1. health and fitness
2. disease
3. cancer

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0 Organizing Committee
Co-President :
William Colmers
Rémi Quirion
International Committee:
Annette Beck Sickinger
Helen Cox
Donald Gehlert
Scientific Advisory Board:
Claudia Cavadas
Herbert Herzog
Akio Inui
Satya Kalra
Dan Larhammar
Alfredo Ribeiro-de-Silva
Local Committee
Yvan Dumont
Brigitte Guérin
Danielle Jacques
i General information
Venue
Hotel OMNI Mont Royal, 1050 Sherbrooke St. W., Montreal QC, Canada, H3A 2R6
Oral presentations: Salon Pierre de Coubertin (Junior Ballroom)
Poster presentations: Salon des Saisons (Ballroom A and B)
Registration Desk
Hotel OMNI Mont Royal, first floor, lobby Printemps
Official Language
All presentations are in English, no simultaneous translation is available
Notice to Speakers
The speakers are requested to bring their Power Point presentation to the session room at
least 30 minutes before their sessions. Please check with the registration desk for details.
Notice to Poster Presenters:
The posters will be located in Salon des Saisons (Ballroom A and B).
Size of poster board: 160 cm (W) x 120 cm (H)
Each poster board is marked with a number referring to the poster number in the program.
All posters should be mounted on July 1st by 12:00 and remove at 9:00 AM on July 4th.
Please note that posters left on the poster board when the removal time is over will be
disposed of by the organizer.
ii Registration
Registration includes admission to scientific sessions (oral and posters). It also includes
continental breakfast, morning and afternoon coffee breaks, the Welcome Reception, four
complimentary lunches and wine and cheese during the two poster sessions.
Welcome Reception
June 30 18:30-21:00
Location Hotel OMNI Mont Royal, first floor, Salon Printemps
Continental breakfasts and coffee breaks
Will be served in Salon des Saisons (Ballroom A and B).
Lunch
Lunches will be served in Salon Printemps
If you are requiring special food, please contact the registration desk, or contact Yvan Dumont
at his e-mail address as soon as possible
Wine and cheese
Wine and cheese will be served during the two poster session on July 1st and 2nd
iii Meeting floor plan
iv Posters room, Salon des Saisons (Ballroom A and B)
v Sponsors of the 10th International NPY-PYY-PP/GIPIO meeting
Many thanks to all sponsors of this scientific and educational event
Terumo Life Science Foundation
Gold Sponsors
vi Silver Sponsors
Other Sponsors
List of Exibitors
PolyPeptide
vii Scientific Program
Oral presentations
In Pierre de Coubertin (Junior Ballroom)
June 30th 2012,
16:00hr Memorial to Kazuhiko Tatemoto and Zofia Zukowska
16:30 Openning of this scientific and educational event
by William Colmers and Rémi Quirion.
Session 1
Implications of Y1, Y2, Y4 and Y5 receptors in anxiety and depression disorders.
Chair by Don Gehlert
Invited speakers will present the evidence supporting the implication of NPY and its
receptors (Y1, Y2, Y4 and Y5 subtypes) in anxiety and depression-like behaviors, and
most importantly their possible therapeutic application.
16:45-17:05 O1. Rémi Quirion (Montréal QC, Canada).
VARIOUS NEUROPEPTIDE Y RECEPTOR SUBTYPES ARE IMPLICATED
IN THE MODULATORY EFFECTS OF NPY IN ANXIETY AND MOOD
DISORDERS.
17:05-17:25 O2. Pascal Bonaventure (San Diego CA, USA).
THERAPEUTIC POTENTIAL OF BRAIN PENETRANT Y2 RECEPTOR
ANTAGONIST
17:25-17:45 O3. Peter Holzer (Graz, Austria).
THERAPEUTIC POTENTIAL OF Y4 RECEPTOR ANTAGONISM IN MOOD
DISORDERS
17:45-18:05 O4. Xinyan Huang (Paramus NJ, USA).
THE NEUROPEPTIDE Y Y5 RECEPTOR ANTAGONIST LU AA33810
EXERTS ANXIOLYTIC AND ANTIDEPRESSANT-LIKE EFFECTS IN
VARIOUS RODENT MODELS OF MOOD DISORDERS.
18:05-18:25 O5. Renu Sah (Cincinnati, USA)
NEUROPEPTIDE Y AND POSTTRAUMATIC STRESS DISORDER (PTSD):
A BENCH AND BEDSIDE VIEW
18:25-18:45 Open discussion and Comments
18:45-19:00
Welcome Reception
Salon Printemps
1 July 1st , 2012
8:45-9:30 Plenary Lecture from Michael Meaney (Montreal QC, Canada).
Effects of environmental factors and epigenetic in association with anxiety
and depressive disorders.
Session 2
9:30-10:20
NPY-PYY-PP: Evolution, Genetic variation and Obesity.
Chair by Dan Larhammar.
9:30-9:55 O6. Dan Larhammar (Uppsala, Sweden).
EVOLUTION OF THE NPY SYSTEM AND GENETIC POLYMORPHISM OF
THE HUMAN Y4 GENE AND ITS ASSOCIATION WITH OBESITY.
9:55-10:20 O7. Cláudia Cavadas (Coimbra, Portugal)
ROLE OF NPY ON AUTOPHAGY IN HYPOTHALAMUS: A CALORIC
RESTRICTION MIMETIC?
10:20-10:45: AM Coffee Break
Coffee and tea
Session 3
10:45-12:30
NPY/PYY/PP in neurogenesis, cell survival and neuroprotection.
Chair by William Gray.
10:45-11:10 O8. William Gray (Southampton, UK).
NPY MODULATION OF NEUROGENESIS: MECHANISMS AND
INTERACTIONS.
11:10-11:35 O09. Joao Malva (Coimbra, Portugal).
MULTIFACETED NPY: NEUROGENESIS, NEUROINFLAMMATION AND
NEUROPROTECTION.
11:35-12:00 O10. Maria Smialowska (Krakov, Poland)
NEUROPROTECTIVE POTENTIAL OF NEUROPEPTIDE Y AND NPY 13-36
IN ISCHEMIC MODELS
12:00-14:00 Lunch, Salon Printemps
2 Session 4
14:00-15:15
NPY-PYY-PP in cardiovascular functions.
Chair by Tom Westfall.
14:00-14:25 O11 Tom Westfall (St-Louis MO, USA)
INTERACTIONS OF NEUROPEPTIDE Y, CATECHOLAMINES AND
ANGIOTENSIN AT THE VASCULAR NEUROEFFECTOR JUNCTION.
14:25-14:50 O12 Jessica Murray (St-Louis MO, USA)
ROLE OF PROSTANOIDS IN THE MODULATION BY ANGIOTENSIN-(1-7)
OF NEUROPEPTIDE Y AND NOREPINEPHRINE RELEASE AT
SYMPATHETIC NEUROEFFECTOR JUNCIONS..
14:50-15:15 O13 Petr Maslyukov (Yaroslavl, Russia)
DEVELOPMENT OF NEUROPEPTIDE Y-CONTAINING NEURONS IN THE
AUTONOMIC GANGLIA
15:15-15-45 PM coffee break
Coffee, tea and juice
Session 5
15:45-16:35
Alcohol-Induced plasticity in brain NPY systems.
Chair by Nicholas Gilpin
15:45-16:10 O14 Nicholas Gilpin (New Orleans LA, USA).
NPY SYSTEMS IN EXTENDED AMYGDALA ARE RECRUITED DURING
THE TRANSITION TO ALCOHOL DEPENDENCE
16:10-16:35 O15 Todd Thiele (Chapel Hill NC, USA).
