control of energy metabolism on reproduction: a mechanism...
TRANSCRIPT
15.11.11
Control of energy metabolism on reproduction:
a mechanism maintained during evolution
Lab. A. Maggi
Center of Excellence in Neurodegenerative Diseases (CEND)
Department of Pharmacological Sciences
University of Milan
Sara Della Torre
Bone Vagina
Breast
Uterus Ovary
Liver
Heart
Brain
Bone Vagina
Breast
Uterus Ovary
Liver
Heart
Brain
The physiological roles of Estrogen
Bone Vagina
Breast
Uterus Ovary
Liver
Heart
Brain
Bone Vagina
Breast
Uterus Ovary
Liver
Heart
Brain
Estrogen and Estrogen Receptors
95% 60%
Estrogen Receptor: mechanisms of action
Enzimatic assay
(luminometer)
Bioluminescence Imaging, BLI
(CCD camera)
INSULATOR
( MAR )
lightluciferin + ATP = oxyluciferin + AMP +
TK
ERE 2x firefly luciferase
+/-+/-
INSULATOR
( MAR )
+/-+/-
ERE-luctransgenic mouse
Activated
Estrogen Receptor
Luciferin + ATP + O2 = oxyluciferin + AMP + LIGHT
Activated
Estrogen Receptor
The ERE-Luc reporter mouse: a paradigmatic model to study of ER transcriptional activity
Ciana et al., 2001
in vivo
Bioluminescence imaging
(CCD camera)
ex vivo
Enzymatic assay
(luminomiter)
Treatment with 17 -estradiol
ERE-Luc mouse: a tool to study ER transcriptional activity
Maggi et al., 2009
Ph
oto
n E
mis
sio
n
evening morning
evening (5.00-6.00 pm)
morning (9.00-10.00 am)
LIVER BLI
n = 15
p < 0.0001
EVIDENCE:
The ingestion of estrogen-free food results in a significant increase in the ER
transcriptional activity in liver. (Ciana et al., 2005)
ERE-Luc ad libitum with estrogen-free food
Giuseppe Arcimboldo, "The market gardener”
We decided to define the nature
of the signalling responsible
for liver ER activity
and its physiological meaning
With 2 different approaches:
Calorie
Restriction
Components
of diet
0 1 2 3 4 weeks
AL = ad libitum
CR = calorie restriction
Effects on ER transcriptional activity
Females ERE-Luc subjected to 40% CR
Acquisitions done in the MORNING
Della Torre et al., Cell Metabolism, 2011
Effects on LIVER ER activity and mRNA expression
Females ERE-Luc subjected to 40% CR
A
0 1 2 3 40
100
200
300
400
500
** ** ***
Time (weeks)
Cts
/s
C
0 1 2 40
100
200
300
400
* *
Time (weeks)
RL
U/
g p
rote
ins
CONCLUSIONS:
1. CR induced a significant decrease in ER
activity in LIVER
2. CR did not affect LIVER ERα mRNA
in vivo ex vivo
B
0 1 2 40
1
2
** **
Time (weeks)
LU
C m
RN
Are
l. e
xp
.
Della Torre et al., Cell Metabolism, 2011
Which component of diet is it responsible for LIVER ER activity?
in vivo - Gavage to ERE-Luc mice
C: carbohydrates AA: amino acids L: lipids
CONCLUSION:
Amino acids are responsible of inducing ER activity in LIVER of ERE-Luc mice
+75%
+60%
Della Torre et al., Cell Metabolism, 2011
AA: amino acids ICI: ICI182,780 (ER antagonist)
CONCLUSION:
Amino acids–dependent luciferase accumulation requires ER activation
Do amino acids have a DIRECT effect on liver ER activity?
Gavage to ERE-Luc mice
in vivo ex vivo
Della Torre et al., Cell Metabolism, 2011
CONCLUSION: Amino acids are responsible of inducing ER activity ALSO
in ERE-Luc hepatocytes in culture.
Do amino acids have a DIRECT effect on ER activity?
in vitro – primary hepatocyte culture from ERE-Luc mice
BCH:
AA-uptake inhibitor
Della Torre et al., Cell Metabolism, 2011
CONCLUSION: Amino acids are responsible of inducing ER activity ALSO
in HepG2 cells co-transfected with ERE-Luc reporter and ERα.
Do amino acids have a DIRECT effect on liver ER activity?
in vitro – HepG2 cells transfected with ERE-Luc reporter +/- ER
Della Torre et al., Cell Metabolism, 2011
CONCLUSIONS: The mechanism of AA induction of ER activity
seems to be mTOR dependent.
Rapa: Rapamycin mTOR inhibitor
Ly: Ly294002 PI3K inhibitor
H-89: PKA inhibitor
About the mechanism…
in vitro – HepG2 cells co-transfected with ERE-Luc reporter and ER
IN PROGRESS
Della Torre et al., Cell Metabolism, 2011
CONCLUSION:
The mechanism of AA
induction of ER activity
requires the phosphorylation
of S167 and/or Y537.
About the mechanism…
in vitro – HepG2 cells co-transfected with ERE-Luc reporter and ER (WT and mutants)
IN PROGRESS
Della Torre et al., Cell Metabolism, 2011
LIVER is a sex steroid-responsive organ (ERα is expressed in liver)
the major site of GH-regulated metabolism
the primary source of circulating IGF-1
the role of ERs in the LIVER remains to be elucidated
B 0 1 2 3 4L
ID/E
RE
-Lu
c E
RE
-Lu
c
CONCLUSION: Calorie restriction decrease ER activity in liver
and induces the blockade of estrous cycle.
