PPAR

IS IT POSSIBLE TO INFLUENCE METABOLIC
PROCESS OF EUROPEAN SEA BASS JUVENILES BY A
NUTRITIONAL CONDITIONING DURING LARVAL
STAGE ?
•
•
•
•
M. Vagner
M
J.L. Zambonino Infante
J.H. Robin
J. Person-Le Ruyet
larvi 2009
5th fish & shellfish larviculture symposium
ghent university, belgium
7 - 10 september 2009
INTRODUCTION
Natural fish stocks
N
Nowadays
d
Carnivorous marine fish are mainly fed with fish
meals and oils
BUT fish aquaculture Ò and natural fish stocks Ô
Substitution of fish lipid sources with vegetable lipid
sources
Disadvantages of vegetable products:
• Not adapted to the requirement in essential fatty acids (HUFA):
rich in C18 fatty acids (i.e. HUFA precursors) BUT poor in HUFA (EPA, DHA)
p
y for marine fish to convert C18 FA into HUFA,, in p
particular because
• Low capacity
of the low expression of the Delta-6 desaturase (D6D)
*
AA
EPA
DHA
INTRODUCTION
Hi h interest
High
i
iin producing
d i fi
fish,
h which
hi h
could better incorporate
p
vegetable
g
products.
BUT HOW ?
Could it be p
possible using
g the concept
p of
metabolic programming ?
INTRODUCTION
Adaptive process at a cellular, molecular and biochemical levels occuring
during young stages, and then maintained in further developmental stages
and potentially transmissible to the offspring (Lucas, 1998)
¾ Is
I it possible
ibl in
i sea b
bass tto orient
i t physiological
h i l gi l process off jjuveniles
il b
by a llarvall
conditioning to dietary vegetable products, characterized by a low HUFA
content?
I- Nutritional conditioning 1
1. Experimental design
Larval sstage
Mouth
openning
d-5-45
LH16
2 diet
C1
2 T°C
HH16
LH22
LH Low HUFA EPA+DHA= 0.8% DM
HH22
HH High HUFA EPA+DHA=2.2% DM
16°C
22°C
Juven
nile stage
90 days
1
commercial IP
diet
1 HUFA
HUFArestricted
diet
R1
ex-LH16
EPA+DHA= 2.7% DM
d-151-211
60 days
ex-HH16
ex-LH22
ex-LH16
LH16
EPA+DHA= 0.5% DM
ex-HH22
19°C
ex-HH16
ex LH22
ex-LH22
19°C
ex-HH22
ex
HH22
I- Nutritional conditioning 1
C1
Indiv
vidual mean weig
ght (mg)
Larval stage
2. Growth performances
d-45
120
T***
100
80
No effect of diet on
the survival rate
diet ***
60
40
20
0
HH16
LH22
HH22
exHH22
exLH22
18
R1
Individual mea
an weight (g)
Juven
nile stage
LH16
exHH16
exLH16
14
100% survival rate
in all groups
No significant effect of
nutritional conditionning
10
6
-10
0
10
20
30
time (days)
40
50
60
J 211
I- Nutritional conditioning 1
5. Lipid metabolism in larvae
D6D Desaturation product at d-45 :
D6D mRNA level at d-45
18:3n-6 in PL
8
6
Relative
R
l ti expression
i iin
relation to an
housekeeping gene and to
a referential condition
HH22
***
***
04
0.4
0.3
18:3n-6 in % FAME
***
***
0.2
4
NS
2
01
0.1
0
0
LH16
HH16
LH22
LH16
HH16
LH22
HH22
Stimulation of desaturation pathways for HUFA synthesis, without any effect of
temperature
I- Nutritional conditioning 1
5. Lipid metabolism in larvae
2
AA % FAME PL
1.6
1.2
***
***
0.8
0.4
0
LH16
10
HH16
LH22
HH22
EPA % FAME PL
8
6
HUFA deficiency
***
***
4
2
0
LH16
30
25
HH16
LH22
HH22
DHA % FAME PL
20
***
15
***
10
5
0
LH16
LH16
HH16
HH16
LH22
LH22
HH22
HH22
I- Nutritional conditioning 1
6. Lipid metabolism in juveniles
D6 mRNA level – 1st part of the challenge
***
6
5
PL final composition in DHA
Relative expression in
relation to an housekeeping gene
24
% FAME
*
*
4
22
***
3
NS
2
20
NS
1
18
0
ex-LH16
ex-HH16
ex-LH22
ex-HH22
ex-LH16
ex-HH16
ex-LH22
ex-HH22
• Significant effect of larval conditionning
• Better capacity of ex
ex-LH
LH groups to
adapt to a low-HUFA diet
• BUT transient (30 days)
• BUT with a low amplitude
• NS for EPA
I- Nutritional conditioning 1
7. Conclusions
POSSIBLE to influence fatty acid desaturation pathways for HUFA synthesis in
juveniles, using a nutritional conditioning during larval stage, without any effect
of temperature on the capacity of FA desaturation (- 30% of HUFA requirement)
BUT this modulation seems to be limited
Is it possible to obtain a response with a larger magnitude?