NPY AGONISTS PROTECT AGAINST BINGE-LIKE ETHANOL DRINKING
IN NON-DEPENDENT MICE: EVIDENCE FOR A ROLE OF THE CENTRAL
AMYGDALA
Session 6
16:45-17:45
Short Oral presentation by students and postdocs posters (3-4 min presentations)
Chair by Claudia Cavadas
16:45 P01 Masanobu Ito
MOOD AND MEMORY-ASSOCIATED BEHAVIORS IN NEUROPEPTIDE Y5
KNOCKOUT MICE.
16:50 P02 Julio-Cesar Morales-Medina
THE SELECTIVE NEUROPEPTIDE Y Y5 AGONIST [CPP1–7,NPY19–23, ALA31,
AIB32, GLN34]HPP DIFFERENTLY MODULATES EMOTIONAL PROCESSES AND
BODY WEIGHT IN THE RAT
3 16:55 P04 Nathalie Trent
THE ROLE OF NEUROPEPTIDE Y Y1 AND Y2 RECEPTORS IN THE LATERAL
SEPTAL REGULATION OF ANXIETY IN THREE ANIMAL MODELS
17:00 P05 Fabio Canneva
NPY ACCUMULATION IN DP4/CD26-DEFICIENT CONGENIC RATS
17:05 P06 Cuihong Jia
NPY MEDIATES INJURY-INDUCED ADULT NEUROREGENERATION
17:10 P07 Ana Santos-Carvalho
NEUROPROTECTIVE ROLE OF NPY IN RETINAL CELLS
17:15 P08 Nima Aghili
POLYMORPHISMS IN NEUROPEPTIDE Y SIGNALLING GENE ARE ASSOCIATED
WITH THE RISK OF MYOCARDIAL INFARCTION IN PATIENTS WITH
ATHEROSCLEROSIS
17:20 P10 Katja Kostelnik
PROTEOME ANALYSIS OF HUMAN Y1-RECEPTOR EXPRESSING BREAST
CANCER CELLS
17:25 P11 Verena Ahrens
CARBABORANE CONTAINING NEUROPEPTIDE Y ANALOGS FOR BREAST
CANCER THERAPY
17:30 P12 Stefanie Babilon
POSTENDOCYTIC FATE OF THE HUMAN Y2-RECEPTOR-LIGAND-SYSTEM
17:35 P13 Sven Hofmann
SWITCHING Y-RECEPTOR SELECTIVITY OF REDUCED-SIZE NPY ANALOGS
17:40 P14 Veronika Mäde
IMPROVED PANCREATIC POLYPEPTIDE-BASED ANALOGS FOR OBESITY
TREATMENT
17:45 P15 Navjett Jolly
LONG CHAIN LIPIDATION OF Y4 RECEPTOR AGONISTS PROLONGS THEIR
ACTIVITY
17:50 P16 Xavier Pedragosa Badia
FROM CHIMERIC RECEPTORS TO SINGLE RESIDUES: CHARACTERISATION OF
NEUROPEPTIDE Y RECEPTOR SELECTIVITY
17:55 P17 Jasna Pruner
INVESTIGATION OF THE HUMAN Y2 RECEPTOR LIGAND-BINDING POCKET
4 18:00 P18 Bo Xu
MODELING, DOCKING AND MUTAGENESIS OF THE HUMAN NPY/PYY
RECEPTOR Y2 IDENTIFIES RESIDUES INVOLVED IN PEPTIDE BINDING AND
RECEPTOR STABILITY
18:10-19:30
Poster session in Salon des Saisons
Wine and Cheese will be served during the poster session
5 July 2nd 2012
8:15-9:00
Plenary Lecture from Annette Beck-Sickinger (Leipzig, Germany)
NPY-PYY-PP selective ligands and their applications in cancer therapy.
.
Session 7
9:00- 10:30
NPY-PYY-PP and cancer.
Chair by Joanna Kitlinska
9:00-9:25 O16 Joanna Kitlinska (Washington, DC, USA).
NPY AND ITS RECEPTORS AS NEW TARGETS IN NEUROBLASTOMA
THERAPY
9:25-9:50 O17 Suliaman Sheriff (Cincinnati, OH, USA).
NPY AND ITS RECEPTORS, A NOVEL THERAPEUTIC TARGET FOR
BREAST CANCER.
9:50-10:15 O18 Brigitte Guerin (Sherbrooke QC, Canada).
RADIOLABELLED NPY PEPTIDES TO IMPROVE CANCER DIAGNOSIS BY
POSITRON EMISSION TOMOGRAPHY.
10:15-10:45 AM Coffee Break
Coffee and tea
Session 8
10:45-12:30
NPY-PYY-PP: Ligand-receptor interactions and drug development.
Chair by Annette Beck-Sickinger.
10:45-11:10 O19 Knud Jensen (Copenhagen, Denmark).
DESIGN OF NOVEL PYY ANALOGUES FOR THE TREATMENT OF
OBESITY.
11:10-11:35 O20 Peter Schmidt (Leipzig, Germany)
THE Y2 RECEPTOR IN INTERACTION WITH NPY – MOVING TOWARDS
STRUCTURAL STUDIES BY NMR.
11:35-12:00 O21 Gregory Sliwoski and Elizabeth Dong (Nashville TN, USA).
COMPUTATIONAL DOCKING OF POTENTIAL THERAPEUTICS IN THE Y4PANCREATIC POLYPEPTIDE COMPARATIVE MODEL
12:00-12:25 O22 Karin Moerl (Leipzig, Germany).
THE ANTEROGRADE TRANSPORT OF HUMAN NEUROPEPTIDE Y
RECEPTORS IS REGULATED BY SEQUENCE MOTIFS IN THE PROXIMAL
C-TERMINUS.
6 12:30-14:00 Lunch, Salon Printemps
Session 9
14:00- 15:40
Diabetes Scourge: Hypothalamic NPY signaling as a new therapeutic target.
Chair by Satya Kalra.
14:00-14:25 O23 Satya Kalra (Gainsville, FL, USA).
LEPTIN-NPY LINK IN THE ETIOLOGY OF DIABETES TYPE 1 AND 2,
INDEPENDENT OF WEIGHT AND APPETITE REGULATION.
14:25-14:50 O24 Yan-Chuan Shi (Sydney, Australia),
THE DIFFERENTIAL ROLE OF CENTRAL AND PERIPHERAL Y2
RECEPTORS IN REGULATING GLUCOSE HOMEOSTASIS.
14:50-15:15 O25 Sheng Bi (Baltimore, MD, USA),
DORSOMEDIAL HYPOTHALAMIC NPY AND GLUCOSE HOMEOSTASIS
15:15-15-45 PM coffee break
Coffee, tea and juice
Session 10
15:45-16:50
NPY-PYY-PP and their implications in Pain and Skin.
Chair by Alfredo Ribeiro-da-Silva
15:45-16:10 O26 Alfredo Ribeiro-da-Silva (Montreal QC, Canada).
CHANGES IN SKIN INNERVATION IN A NEUROPATHIC PAIN MODEL
16:10-16:35 O27 Bradley Taylor (Lexington KY, USA).
TONIC INHIBITON OF CHRONIC PAIN BY NEUROPEPTIDE Y.
