Control of energy metabolism on reproduction: a mechanism maintained during evolution Sara Della Torre Lab. A. Maggi Center of Excellence in Neurodegenerative Diseases (CEND) Department of Pharmacological Sciences University of Milan 15.11.11
The physiological roles of Estrogen Brain Heart Liver Breast Ovary Uterus Vagina Bone
Estrogen and Estrogen Receptors 95% 60%
Estrogen Receptor: mechanisms of action
The ERE-Luc reporter mouse: a paradigmatic model to study of ER transcriptional activity Luciferin + ATP + O 2 = oxyluciferin + AMP + LIGHT Activated Estrogen Receptor ERE-luc transgenic mouse Activated Estrogen Receptor TK +/- +/- INSULATOR ( MAR ) ERE 2x firefly luciferase luciferin + ATP = oxyluciferin + AMP + light INSULATOR ( MAR ) in vivo Bioluminescence Imaging, imaging BLI (CCD (CCD camera) ex vivo Enzymatic Enzimatic assay (luminometer) (luminomiter) Ciana et al., 2001
Photon Emission ERE-Luc mouse: a tool to study ER transcriptional activity Treatment with 17 -estradiol Maggi et al., 2009
ERE-Luc ad libitum with estrogen-free food evening morning LIVER BLI n = 15 p < 0.0001 evening (5.00-6.00 pm) morning (9.00-10.00 am) EVIDENCE: The ingestion of estrogen-free food results in a significant increase in the ER transcriptional activity in liver. (Ciana et al., 2005)
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 Giuseppe Arcimboldo, "The market gardener
Effects on ER transcriptional activity Females ERE-Luc subjected to 40% CR 0 1 2 3 4 weeks AL = ad libitum CR = calorie restriction Acquisitions done in the MORNING
Cts/s Effects on LIVER ER activity and mrna expression Females ERE-Luc subjected to 40% CR RLU/ g proteins LUC mrna rel. exp. A 500 400 300 200 100 0 in vivo * ** ** ** 0 1 2 3 4 Time (weeks) C 400 300 200 100 ex vivo * * 0 0 1 2 4 Time (weeks) B 2 1 ** ** 0 0 1 2 4 Time (weeks) CONCLUSIONS: 1. CR induced a significant decrease in ER activity in LIVER 2. CR did not affect LIVER ERα mrna
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 +75% +60% CONCLUSION: Amino acids are responsible of inducing ER activity in LIVER of ERE-Luc mice
Do amino acids have a DIRECT effect on liver ER activity? Gavage to ERE-Luc mice AA: amino acids ICI: ICI182,780 (ER antagonist) in vivo ex vivo CONCLUSION: Amino acids dependent luciferase accumulation requires ER activation
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 ERE-Luc hepatocytes in culture.
Do amino acids have a DIRECT effect on liver ER activity? in vitro HepG2 cells transfected with ERE-Luc reporter +/- ER CONCLUSION: Amino acids are responsible of inducing ER activity ALSO in HepG2 cells co-transfected with ERE-Luc reporter and ERα.
About the mechanism in vitro HepG2 cells co-transfected with ERE-Luc reporter and ER IN PROGRESS Rapa: Rapamycin Ly: Ly294002 H-89: mtor inhibitor PI3K inhibitor PKA inhibitor CONCLUSIONS: The mechanism of AA induction of ER activity seems to be mtor dependent.
About the mechanism in vitro HepG2 cells co-transfected with ERE-Luc reporter and ER (WT and mutants) IN PROGRESS CONCLUSION: The mechanism of AA induction of ER activity requires the phosphorylation of S167 and/or Y537.
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
LID/ERE-Luc ERE-Luc Effects of 40% CR on estrous cycle progression Females ERE-Luc B 0 1 2 3 4 Weeks of CR CONCLUSION: Calorie restriction decrease ER activity in liver and induces the blockade of estrous cycle.
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) Females ERE-Luc subjected to 40% CR 66% 14% regular diet AL regular diet CR hyperproteic diet AL hyperproteic diet CR CONCLUSION: Dietary proteins rescue mice from CR-induced blockade of the estrous cycle.
Hyp: Liver ER activation could represent a permissive signal for the reproductive organs. HOW??? IGF-1 IGF-1
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) CONCLUSION: Amino acids are important to maintain liver IGF-1 production
Circulating IGF-1 levels in vivo - Gavage to ERE-Luc mice CONCLUSIONS: 1. Amino acids increase liver IGF-1 output 2. this effect require transcriptional activation of ER
LERKO: liver ERα KO mice
Circulating IGF-1 levels in LERKO mice Physiological conditions and after Calorie Restriction 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 ERα flox/flox AL days Proestrus Estrus Metestrus Diestrus ERα flox/flox CR days LERKO AL after CR LERKO mice: IGF-1 Susceptibility to CR days after CR
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)
ERE ERE TK ERE ERE TK LID: liver IGF-1 KO mice IGF-1 floxed Alb-Cre recombinase loxp ERE-Luc LID Liver IGF-1 deficient 75% less IGF-1 loxp LID-ERE-Luc Della Torre et al., Cell Metabolism, 2011
Impaired IGF-1 signaling is associated to altered estrous cycle ERE-Luc (controls) 4 days cycle ERE-Luc + JB3 7 days cycle LID-ERE-Luc Della Torre et al., Cell Metabolism, 2011
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 Weeks of CR CONCLUSION: Circulating IGF-1 has a role in the communication of the energetic status to the reproductive tissues.
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
ER and IGF activity in the UTERUS IHC E 2 max IGF-1 max E 2 min IGF-1 min E 2 min IGF-1 P E M D E 2 IGF-1 CONCLUSION: IGF-1 and IGF1-R are strongly involved in the progression of the reproductive cycle.
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. Dietary amino acids Liver ER
FOOD INTAKE and REPRODUCTION C. elegans
(insulin) (IGF1) DAUER PATHWAY in C. elegans (PI3K) (IR/IGF1R) (PTEN) (AKT) (CYP450) (FOXO1) (NHR) (FOXO1) Modified from Von Stetina et al., Genome Biology, 2007.
Fontana et al., Science 2010
ACKNOWLEDGMENTS 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 Department of Endocrinology, Pathophysiology and Applied Biology, University of Milan Paolo Magni Institut de Genetique et de Biologie Moleculaire et Cellulaire Centre National de la Recherche Scientifique, France Pierre Chambon and Andrée Krust Alessia Stell Cristian Ibarra (now Karolinska Institutet) Paolo Ciana Mount Sinai School of Medecine, USA Derek LeRoith Valeria Benedusi Elisa Faggiani Giusy Monteleone Paolo Sparaciari Cristina Vantaggiato Elisabetta Vegeto University of Calabria Marcello Maggiolini Department of Structural and Cellular Biology, Tulane University School of Medicine, New Orleans, USA Brian Rowan