II. Nutritional conditioning 2
Larval stage
2. Experimental design
Mouth
opening
d-5-45
XLH1
= C1
4 diets
C2
XLH1= 0.5%
HH1
LH1
LH1 = 0.7% HH1 = 1.7%
XH1
XH1 =3.7%
19°C
Juve
enile stag
ge
30 days
≠ 90 (C1)
1
PI
commercial
diet
XLH2
LH2
EPA+DHA=
EPA
DHA 2.7% DM
HH2
XH2
HH2
XH2
19°C
d-83- 118
1 more HUFA
≠d151-211 XLH2
LH2
R2
(R1)
restricted
diet
35 days
EPA+DHA= 0.3% DM
19°C
II. Nutritional conditioning 2
3. Growth performances
Mean individual weig
ght (mg)
Larval stag
ge
C2
b
b
60
***
50
a
a
XLH1
LH1
No effect of diet on
the survival rate
40
30
20
Idem C1
R2
Mean individual weight (g)
Juven
nile stage
10
HH1
XH1
4.5
100% of survival
rate in all groups
3.5
No significant effect of
larval conditionning
2.5
1.5
Idem R1
0.5
0
5
10
15
20
25
30
35
II. Nutritional conditioning 2
4. Lipid metabolism in larvae
D6D desaturation product at d-45 in
PL
D6D mRNA level at d-45
6
5
Relative expression in relation to an
house keeping gene
house-keeping
a
18:3n-6 in % FAME
0 6%
0.6%
***
a
***
0.5%
a
4
0.4%
b
3
0.3%
b
2
c
c
0.2%
0.1%
1
c
0.0%
0
XLH1
LH1
HH1
XH1
XLH
LH
HH
XH
Stimulation of desaturation pathways for HUFA synthesis
Idem C1
II. Nutritional conditioning 2
4. Lipid metabolism in larvae
3.5%
d
3.0%
Idem C1
2.5%
AA (% FAME in PL)
***
2.0%
c
1.5%
b
1.0%
a
0.5%
0.0%
12%
10%
8%
XLH1
LH1
HH1
EPA (% FAME in PL)
***
c
XH1
d
HUFA deficiency
6%
b
4%
a
2%
0%
40%
35%
30%
25%
XLH1
LH1
HH1
DHA (% FAME in PL)
***
c
XH1
d
20%
b
15%
10%
a
5%
0%
XLH1
XLH1
LH1
LH1
HH1
HH1
XH1
XH1
II. Nutritional conditioning 2
5. Lipid metabolism in juveniles
D6D mRNA level
7
PL final composition in DHA
Relative expression in relation to an house-keeping gene
XLH2
LH2
HH2
XH2
% FAME
NS
a
6
18
Diet ***
ab
a
5
4
b
a
a
3
IDEM for EPA
a
ab
2
c
b b
1
16
b
a
14
b
bc c
12
0
J-83
J-90
J-107
• Significant
g
effect of larval
conditionning
• D6D stimulation NON transient
≠ R1
J-118
ex-XLH
ex-LH
ex-HH
ex-XH
• Non significant effect of larval
conditioning
≠ R1
Summary
Although no beneficial effect on growth performances and no clear higher
HUFA content in PL, POSSIBLE to influence FA desaturation pathways for HUFA
synthesis
y
for a better incorporation
p
of vegetable
g
products,
p
, using
g a nutritional
conditioning during larval stage (-60% of the HUFA requirement)
Possible to use the concept of metabolic programming in sea bass
BUT is it a:
Long lasting adaptation?
Long-lasting
Transient acclimatation of juveniles to their nutritional environment ?
Need to better investigate mechanisms involved in desaturation pathways in
response to a low HUFA dietary content in larvae and in juveniles
PPAR
XLH1
LH1
0.5
0.7
+
PPAR
2.2
0.8 1.7%
Stimulation of desaturation pathways for HUFA synthesis
+ D6D
HH
LH HH1
+ D6D - PPAR
XH1
3.7%
Inhibition
- D6D
- PPAR
Implementation of metabolic programming using a nutritional conditioning
during larval stage
Stimulation of desaturation pathways for HUFA synthesis
+ D6D
+
PPAR
Inhibition
+ D6D - PPAR
- D6D
- PPAR
Possible to influence the FA desaturation pathways for HUFA synthesis for a
better incorporation of vegetable products
R2
BUT
R1
0,3
NO transient stimulation
DHA content in PL
0,5
HUFA
requirement at
0,7%
Transient stimulation
DHA content in PL
Additional factors: HUFA n-3 from the juvenile environment
Control of desaturation mechanisms previously gained
Control of the fish lipid composition
IV- Perspectives
Was the metabolic programming really present ?
Intermediate
period: Stop
or Non stop
?
Challenge extension:
what will happen during
adulthood
?
IF
YES
Application to other carnivorous species
with high commercial value in aquaculture
Transmission to
the offspring
?
Thanks to the financiallyy support:
pp
Ifremer,, INRA,, RAQ,
Q, Larvi.
Thanks
Th
k to the
h technical
h i l support: M
M.M
ML
Le G
Gall,
ll A
A. Sé
Sévère,
è
H
H.