Session 11
16:45-18:00
Short Oral presentation by students and postdocs posters (3-4 min presentations)
Chair by Karin Moerl
16:45 P19 Cecilia Alves
ALTERED NPY SYSTEM ACTIVITY THROUGHOUT BONE REGENERATION
16:50 P20 Joana Rosmaninho-Salgado,
NPY CLEAVED BY DIPEPTIDYL-PEPTIDASE IV (DPPIV) INDUCES PREADIPOCYTES DIFFERENTIATION
7 16:55 P21 Shu Lin
CENTRAL ROLE OF NPY IN THE REGULATION OF SYMPATHETIC TONE AND
ENERGY EXPENDITURE
17:00 P22 Yan-Chuan Shi
LIPID METABOLISM IN SKELETAL MUSCLE IS CONTROLLED BY ARCUATE
NEUROPEPTIDE Y
17:05 P23 Amy Nguyen
Y1 AND Y5 RECEPTORS ARE BOTH CRITICAL FOR THE REGULATION OF FOOD
INTAKE AND ENERGY HOMEOSTASIS IN MICE
17:10 P24 Lei Zhang
ADRENERGIC NEURON-ONLY EXPRESSION OF NPY EXACERBATS STRESSINDUCED FAT GAIN IN CHOW-FED MICE
17:15 P25 Laura Vähätalo
INVOLVEMENT OF PERIPHERAL Y1 AND Y2 RECEPTORS IN THE METABOLIC
PHENOTYPE OF TRANSGENIC MICE OVEREXPRESSING NPY IN THE
SYMPATHETIC NERVOUS SYSTEM
17:20 P26 Liisa Ailanen
HEPATIC FATTY ACID AND CHOLESTEROL METABOLISM IN NPY
OVEREXPRESSING MICE
17:25 P27 Sarah Forbes
ROLE OF ENDOGENOUS PEPTITE YY IN GASTROINTESTINAL AND FEEDING
RESPONSES TO STRESS
17:30 P28 Iain R. Tough
DIFFERENTIAL EFFECTS OF GPR119 AGONISTS IN WILD TYPE AND PEPTIDE
YY KNOCKOUT MOUSE AND HUMAN COLON MUCOSAE.
17:35 P29 Maria Daniela Hurtado
THE NPY SYSTEM: A NOVEL PHYSIOLOGICAL DOMAIN
17:40 P32 Sylvia Els
A TRIPEPTIDE SWITCH REGION TO INDUCE AGONISM AND INVERSE AGONISM
AT THE GHRELIN RECEPTOR
17:45 P33 Robert Tolhurst
THE EFFECT OF NOVEL GHRELIN RECEPTOR AGONISTS ON CELL GROWTH
18:00-19:30
Poster session
Wine and cheese will be served during the poster session
8 July 3rd 2012
8:15-9:00
Plenary Lecture from Dr. Scott Sternson (Ashburn VA, USA)
The application of optogenetic for dissecting neuronal pathways involved in
feeding behaviors.
Session 12
9:00-10:30
NPY-PYY-PP and the hypothalamic network
Chair by William Colmers
9:15-9:40 O28 William F. Colmers (Edmonton, AB, Canada).
YOU EAT WITH YOUR BRAIN -NPY AND ENERGY BALANCE.
9:40-10:05 O29 Michael Cowley (Melbourne, Australia).
NPY NEURONS IN THE DORSOMEDIAL HYPOTHALAMUS REGULATE
ENERGY BALANCE AND BLOOD PRESSURE.
10:05-10:30 O30 Donald Gehlert (Indianapolis, IN USA).
TRANSCRIPTOME OF THE HYPOTHALAMIC ARCUATE NUCLEUS OF
THE RAT: A COMPARISON OF MICROARRAY AND NEXT GENERATION
RNA SEQUENCING.
10:30-10:45 AM Coffee Break
Coffee and tea
Session 13
10:45-12:30
Therapeutic application of NPY-PYY-PP in obesity and anorexia: CNS implications.
Chair by Akio Inui.
10:45-11:10 O31 Seiji Shioda (Tokyo, Japan).
NEUROPEPTIDES IN THE REGULATION OF FOOD INTAKE AND
ENERGY METABOLISM.
11:10-11:35 O32 Toshihiko Yada (Tochigi, Japan).
FEEDING-RELATED PANCREATIC AND GASTROINTESTINAL
HOROMONES INCULUDING NPY FAMILY PEPTIDES DIRECTLY
REGULATE VAGAL AFFERET NEURONS.
11:35-12:00 O33 Hideo Yukioka (Shionogi, Japan).
S-2367, A NOVEL NPY Y5 RECEPTOR ANTAGONIST BEING
INVESTIGATED FOR THE TREATMENT OF OBESITY.
12:00-12:25 O34 Akihiro Asakawa (Kagoshima, Japan).
PANCREATIC POLYPEPTIDE AND BODY WEIGHT REGULATION
9 12:30-14:15 Lunch, Salon Printemps
Session 14
14:15-15:05
NPY and its receptors in seizure and epilepsy and memory.
Chair by Guenther Sperk.
14:15-14:40 O35 Guenther Sperk (Innsbruck, Austria).
NPY IN EXPERIMENTAL AND HUMAN EPILEPTIC TISSUE.
14:40-15:05 O36 Ramon Tasan (Innsbruck, Austria)
NEUROPEPTIDE Y MODULATES EMOTIONAL MEMORY PROCESSES IN
THE DORSAL HIPPOCAMPUS
15:05-15:35 PM coffee break
Coffee, tea and juice
Session 15
15:35-17:15
NPY-PYY-PP transgenic animals: What have we learned from them.
Chair by Herbert Herzog.
15:35-16:00 O37 Herbert Herzog (Sydney, Australia).
NPY and NPY receptor KO mice: What’s new?
16:00-16:25 O38 Eriika Savontaus (Turku, Finnland).
NEW INSIGHTS TO THE REGULATION OF METABOLISM FROM A
SYMPATHETIC NERVOUS SYSTEM SPECIFIC NPY TRANSGENIC
MOUSE MODEL.
16:25- 16:50 O39 Serge Zolotukhin (Gainesville, USA).
LONG-TERM MODULATION OF INGESTIVE AND SOCIAL BEHAVIOURS
USING VIRAL VECTORS EXPRESSING PYY IN THE SALIVARY GLAND.
16:50-17:15 O40 Paul A Baldock (Sydney, Australia)
NEUROPEPTIDE Y RECEPTOR SIGNALLING IN BONE TISSUE IS
REQUIRED FOR NORMAL GLUCOSE HOMEOSTASIS IN MICE.
17:30 Free for the Jazz festival
10 July 4th 2012
Session 16
9:00-10:15
NPY-PYY-PP and Others
Chair by Danielle Jacques
9:00-9:25 O41 Danielle Jacques (Sherbrooke QC, Canada).
HUMAN ENDOCARDIAL ENTHELIAL CELLS HYPERTROPHY AND
RELEASE OF ENDOTHELIN-1: ROLE OF NEUROPEPTIDE y AND
ANGIOTENSIN II.
9:25-9:50 O42 David PD Woldbye (Copenhagen, Denmark)
VECTOR-INDUCED OVEREXPRESSION OF NPY REDUCES
PARKINSONIAN SYMPTOMS IN MICE
9:50-10:15 O43 Ning Lee (Pennington, NJ, USA)
THE BETA-ARRESTIN-2-MEDIATED PATHWAY IS NOT REQUIRED FOR
PANCREATIC POLYPEPTIDE (PP)-MEDIATED REDUCTION IN FOOD
INTAKE AND BODY WEIGHT
10:15-10:45 AM Coffee Break
Coffee and tea
Session 17
10:45-12:25
PYY activities in the gastrointestinal tract.
Chair by Helen Cox.
10:45-11:10 O44 Helen Cox (London, UK).
