : comparisons between the constants of MichaelisMichaelis

and the effect of inhibitorsand the effect of inhibitors

0,050,05

0,050,05

DiffusionDiffusion

(nM/h/10(nM/h/1066

cells)cells)

0,1170,1170,950,95AprilApril

0,0330,0331,781,78FebruaryFebruary

Vmax Vmax (nmol//h/10(nmol//h/1066 cells)cells)

Km Km ((mMmM))

DD--GlucoseGlucose

Results are coherentResults are coherent : stronger : stronger

effect of cytochalasin B during April effect of cytochalasin B during April

(GLUT=high affinity for glucose) (GLUT=high affinity for glucose)

than Februarythan February--March.March.

28,6 %

71,3 %

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2. 2. SaturableSaturable

Diffusion Diffusion

(GLUT)(GLUT)

Hypothesis : influence of glucose concentration on the Hypothesis : influence of glucose concentration on the

uptake ?uptake ?

1. Passive 1. Passive

DiffusionDiffusion

GlucoseGlucose

GLUT transporterGLUT transporter

sodiumsodium--potassium potassium PumpPump

SGLT (Sodium Glucose transporter)SGLT (Sodium Glucose transporter)

K+

K+

3. Na+/glucose 3. Na+/glucose

transport (SGLT)transport (SGLT)Na+

Na+

Na+

Na+

SGLT SequenceSGLT Sequence

available available

(AY551089, (AY551089, HuvetHuvet

et al.,et al., 2004) 2004)

1st step of regulation1st step of regulation

on transport of on transport of

glucoseglucose

GlucoseGlucose

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Results : influence of the concentration of glucose on his Results : influence of the concentration of glucose on his

uptake and the expression of SGLT ?uptake and the expression of SGLT ?

Method : Different concentrations of glucose during 2 days of inMethod : Different concentrations of glucose during 2 days of incubation cubation

(0 to 10 (0 to 10 mMmM) of vesicular cells) of vesicular cells

This analyze is in processThis analyze is in process

Expression of SGLT is regulated by the Expression of SGLT is regulated by the extracellularextracellular concentration of concentration of

glucose. The maximum of expression is obtained for the cells whiglucose. The maximum of expression is obtained for the cells which were in ch were in

a low concentration of glucose a low concentration of glucose

�� The high concentrations of glucose regulate negatively the exprThe high concentrations of glucose regulate negatively the expression of ession of

SGLT transportersSGLT transporters

Expression of SGLTExpression of SGLT

0

0,005

0,01

0,015

0,02

To 0 0,10 0,50

[glucose extracellulaire] (mM)

QR

SG

LT

* : significant difference p<0,05 (test Student-Newman-Keuls)

*

*

*

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K+

K+

Na+

Na+

Na+

Na+

III. Experimental Trophic ConditioningIII. Experimental Trophic Conditioning

Effect of the food on glucose Effect of the food on glucose

metabolismmetabolism

IFREMER (Brest) IFREMER (Brest) –– University (University (CaenCaen) Collaboration, with ) Collaboration, with

H. Bacca H. Bacca

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Reproductive cycle of Reproductive cycle of Crassostrea gigasCrassostrea gigas

�� Aim : Study reserves management during Aim : Study reserves management during

these 3 periodsthese 3 periods

GametogenesisGametogenesis

ReservesReserves

Rest + Rest + lysislysis ReinitiatingReinitiating MaturationMaturation

StorageStorage MobilizationMobilizationMobilizationMobilization

maturitymaturity

JuneJune--JulyJuly OctoberOctober--NovemberNovember MarchMarch--AprilApril

MMAAMMFFJJDDNNOOSSAAJJJJ

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Materials and MethodsMaterials and Methods

Fast (AFast (A--) or Abundant diet (A+)) or Abundant diet (A+)((SkeletonemaSkeletonema costatumcostatum : 120 : 120 cells/µL or fast)cells/µL or fast)