Le Delliou, P. Quazuguel, N. Le Bayon, J. Moriceau, C.
Huelvan & E. Desbruyères.
y
Thanks for your attention
larvi 2009
5th fish & shellfish larviculture symposium
ghent university, belgium
7 - 10 september 2009
Récapitulatif
XLH1
0 5%
0,5%
LH1
0 7%
0,7%
LH
0 8%
0,8%
HH1
HH
1 7%
1,7%
2 2%
2,2%
XH1
3 7%
3,7%
LARVES
Stimulation des mécanismes de désaturation
Adaptation à un régime carencé en HUFA n-3
R2
0,3
Niveau ARNm D6 continue
R1
0,5
Niveau ARNm D6 transitoire
D6 fonctionnelle ? D6 non fonctionnelle ?
-
Synthèse d’HUFA
R1 trop proche du besoin
(0,7% EPA+DHA)
dé i é
dérivées
teneur en DHA
Faible en DHA
JUVENILES
XLH1
0 5%
0,5%
LH1
0 7%
0,7%
LH
0 8%
0,8%
HH1
HH
1 7%
1,7%
2 2%
2,2%
+ +
D6D PPAR
+ D6D PPAR
D6D
PPAR
Adaptation à un régime carencé en HUFA n-3
0,3
3 7%
3,7%
LARVES
Stimulation des mécanismes de désaturation
R2
XH1
R1
0,5
HUFA = AGLPI (n-9, n-6 et n-3) = Acides gras insaturés à 20-22 atomes de
carbone et au moins 4 doubles liaisons = Acides g
gras essentiels
Constituants principaux des PL de la bi-couche
membranaire
C:X n-Y
Chez les poissons, majoritairement les HUFA n-3:
O
OH
-Acide docosahéxaénoique 22:6n-3 (DHA)
2
-Acide écosapentaénoique 20:5n-3 (EPA)
4
5
3
6
1
Acide arachidonique 20:4n-6 (AA)
• Croissance et survie larvaire
• Développement de la vision et du cerveau
• Résistance au stress et aux maladies
• Qualité de la chair, …
III- Hypothesis concerning the regulation of lipid metabolism in sea bass
PPARs
d-45
Larval sstage
0.8%
0.7%
0.5%
Juvenile sttage
The end of
the challenge
LH
LH1
XLH1
HH1
HH
1.7%
2.2%
+
+
-
D6D
D6D
D6D
+
-
PPAR PPAR
PPAR
+
+
-
D6D
D6D
D6D
+
-
PPAR PPAR
PPAR
XH1
3.7%
III- Hypothèses de régulation du métabolisme lipidique du bar
Étude des PPAR
P
Peroxisome
i
P
Proliferator-Activated
lif
t A ti t d R
Receptor,
t
récepteurs
é
t
nucléaires
lé i
Trois isoformes: α, β et γ
Décrits chez les mammifères et le bar
Facteur de transcription de la delta 6 chez les mammifères
Dans le contexte de programmation métabolique, les PPAR sont-ils impliqués
dans la stimulation de la transcription du gène de la D6D chez le bar?
III- Hypothèses de régulation du métabolisme lipidique du bar
+
+
PPAR
D6D
PPAR
D6D
Mise en p
place d’une mémorisation au niveau moléculaire des
mécanismes de désaturation des AG
IV- Conclusions & Perspectives
1. General conclusion
1
Nutritional
environment
Nutritional
conditionning
during larval
stage
POSSIBLE to influence the fatty
acid desaturation pathways for
HUFA synthesis for a better
incorporation of vegetable meals
3
+
Previously gained
mechanisms
Fish lipid composition
0 5 < HUFA dietary
0.5
di
content
PPAR
2
<
07
0.7
D6D transcription
0,5
0,7
0,8
1,7 2,2 3,7
Teneur en EPA+DHA du régime (%MS)
Stimulation des mécanismes de désaturation
HUFA
HUFA
HUFA
HUFA
HUFA
+
+
Inhibition
HUFA
A
HUFA
HUFA
HUFA
HUFA
HUFA
-
PPAR
?
?
Stad
de larvaire
e
Gène de la delta 6
+
-
+
+
Transcription du gène de laΔ6-D
+
-
Traduction de l’ARNm en protéine
+
+
Synthèse d’AG précurseurs des HUFA
-
Mise en place d’une programmation métabolique par le
conditionnement larvaire
Présence de cette programmation chez les juvéniles
Facteurs additionnels: teneur en HUFA n-3 de l’environnement
nutritionnel
g
(%MS)
(
)
R2 = 0,3%
0 3% Teneur en EPA+DHA du régime
R1 = 0,5%
0 5%
07
0.7
Stade
e juvénile
Contrôle des mécanismes de désaturation préalablement acquis
- Fin du challenge
+
+
Début du challenge
Transcription du gène de la D6D
Niveau ARNm D6 continue
D6
fonctionnelle ?
-
Ni
Niveau
ARNm
ARN D6 transitoire
t
it i
D6 non
fonctionnelle ?
+
-
+
-
Synthèse d’HUFA
dérivées
teneur en DHA
Faible en DHA