PYY MEDIATES GPR119 ACTIVITY IN THE INTESTINE.
11:10-11:35 O45 Thue Schwartz (Copenhagen, Denmark).
TWO-WAY COMMUNICATION BETWEEN ENTEROCYTES AND
ENTEROENDOCRINE CELLS.
11:35-12:00 O46 Christoph Beglinger (Basel, Switzerland).
BILE ACID INDUCED SECRETION OF PYY; PATHOPHYSIOLOGICAL
IMPLICATIONS
12:00-13:30 Lunch, Salon Printemps
11 Session 18
13:30-16:05
Therapeutic application of gastro-intestinal peptides in obesityChair by Michael Stumvoll
13:30-13:55 O47 Timo Müller (Munich, Germany).
GPR83 – A NOVEL REGULATOR OF SYSTEMIC ENERGY METABOLISM
CONTROL
13:55-14:20 O48 Michael Stumvoll (Leipzig, Germany).
DIRECT INFLUENCES OF GASTRO-INTESTINAL PEPTIDES ON BRAIN
NETWORKS AS REVEALED BY NEUROIMAGING IN HUMANS.
14:20-14:45 O49 Thomas A. Lutz (Zurich, Switzerland).
ROLE OF GI HORMONES IN THE TREATMENT SUCCESS OF BARIATRIC
SURGERY
14:45-15:10 O50 Magdy El-Salhy (Stord, Norway).
HIGH DENSITIES OF PEPTIDE YY AND SEROTONIN CELLS IN THE
COLON OF PATIRNTS LYMPHOCYTIC COLITIS
15:10-15:30 Closing remarks
William Colmers and Rémi Quirion
12 Scientific Program -- Poster Session-P01 Ito, M., Dumont Y., Quirion R.
MOOD AND MEMORY-ASSOCIATED BEHAVIORS IN NEUROPEPTIDE Y5
KNOCKOUT MICE.
P02 Morales-Medina JC, Dominguez-Lopez S, Gobbi G, Beck-Sickinger AG and Quirion R
THE SELECTIVE NEUROPEPTIDE Y Y5 AGONIST [CPP1–7,NPY19–23, ALA31,
AIB32, GLN34]HPP DIFFERENTLY MODULATES EMOTIONAL PROCESSES AND
BODY WEIGHT IN THE RAT
P03 Morales-Medina J.C., Dumont Y., Bonaventure P., Quirion R.
CHRONIC ADMINISTRATION OF THE Y2 RECEPTOR ANTAGONIST JNJ-31020028
INDUCED ANTI-DEPRESSANT LIKE-BEHAVIORS IN OLFACTORY
BULBECTOMIZED RAT
P04 Trent, N.L., Menard, J.L.
THE ROLE OF NEUROPEPTIDE Y Y1 AND Y2 RECEPTORS IN THE LATERAL
SEPTAL REGULATION OF ANXIETY IN THREE ANIMAL MODELS
P05 Canneva F., Distler J., Meyer S., Dobner J. and von Hörsten S
NPY ACCUMULATION IN DP4/CD26-DEFICIENT CONGENIC RATS
P06 Jia C., Hayoz S., Hegg CC
NPY MEDIATES INJURY-INDUCED ADULT NEUROREGENERATION
P07 Santos-Carvalho, A.; Álvaro, A. R.; Ambrósio, A. F.; Cavadas, C.
NEUROPROTECTIVE ROLE OF NPY IN RETINAL CELLS
P08 Nima Aghili, Joseph M. Devaney, Mingyao Li, Muredach Reilly, Daniel Rader, Zofia
Zukowska, Mary Susan Burnett, Stephen Epstein
POLYMORPHISMS IN NEUROPEPTIDE Y SIGNALLING GENE ARE ASSOCIATED
WITH THE RISK OF MYOCARDIAL INFARCTION IN PATIENTS WITH
ATHEROSCLEROSIS
P09 Nima Aghili, Roberta Lassances-Soares, Subeena Sood, Lee O Alderman, Amir Najafi,
Hajra Nashin, Sunny S Jhamnani, Lijun Li, Zofia Zukowska, Stephen E Epstein, Mary
Susan Burnett
PRENATAL STRESS SUPPRESSES NEUROPEPTIDE Y LEVELS IN ADULT
OFFSPRING
P10 Kostelnik K., Baumann S , Mörl K., von Bergen M., Beck-Sickinger A.G.
PROTEOME ANALYSIS OF HUMAN Y1-RECEPTOR EXPRESSING BREAST
CANCER CELLS
P11 Ahrens, V.M., Frank, R., Hey-Hawkins, E., Beck-Sickinger, A. G.
CARBABORANE CONTAINING NEUROPEPTIDE Y ANALOGS FOR BREAST
CANCER THERAPY
13 P12 Babilon S., Ahrens V., Mörl K., Beck-Sickinger A.G.
POSTENDOCYTIC FATE OF THE HUMAN Y2-RECEPTOR-LIGAND-SYSTEM
P13 Hofmann, S, Frank, R., Hey-Hawkins, E, Beck-Sickinger, A. G
SWITCHING Y-RECEPTOR SELECTIVITY OF REDUCED-SIZE NPY ANALOGS
P14 Mäde V., Bellmann-Sickert K., Beck-Sickinger A. G.
IMPROVED PANCREATIC POLYPEPTIDE-BASED ANALOGS FOR OBESITY
TREATMENT
P15 Jolly N., Mäde V., Elling C., Little P., Beck-Sickinger A., Schwartz T. & Cox H.
LONG CHAIN LIPIDATION OF Y4 RECEPTOR AGONISTS PROLONGS THEIR
ACTIVITY
P16 Pedragosa Badia X., Stichel J., Beck-Sickinger A. G.
FROM CHIMERIC RECEPTORS TO SINGLE RESIDUES: CHARACTERISATION OF
NEUROPEPTIDE Y RECEPTOR SELECTIVITY
P17 Pruner J., Xu B., Fällmar H., Boukharta L., Keränen H., Gutiérrez-de-Terán H., Lundell I.,
Mohell N., Åqvist J., Larhammar D.
INVESTIGATION OF THE HUMAN Y2 RECEPTOR LIGAND-BINDING POCKET
P18 Xu B., Fällmar H., Boukharta L., Gutiérrez-de-Terán H. , Pruner J., Lundell I., Mohell N.,
Åqvist J., Larhammar D.
MODELING, DOCKING AND MUTAGENESIS OF THE HUMAN NPY/PYY
RECEPTOR Y2 IDENTIFIES RESIDUES INVOLVED IN PEPTIDE BINDING AND
RECEPTOR STABILITY
P19 Alves C.J., Ribas J., Neto E., Ferreira S., Summavielle T., Lamghari M.
ALTERED NPY SYSTEM ACTIVITY THROUGHOUT BONE REGENERATION
P20 Joana Rosmaninho-Salgado, Vera Cortez, Maria Alexandra Gonçalves, Magda Santana,
Patrícia Marques, Marta Estrada, Cláudia Cavadas.
NPY CLEAVED BY DIPEPTIDYL-PEPTIDASE IV (DPPIV) INDUCES PREADIPOCYTES DIFFERENTIATION
P21 Shu Lin, Yanchuan Shi, Lesley Castillo, Jackie Lau, A. Sainsbury and Herbert Herzog
CENTRAL ROLE OF NPY IN THE REGULATION OF SYMPATHETIC TONE AND
ENERGY EXPENDITURE
P22 Yan-Chuan Shi, Shu Lin, Lesley Castillo, Hui Zhang, Jackie Lau, Amanda Sainsbury &
Herbert Herzog
LIPID METABOLISM IN SKELETAL MUSCLE IS CONTROLLED BY ARCUATE
NEUROPEPTIDE Y
P23 Nguyen AD, Mitchell NF, Lin S, Macia L, Yulyaningsih E, Baldock PA, Enriquez RF,
Zhang L, Shi YC, Zolotukhin S, Herzog H and Sainsbury A.