SYstèmeSYstème Contrôlé Contrôlé

d’Ambiance d’Ambiance MARineMARine

T = 0T = 0

A+A+

•• CryopreservationCryopreservation of cells of cells

••Tests of cellTests of cell’’s survey and s survey and

metabolic tests metabolic tests

•• Measurement of quantity Measurement of quantity

of glycogen reservesof glycogen reserves

•• study of the gonad study of the gonad

development development

•• expressionexpression of SGLTof SGLTT = 30 T = 30 daysdays

AA--

Natural Natural

reference reference

= control= control

RR SS

SystSystèème Contrôlme Contrôléé dd’’Ambiance Ambiance MARineMARine

aération

Alimentation eau de mer+phytoplancton

évacuation

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Results :Results : Evolution of glycogen reservesEvolution of glycogen reserves

Conditionnement octobre 2004

mg de gln par prélèvement (36 animaux)

Huîtres S

0

100

200

300

400

500

600

T0E Tf E Tf A+ TfA-

palpes

GM

Conditionnement octobre 2004

mg de gln par prélèvement (36 animaux)

Huîtres R

0

100

200

300

400

500

600

T0E TfE TfA+ Tf A-

palpes

GM

RR : no significant differences between : no significant differences between

T0=“beginning of the experiment” T0=“beginning of the experiment”

(natural reference) and TF=“End of the (natural reference) and TF=“End of the

experiment” ; but for the fast diet the experiment” ; but for the fast diet the

glycogen decreased significantly (Aglycogen decreased significantly (A-- with with

p=0,0001p=0,0001) )

SS : no differences for each : no differences for each

condition in term of glycogen condition in term of glycogen

storagestorage

RR SS�� At fast diet : ability to mobilize At fast diet : ability to mobilize

the reserves of glycogen ?the reserves of glycogen ?Do n

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uthor

autho

rizati

on

Results : Expression of SGLT by realResults : Expression of SGLT by real--Time PCRTime PCR

SGLT (gonad-Mantel region) oct 2004

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

0,50

T0 E TF E TF A+ TF A-

QR

SG

LT

R

S

SGLT (palps) oct 2004

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

1,80

T0 E TF E TF A+ TF A-

QR

SG

LT

R

S

�� R and S showed no significant differences of SGLT expression. R and S showed no significant differences of SGLT expression.

BUTBUT

�� the level of SGLT expression is linked to trophic level the level of SGLT expression is linked to trophic level : :

higher for fast diets : facilitating the glucose uptake and the higher for fast diets : facilitating the glucose uptake and the storage of glycogen during storage of glycogen during

the autumnal period when the nutritive resources are poor.the autumnal period when the nutritive resources are poor.

RR SS

R = S

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CONCLUSION CONCLUSION

Characterization of glucidic metabolism :Characterization of glucidic metabolism :

•• Existence of two components : a Existence of two components : a passivepassive diffusion diffusion andand aa saturablesaturable component.component.

••The Characteristics of The Characteristics of saturablesaturable component can be obtained by component can be obtained by michaelismichaelis--mentenmenten

transformation. transformation.

Seasonal variationsSeasonal variations ::

Difference of the capacity of glucose transport during the year.Difference of the capacity of glucose transport during the year.

Interpretation : Interpretation : The system of glucose transport is modifiedThe system of glucose transport is modified. (variability of the . (variability of the

number of glucose transporters ? Modification of the affinity ?)number of glucose transporters ? Modification of the affinity ?)

Experimental trophic conditioning :Experimental trophic conditioning :

R oysters are able to use their reserves during fast period contR oysters are able to use their reserves during fast period contrary to S oystersrary to S oysters

R and S have not significant differences of SGLT expression butR and S have not significant differences of SGLT expression but the SGLT the SGLT

expression is linked to trophic level expression is linked to trophic level : higher for fast diet : facilitating the glucose : higher for fast diet : facilitating the glucose

uptake and the storage of glycogen during the autumnal period whuptake and the storage of glycogen during the autumnal period when the nutritive en the nutritive

resources are poor.resources are poor.

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ThanksThanks

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