control of energy metabolism on reproduction: a mechanism...

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


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

.


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%


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


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


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


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


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


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


1. CR ER activity in liver

2. CR fertility

3. AA ER activity in liver

4. CR + AA fertility?


Regular diet versus hyper-proteic diet (+40% proteins)


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 (

%)


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)


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


LERKO: liver ERα KO mice


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


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


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

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


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


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


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

control of energy metabolism on reproduction: a mechanism...

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