14 Y1 AND Y5 RECEPTORS ARE BOTH CRITICAL FOR THE REGULATION OF FOOD
INTAKE AND ENERGY HOMEOSTASIS IN MICE
P24 Lei Zhang, I-Chieh J Lee, Rondaldo F Enriquez, Shu Lin, Paul Baldock, Errika
Savontaus, Herbert Herzog
ADRENERGIC NEURON-ONLY EXPRESSION OF NPY EXACERBATS STRESSINDUCED FAT GAIN IN CHOW-FED MICE
P25 Vähätalo LH, Ailanen L, Ruohonen ST, Savontaus E.
INVOLVEMENT OF PERIPHERAL Y1 AND Y2 RECEPTORS IN THE METABOLIC
PHENOTYPE OF TRANSGENIC MICE OVEREXPRESSING NPY IN THE
SYMPATHETIC NERVOUS SYSTEM
P26 Ailanen L, Vähätalo L, Ruohonen ST, Gylling H, Miettinen TA and Savontaus E.
HEPATIC FATTY ACID AND CHOLESTEROL METABOLISM IN NPY
OVEREXPRESSING MICE
P27 Sarah Forbes, Herbert Herzog, Helen Cox
ROLE OF ENDOGENOUS PEPTITE YY IN GASTROINTESTINAL AND FEEDING
RESPONSES TO STRESS
P28 Iain R. Tough, Herbert Herzog and Helen M. Cox
DIFFERENTIAL EFFECTS OF GPR119 AGONISTS IN WILD TYPE AND PEPTIDE
YY KNOCKOUT MOUSE AND HUMAN COLON MUCOSAE.
P29 Maria Daniela Hurtado, Alicia Brown, Shawn Dotson, Herbert Herzog, Paola Riveros and
Sergei Zolotukhin
THE NPY SYSTEM: A NOVEL PHYSIOLOGICAL DOMAIN
P30 Maria Daniela Hurtado, Andres Acosta, Michael La Sala, David Duncan, C. Shawn
Dotson, Herbert Herzog, Bruce J. Baum, and Sergei Zolotukhin
SALIVARY PYY: A NOVEL PHYSIOLOGICAL ROLE
P31 Maria Daniela Hurtado, Andres Acosta, Oleg Gorbatyuk, Valeriy G. Sergeyev, Cedrick D.
Dotson, and Sergei Zolotukhin.
SALIVARY PYY ACTIVATES CNS NUCLEI INVOLVED IN FEEDING BEHAVIOR AND
TASTE PERCEPTION
P32 Els S., Schild E., Peterson P.S., Chollet C., Holst B., Schwartz T.W., Beck-Sickinger A.G
A TRIPEPTIDE SWITCH REGION TO INDUCE AGONISM AND INVERSE AGONISM
AT THE GHRELIN RECEPTOR
P33 Tolhurst R., Els S., Beck-Sickinger A., Holst B., Schwartz T., Cox, H.
THE EFFECT OF NOVEL GHRELIN RECEPTOR AGONISTS ON CELL GROWTH
15 P34 Ahmarani L., Perreault C., Bkaily G., Magder S., D’Orléans-Juste P. and Jacques D
NEUROPEPTIDE Y RECEPTORS AND NEUROPEPTIDE Y-INDUCED SECRETION
ARE DIFFERENT IN LEFT COMPARED TO RIGHT VENTRICULAR HUMAN
ENDOCARDIAL ENDOTHELIAL CELLS.
P35 Oscar Lui, Simon Mountford, Lei Zhang, Herbert Herzog, Philip Thompson
EXAMINATION OF BVD15 AS A TEMPLATE FOR NEW Y1 RECEPTOR IMAGING
AGENTS.
P36 Duranton, A., Dumont, Y, Bastianetto, S, Quirion R, Breton, L.,.
EXPRESION OF PEPTIDE YY, PANCREATIC POLYPEPTIDE, Y1 AND Y4
RECEPTORS IN HUMAN SKIN AND RECONSTRUCTED HUMAN EPIDERMIS.
P37 Veyrat-Durebex C., Gaudreau P., Dumont Y., Ferland G., Quirion R.
EFFECT OF AGING AND LONG-TERM CALORIC RESTRICTION ON
NEUROPEPTIDE Y RECEPTOR SUBTYPES
P38 Takenoya F., Kageyama H., Ogawa T., Shioda S.
NPW INDUCED HYPOPHAGIA IS MEDIATED THROUGH STRESS RESPONSE.
P39 Tasan RO., Verma D., Herzog H. & Sperk G.
NEUROPEPTIDE Y MODULATES FEAR, ANXIETY AND DEPRESSION-LIKE
BEHAVIOR IN DISTINCT NUCLEI OF THE AMYGDALA
P40 Jason Tilan, Ewa Izycka-Swieszewska, Joshua Patrick Earnest, Susanna Galli, Asim
Shabir, Congyi Lu, Magdalena Czarnecka, Olga Rodriguez, Chris Albanese, and
Joanna Kitlinska.
NEUROPEPTIDE Y AS A MEDIATOR OF STRESS INDUCED EFFECTS ON
CANCER INITIATION AND PROGRESSION.
P41 Magdalena Czarnecka, Congyi Lu, Zofia Zukowska, Joanna Kitlinska
NEUROPEPTIDE Y RECEPTOR TYPE 5 – INTERACTIONS WITH G PROTEINCOUPLED AND TYROSINE KINASE RECEPTORS AND THEIR FUNCTIONAL
CONSEQUENCES.
P42 Larhammar D. and Bergqvist C.
ANCIENT GRANDEUR OF THE NPY SYSTEM CONFIRMED BY DISTANTLY
RELATED VERTEBRATES.
P43 Brothers, S.P. and Wahlestedt C.
HIGH THROUGHPUT DISCOVERY OF NEUROPEPTIDE Y Y2 RECEPTOR
ANTAGONISTS
P44 Janice H. Urban, Mary R. De Joseph, and Randy J. Leitermann.
IDENTIFICATION OF EXTRINSIC SOURCES OF NEUROPEPTIDE Y INPUT TO THE
RAT BASOLATERAL AMYGDALOID COMPLEX AND REGULATION BY
CONDITIONED CONTEXTUAL FEAR.
16 Plenary speakers abstracts
Michael Meaney
17 Annette G. Beck-Sickinger
NPY-PYY-PP selective ligands and their applications in cancer therapy
Institute of Biochemistry, Leipzig University, Brüderstr. 34, Leipzig
Peptides hormones play an important role in the regulation of manifold activities in the body. In
addition to their physiological function many peptides are involved in the regulation of tumour growths
as tumours frequently express their receptors. Interestingly, as agonist-induced internalization of G
protein-coupled receptors plays an important role in signal regulation, these properties can be easily
used to shuttle agonist-conjugates selectively into tumour cells.