Weeks of CR
Effects of 40% CR on estrous cycle progression
Females ERE-Luc
Della Torre et al., Cell Metabolism, 2011
1. CR ER activity in liver
2. CR fertility
3. AA ER activity in liver
4. CR + AA fertility?
Della Torre et al., Cell Metabolism, 2011
Regular diet versus hyper-proteic diet (+40% proteins)
Females ERE-Luc subjected to 40% CR
14%
66%
CONCLUSION: Dietary proteins rescue mice from CR-induced blockade
of the estrous cycle.
B
0 1 20
50
100
150regular diet AL
regular diet CR
hyperproteic diet AL
hyperproteic diet CR
Time (weeks)
Pro
estr
us (
%)
Della Torre et al., Cell Metabolism, 2011
Hyp: Liver ER activation could represent a permissive signal
for the reproductive organs.
HOW???
IGF-1 IGF-1
CONCLUSION: Amino acids are important to maintain liver IGF-1 production
Circulating IGF-1 levels
in vivo - Females ERE-Luc
*P<0.05; **P<0.01; ***P<0.001 (vs .P);
P<0.05 (vs. time 0) *P<0.05 (vs reg diet AL)
Della Torre et al., Cell Metabolism, 2011
CONCLUSIONS: 1. Amino acids increase liver IGF-1 output
2. this effect require transcriptional activation of ER
Circulating IGF-1 levels
in vivo - Gavage to ERE-Luc mice
Della Torre et al., Cell Metabolism, 2011
LERKO: liver ERα KO mice
Della Torre et al., Cell Metabolism, 2011
Physiological conditions
and after Calorie Restriction
Circulating IGF-1 levels in LERKO mice
CONCLUSIONS: 1. ER is involved in liver IGF-1 output
2. in LERKO CR sligthly decrease IGF-1 synthesis
Della Torre et al., Cell Metabolism, 2011
Effect on estrous cycle progression
Females LERKO subjected to 40% CR
LERKO mice:
IGF-1
Susceptibility to CR
after CR
after CR
days
days
days
ERαflox/flox AL
ERαflox/flox CR
LERKO AL
Proestrus
Estrus
Metestrus
Diestrus
Della Torre et al., Cell Metabolism, 2011
WHAT is the role of circulating IGF-1???
Is IGF-1 signaling involved
in the progression of the estrous cycle??
2 different approaches:
- a pharmacological blockade of IGF-1 signaling
(JB3, IGF1-R antagonist)
- a genetic mouse model (LID = liver IGF-1 KO)
Della Torre et al., Cell Metabolism, 2011
Della Torre et al.,
Cell Metabolism, 2011
LID Liver IGF-1 deficient
75% less IGF-1
IGF-1 floxed Alb-Cre recombinase
ERE-Luc
LID-ERE-Luc
loxP
loxP
ER
E
ER
E
ER
E
ER
E
TK
TK
LID: liver IGF-1 KO mice
Della Torre et al.,
Cell Metabolism, 2011
4 days cycle
ERE-Luc + JB3
ERE-Luc
(controls)
7 days cycle
LID-ERE-Luc
Impaired IGF-1 signaling is associated to altered estrous cycle
Circulating IGF-1 is necessary
for a proper progression
of the estrous cycle
Hypothesis:
Circulating IGF-1 could participate in the
control of the activity of reproductive organs
Effect on estrous cycle progression
Females ERE-Luc and LID/ERE-Luc subjected to 40% CR
CONCLUSION: Circulating IGF-1 has a role in the communication of
the energetic status to the reproductive tissues.
Weeks of CR
Della Torre et al., Cell Metabolism, 2011
Estrous cycle progression: focus on UTERUS
ISHIKAWA cells as model of uterine proliferation
CONCLUSIONS: 1. Uterine cell proliferation requires IGF-1
2. Uterine cell proliferation is ER-dependent
Della Torre et al., Cell Metabolism, 2011
CONCLUSION: IGF-1 and IGF1-R are strongly involved in the progression
of the reproductive cycle.
ER and IGF activity in the UTERUS
IHC
E2 max
IGF-1 max
E2 min
IGF-1 min
E2 min
IGF-1 ↑
E2
IGF-1 P E M D
Della Torre et al., Cell Metabolism, 2011
Liver ER
Dietary
amino acids
CONCLUSIONS
1. In mice liver ER is a sensor of amino acids availability
necessary for the control of fertility.
2. Liver ER controls fertility regulating the levels of
circulating IGF-1.
FOOD INTAKE and REPRODUCTION
C. elegans
(IR/IGF1R)
(insulin) (IGF1)
(NHR)
(CYP450)
(FOXO1)
(FOXO1)
DAUER PATHWAY in C. elegans
(PTEN)
(PI3K)
(AKT)
Modified from Von Stetina et al.,
Genome Biology, 2007.
Fontana et al., Science 2010
A. MAGGI Lab
Center of Excellence on Neurodegenerative Diseases
Department of Pharmacological Sciences
University of Milan
Adriana Maggi
Gianpaolo Rando (now University of Lausanne)
Clara Meda
Alessia Stell
Cristian Ibarra (now Karolinska Institutet)
Paolo Ciana
Valeria Benedusi
Elisa Faggiani
Giusy Monteleone
Paolo Sparaciari
Cristina Vantaggiato
Elisabetta Vegeto
ACKNOWLEDGMENTS
Institut de Genetique et de Biologie Moleculaire
et Cellulaire Centre National de la Recherche
Scientifique, France
Pierre Chambon and Andrée Krust
Department of Endocrinology,
Pathophysiology and Applied Biology,
University of Milan
Paolo Magni
Department of Structural and Cellular Biology,
Tulane University School of Medicine,
New Orleans, USA
Brian Rowan
Mount Sinai School of Medecine, USA
Derek LeRoith
University of Calabria
Marcello Maggiolini