In the past, it has been shown, that neuropeptide Y receptors are overexpressed in several different type
of tumours, including breast cancer (Y1), neuroblastoma (Y2) or colon cancer (Y4). Whereas normal
breast tissue expresses exclusively Y2 receptor subtypes, it has been shown that tumour cells and breast
cell metastasis express Y1 receptors in high numbers 1. Neoplastic changes accordingly correlate with a
change in receptor subtype expression. We recently could show that this system can be used to
selectively label tumors in patients2. By developing Y1 receptor selective peptides3 and subsequent
labeling with radioactive conjugates4 or chemotherapeutics5 this approach suggests a novel tumour
specific targeting of breast cancer6. The underlying mechanisms of the internalization of the human
neuropeptide Y receptors7 as well as their desensitization, endocytosis and resensitization is however
mainly unknown and suggests that the biophysical properties of cells may influence the success of this
approach8 Accordingly, internalization studies have been performed on different cells and tissues in
order to understand the underlying mechanism and to explore of the general relevance of this concept
for Y1, Y2, Y4 and Y5 receptors. Interestingly, different YR show different internalization properties,
which suggests that internalization is a highly regulated process.
Novel concepts on internalization strategies in combination with selective peptide-conjugate to target
tumours will be shown and discussed for Y1, Y2 and Y4 receptor subtypes. 1
Körner M, Reubi JC. NPY receptors in human cancer: a review of current knowledge. Peptides. 2007,
28:419-25.
2
Khan IU, Zwanziger D, Böhme I, Javed M, Naseer H, Hyder SW, Beck-Sickinger AG. Breast-cancer
diagnosis by neuropeptide Y analogues: from synthesis to clinical application. Angew Chem Int Ed
Engl. 2010,49:1155-8.
3
Zwanziger D, Böhme I, Lindner D, Beck-Sickinger AG. First selective agonist of the neuropeptide
Y1-receptor with reduced size. J Pept Sci. 2009, 15:856-66.
4
Zwanziger D, Khan IU, Neundorf I, Sieger S, Lehmann L, Friebe M, Dinkelborg L, Beck-Sickinger
AG. Novel chemically modified analogues of neuropeptide Y for tumor targeting. Bioconjug Chem.
2008,19:1430-8.
5
Ahrens VM, Frank R, Stadlbauer S, Beck-Sickinger AG, Hey-Hawkins E. Incorporation of orthocarbaboranyl-Nε-modified L-lysine into neuropeptide Y receptor Y1- and Y2-selective analogues. J
Med Chem. 2011, 54: 2368-77.
6
Zwanziger D, Beck-Sickinger AG. Radiometal targeted tumor diagnosis and therapy with peptide
hormones. Curr Pharm Des. 2008,14:2385-400.
7
Walther C, Mörl K, Beck-Sickinger AG. Neuropeptide Y receptors: ligand binding and trafficking
suggest novel approaches in drug development. J Pept Sci. 2011, 17:233-46.
8
Walther C, Nagel S, Gimenez LE, Mörl K, Gurevich VV, Beck-Sickinger AG. Ligand-induced
internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxylterminal tail. J Biol Chem. 2010, 285:41578-90.
18 Scott Sternson
The application of optogenetic for dissecting neuronal pathways involved in feeding behaviors.
Hunger is a complex behavioral state that elicits intense food seeking and consumption. These
behaviors are recapitulated in well-fed mice by activation of starvation-sensitive AGRP neurons, but
the relative contribution of different AGRP neuron projection targets to acute feeding behavior is
poorly understood. To investigate the neural circuits underlying hunger, we mapped synaptic
interactions of AGRP neurons with multiple cell types and probed the contribution of these distinct
circuits to feeding behavior using optogenetic and pharmacogenetic techniques. These experiments
establish functional contributions of specific neural circuits that regulate hunger and pathways
associated with overeating disorders.
19 Session 1
O1
VARIOUS NEUROPEPTIDE Y RECEPTOR SUBTYPES ARE IMPLICATED IN THE MODULATORY
EFFECTS OF NPY IN ANXIETY AND MOOD DISORDERS.
Quirion R., Morales J., Ito M., Dumont Y.
Douglas Mental Health University Institute, McGill University, Dept. Psychiatry, Montreal QC, Canada
Pre-clinical and clinical investigations suggest that neuropeptide Y (NPY) plays an important role in various
psychiatric disorders such as depression and anxiety (Morales et al., Brain Res. 1314:194-205, 2010). Indeed, Y1
and Y2 receptor subtypes have been particularly associated with these behaviors in naïve, knockout and
transgenic animals. Recently, the Y4 and Y5 subtypes were also proposed to be involved in the regulation of
mood-related behaviors
Using animal models that display behavioral and neurochemical alterations similar to those observed in anxiety
and depression, we have investigated the effects of chronic administration of [Leu31, Pro34]PYY (Y1 agonist),
BIBO3304 (Y1 antagonist), PYY3-36 (Y2 agonist), BIIE0246 (Y2 antagonist), JNJ31020028 (Y2 antagonist)
and [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]hPP (Y5 agonist) in several behavioral tests including the open
field (OF), elevated plus maze (EPM), social interactions SI), forced swim test (FST) and Morris water maze
(MWM). In the olfactory bulbectomized rat (OBX), [Leu31, Pro34]PYY decreases depression- and anxiety- like
behaviors.. On the other hand, the Y1 antagonist had no effect in these tests in either naive or OBX animals. .
Interestingly, a treatment with the Y2 antagonist reversed depression-related behaviors in OBX rats, but had no
effects on anxiety. In the corticosterone treated rat model which displays mainly anxiety-like behaviors, the Y5
agonist reversed these behaviors. These data suggest that the Y1, Y2 and Y5 receptors differentially modulate
traits of anxiety- and depression-like behaviors in animal models and further demonstrate the potential
therapeutic potential of Y2 antagonists in the treatment of mood disorders.
Supported by CIHR
O2
THERAPEUTIC POTENTIAL OF BRAIN PENETRANT Y2 RECEPTOR ANTAGONIST.
Bonaventure P, Shoblock J, Welty N, Nepomuceno D, Lord B, Aluisio L, Fraser I, Motley T, Sutton S, Dvorak
C, Jablonowski J, Carruthers N, Lovenberg T. Janssen Research & Development, L.L.C. San Diego, CA, USA
Y2 receptors are abundantly expressed in the brain, mainly in the limbic system and, in contrast to other NPY
receptor subtypes, Y2 receptors exist as a pre and post-synaptic receptor. Pre-synaptic Y2 receptors are located
on NPY-ergic neurons and control the release of endogenous NPY. Antagonism at central Y2 receptors should
therefore potentiate endogenous NPY release, which might activate the Y1 receptor and result in enhanced NPYmediated effects, such as anxiolysis. Because of the lack of selective brain penetrant Y2 receptor antagonists,
the number of pharmacological studies supporting this hypothesis is limited. In a program directed towards the
discovery of novel Y2 receptor ligands, we have discovered JNJ-5207787 (N-(1-Acetyl-2,3-dihydro-1H-indol-6yl)-3-(3-cyano-phenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]-acrylamide) and JNJ-31020028 ((N-(4-{4-[2(Diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide)), non-peptidic,
low molecular weight, selective Y2 ligands. The modest affinity of JNJ-5207787 for the Y2 receptor and its
moderate level of receptor occupancy in rat brain after systemic administration have limited the use of this
compound. In contrast, JNJ-31020028 after subcutaneous administration of a 10 mg/kg dose fully occupied Y2
receptor binding sites as revealed by ex vivo receptor autoradiography. The in vivo activity of JNJ-31020028
was confirmed by demonstrating that JNJ-31020028 increased norepinephrine (NE) release in the hypothalamus,
consistent with the colocalization of NE and NPY. In a variety of in vivo anxiety models, JNJ-31020028 was
found to be ineffective, although it did block a stress-induced elevation in plasma on corticosterone without
altering basal levels. Further investigation of stress-related responses showed that JNJ-31020028 had no effects
on basal food intake but normalized food intake in stressed animals. These results suggest that Y2 receptors may
not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under
specific situational conditions.
20 O3
THERAPEUTIC POTENTIAL OF Y4 RECEPTOR ANTAGONISM IN MOOD DISORDERS
Holzer P. (1), Painsipp E. (1), Herzog H. (2), Singewald N. (3), Tasan R. (4), Sperk G (1).
(1) Medical University of Graz, Austria, (2) Garvan Institute of Medical Research, Sydney, Australia, (3)
University of Innsbruck and (4) Medical University of Innsbruck, Austria
Pancreatic polypeptide (PP) and peptide YY (PYY) are almost exclusively expressed in the pancreas and lower
gut, respectively, and their biological effects are predominantly mediated by Y4 and Y2 receptors. Although
considered to be hormones primarily involved in the regulation of digestion, these peptides also influence brain
functions related to energy homeostasis and emotional-affective processes. Consistent with these implications,
peripheral administration of PP and PYY activates distinct neurons in brainstem, hypothalamic, limbic and
corticolimbic areas.
Gene knockout experiments show that Y4 receptors play a role in the regulation of exploration, anxiety and
stress-coping behaviour. While locomotion in a familiar environment is unchanged, locomotion in a novel
environment is enhanced in Y4 knockout mice. In addition, the circadian pattern of exploration, locomotion,
ingestion and rectal temperature is altered by Y4 gene deletion. The anxiety-related behaviour of Y4 knockout
mice is blunted in a number of behavioural tests, and their depression-like behaviour is reduced as shown by
improved stress-coping in the forced swim and tail suspension tests. Learning and memory in the novel object
recognition test remain unaltered by Y4 gene deletion.
The anxiolytic-like phenotype of Y4 knockout mice is in keeping with the anxiogenic phenotype of PPoverexpressing mice. It remains to be explored whether Y4 receptor antagonism replicates the findings of Y4
gene deletion and whether Y4 receptor antagonists may have a therapeutic potential in anxiety and mood
disorders.
O4
THE NEUROPEPTIDE Y Y5 RECEPTOR ANTAGONIST LU AA33810 EXERTS ANXIOLYTIC AND
ANTIDEPRESSANT-LIKE EFFECTS IN VARIOUS RODENT MODELS OF MOOD DISORDERS.
Huang X.1, Lee AW1, Walker MW1, Wolinsky TD1, Jubian V1, Chandrasena G1, Zhong H1, Miller S1, Hegde LG1,
Marsteller DA1, Marzabadi MR1, Papp M2, Overstreet DH3, Gerald CPG1 and Craig DA1
1
Lundbeck Research USA, Paramus, New Jersey; 2Institute of Pharmacology, Polish Academy of Sciences, Krakow,
Poland; 3University of North Carolina, Chapel Hill, North Carolina
Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y1, Y2, Y4, Y5, and y6). Based on its
expression in the limbic system, we proposed Y5 receptors in the CNS might be novel targets for the pharmacological
treatment of mood-related disorders. We identified Lu AA33810 [N-[[trans-4-[(4,5-dihydro[1]-benzothiepino[5,4-d]thiazol2-yl)amino]cyclohexyl]methyl]-methanesulfonamide] as a Y5 receptor-selective antagonist with high affinity to cloned rat
Y5 receptors (Ki = 1.5 nM) and able to antagonize NPY-evoked cAMP and calcium mobilization in vitro. Lu AA33810 (330 mg/kg p.o.) blocked feeding elicited by intracerebroventricular injection of the Y5 receptor-selective agonist [cPP17
,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide (cPP) in Sprague-Dawley rats. In vivo effects of Lu AA33810 were
correlated with brain exposure above 50 ng/g and ex vivo Y5 receptor occupancy of 22 to 95%. Lu AA33810 was
subsequently evaluated in various in vivo rodent models. In Fischer 344 rats, Lu AA33810 (30 mg/kg p.o.) attenuated
increases in plasma ACTH and corticosterone elicited by intracerebroventricular injection of cPP. In Sprague-Dawley rats
subjected to the social interaction test, Lu AA33810 (3-30 mg/kg p.o.) produced anxiolytic-like effects after acute or
chronic treatment. In Flinders sensitive line rats, chronic dosing of Lu AA33810 (10 mg/kg/day i.p.) produced anxiolyticlike effects in the social interaction test, plus antidepressant-like effects in the forced swim test. In Wistar rats exposed to
chronic mild stress, chronic dosing of Lu AA33810 (3 and 10 mg/kg/day i.p.) produced antidepressant-like activity, i.e.,
normalization of stress-induced decrease in sucrose consumption. To determine which populations of NPY Y5 receptors
may contribute to the observed behavioral effects, we evaluated whether activation of specific populations of these receptors
would impact the activity state of limbic-associated circuits using c-fos as a surrogate measure. Intracranial injection of cPP
into the medial prefrontal cortex induced c-fos expression throughout the basolateral amygdala as well as the ventral
posterior striatum. Furthermore, when cPP was infused into the striatum, induction of c-fos was observed in the nucleus
accumbens and anterior cingulate cortex. In each case where measured, the induction of c-fos was prevented by Lu
AA33810. In summary, we report that a selective Y5 antagonist, Lu AA33810 produces anxiolytic- and antidepressant-like
effects in various rodent models of mood disorders and add support to the hypothesis that NPY Y5 receptors may regulate
the activity state of limbic-associated structures important for manifesting these behaviors.
21 O5
NEUROPEPTIDE Y AND POSTTRAUMATIC STRESS DISORDER (PTSD): A BENCH AND BEDSIDE
VIEW
Renu Sah, Department of Psychiatry & Behavioral Neuroscience, University of Cincinnati &
VA Medical Center, Cincinnati, Ohio
PTSD is a chronic anxiety disorder that can occur in individuals who experience a traumatic event. Resiliency to
the adverse effects of extraordinary emotional trauma on the brain varies within the human population. Evidence
from preclinical and human studies demonstrate a role for NPY in stress coping, conferring that it may be a
potential “resilience-to-stress” factor. We hypothesized that central NPY is dysregulated in PTSD, where stress
resiliency may be important. Significant reduction in cerebrospinal fluid NPY concentrations were reported by
us in combat veterans with PTSD (Sah et al 2009). To investigate the mechanistic association of NPY with
PTSD, translational studies were conducted in a rodent model of chronic variable stress (CVS) with PTSD-like
outcomes, developed by us (McGuire et.al, 2010). A significant and enduring reduction in NPY selective to the
amygdala was observed in CVS animals (McGuire et al. 2011). Interestingly, NPY in the prefrontal cortex was
significantly upregulated by CVS indicative of regional heterogeneity in NPY regulation by chronic trauma.
Recently, we observed significant impairment of extinction recall following NPY administration in the
infralimbic cortex, indicative of fear memory modulation by PFC NPY. Collectively our data suggest that
maladaptive functioning of the NPY system may be associated with PTSD relevant outcomes such as fear
memory, arousal and neuroendocrine abnormalities. Relevance of NPY receptor subtypes in the CVS model is
being studied. The presentation will collate clinical and preclinical findings to discuss the potential link between
PTSD and NPY. In pursuit of understanding the physiological basis and treatment of PTSD, the NPY system is
an attractive target.
Funded by VA Merit Grant (Sah).
Session 2
O6
EVOLUTION OF THE NPY SYSTEM AND GENETIC POLYMORPHISM OF THE HUMAN Y4 GENE
AND ITS ASSOCIATION WITH OBESITY.
Larhammar D., Lapshyna K., Bergqvist C., Xu, B., Lundell I., Larsson T. A., Sundström G. Department of
Neuroscience, Uppsala University, Uppsala, Sweden.
The NPY system displayed extensive complexity already at the origin of the vertebrates with seven receptor
subtypes and the two peptides NPY and PYY. The three receptor subtypes Y1, Y2 and Y5 existed before the
vertebrate diversification. We have found representatives for all three subtypes in the sea lamprey. The two
vertebrate genome doublings copied Y1 to give rise to Y4, Y6 and Y8. The Y2 gene was duplicated to Y7,
whereas Y5 remains alone. This impressive vertebrate beginning has been confirmed by our studies in a shark
and in the famous coelacanth Latimeria chalumnae, both of which still retain all seven ancestral vertebrate
receptors. Mammals, in contrast, have lost 2-3 receptors. The teleost fish ancestor doubled its genome one more
time, but only one receptor duplicate has survived. Some fish species have duplicates of NPY or PYY or both.
Our recent studies show that the ‘cousins’ of the NPY receptors, the receptors for NPFF, PRLH and QRFP, also
displayed great complexity in the vertebrate ancestor, providing a useful resource for receptor modeling.
We have discovered that the pancreatic polypeptide receptor Y4 (gene called PPYR1) displays copy number
variation (CNV) in humans, ranging from 3 to as many as 8 copies. Interestingly, the Y4 copy number correlates
with body mass index, implying a role in appetite regulation. Furthermore, SNPs affect the signal transduction
efficiency. We are presently characterizing the Y4 gene duplicates and determining the organization of
noncoding exons and promoter regions. If more precise genotyping can be developed, this will facilitate
correlation studies of Y4 CNV with obesity, type 2 diabetes and anorexia.
Supported by: The Swedish Research Council.
22 O7
ROLE OF NPY ON AUTOPHAGY IN HYPOTHALAMUS: A CALORIC RESTRICTION MIMETIC?
Mariana Botelho, Célia Aveleira, Lígia Sousa-Ferreira, Jorge Pascoal, Clévio Nóbrega, Ana Rita Álvaro, Luís
Pereira de Almeida, Cláudia Cavadas, CNC - Center for Neuroscience and Cell Biology, Univ. Coimbra; Dep.
Biology and Envir, Univ. of Trás-os-Montes & Alto Douro, Vila Real, Portugal; Fac. Pharmacy, Univ. Coimbra,
Portugal.
Caloric restriction (CR) is a robust anti-aging intervention known to extend lifespan in many different
organisms. Increase evidence shows that autophagy is an essential mechanism on the anti-aging effect of CR. In
addition, one the major neuroendocrine effect of CR is the increase of NPY in the hypothalamic arcuate nucleus.
The aim of this study was to investigate the role of NPY and CR on autophagy regulation in the hypothalamus.
The murine hypothalamic cell line N42 and rat primary hypothalamic neural cultures were used as cell models.
We also evaluated the “in vivo” role of NPY on the regulation of autophagy, by overexpressing NPY in the
arcuate nucleus of hypothalamus of C56BL/6 normal mice, using adeno-associated viral (AAV) gene transfer
technology. Autophagy activation was evaluated by measuring the conversion LC-3BI to LC-3BII and p62,
autophagy biomarkers, by Western blotting and immunofluorescence. NPY (100 nM) or nutrient deprivation
induced an increase of the autophagic flux in N42 cells and rat hypothalamic neural cells. NPY Y1, Y2 and Y5
receptor antagonists inhibited these effects. Mice overexpressing NPY in the hypothalamus showed an increase
of LC-3B ratio and a decrease of p62 in hypothalamus. Overall, these results show that autophagy activation
under nutrient deprivation is, at least in part, mediated by NPY, in hypothalamic neurons. These observations
support the hypothesis that NPY may act as a CR mimetic to extend lifespan.
Supported by: FCT (PTDC/SAU-FCF/099082/2008, SFRH/BPD/73942/2010, SFRH/BD/73004/2010,
SFRH/BPD/78424/2011) and Feder.
Session 3
O8
NPY MODULATION OF NEUROGENESIS: MECHANISMS AND INTERACTIONS
Angela Cheung, Malik Zaben, Philip L. Newland, and William P. Gray.
Division of Clinical Neurosciences University of Southampton and National Institute for Neuroscience and
Mental Health Research, Cardiff University, Cardiff, UK
Neuropeptide Y (NPY) is widely expressed in the central and peripheral nervous systems and is proliferative for
a range of cells types in vitro. NPY plays a key role in regulating adult hippocampal neurogenesis in vivo under
both basal and pathological conditions, although the underlying mechanisms are largely unknown.
We have investigated the mechanisms of the neurogenic effects of NPY and its interactions with other peptide
neurotransmitters. Using postnatal rat hippocampal cultures, we show that the proliferative effect of NPY on
nestin+ precursor cells is NO-dependent. As well as the involvement of neuronal nitric oxide synthase, the
proliferative effect is mediated via a NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein
kinase (PKG) and extracellular signal-regulated kinase (ERK) 1/2 signalling pathway. We show that NPYmediated intracellular NO signalling results in an increase in neuroproliferation. By contrast, extracellular NO
had an opposite, inhibitory effect, on proliferation. The importance of the NO-cGMP-PKG signalling pathway in
ERK 1/2 activation was confirmed using western blotting. We also show that the proliferative effect of NPY is
abolished by VIP but with significant and specific enhancement of neurogenesis.
This work unites two significant modulators of hippocampal neurogenesis within a common signalling
framework and provides a mechanism for the independent extra- and intracellular regulation of postnatal neural
precursors by NO. It also identifies an additional layer of complex control, mediated via its interactions with
other peptie neurotransmitters.
Supported by the Kerkut Trust and MRC.
23 O9
MULTIFACETED NPY: NEUROGENESIS, NEUROINFLAMMATION AND NEUROPROTECTION
João O. Malva*, Raquel Ferreira, Jorge Valero, Sara Xapelli, Fabienne Agasse, and Ana Paula Silva*
Center for Neuroscience and Cell Biology and *Faculty of Medicine, University of Coimbra, 3004-504 Coimbra,
Portugal.
Neuropeptide Y (NPY) has been implicated in the modulation of important features of neuronal physiology in
the hippocampal formation, including calcium homeostasis, neurotransmitter release and excitability.
Consistently, NPY received a particular attention in face of its potential antiepileptic properties. NPY not only
inhibits excessive glutamate release (decreasing circuitry hyperexcitability) but also protects neurons from
excitotoxic cell death. Furthermore, NPY has been involved in the modulation of the dynamic of dentate and
subventricular zone neural stem cell niche. In both regions, NPY is a member of the chemical resource of the
neurogenic niche and acts through NPY Y1 receptors to promote neuronal differentiation.
Recently, we revealed that NPY can play a key role in mediating the innate neuroimmune response by
modulating microglial cell activity. Accordingly, using a microglial cell line, we unraveled a new NPY Y1
receptor-mediated anti-inflammatory action. Consistently, NPY inhibits lipopolysaccharide (LPS) or interleukin1 beta -induced NFkB nuclear translocation, inducible nitric oxide synthase expression and nitric oxide
production. Furthermore, we also observed that the activation of Y1 receptors inhibits LPS- or interleukin-1
beta-induced motility and cytoskeletal reorganization. The inhibition of microglial activation by NPY (via Y1
receptor activation) also resulted in inhibition of the phagocytic efficiency.
Taken together, these observations highlight a central role for NPY as a key modulator of the neuron-microglial
crosstalk, with critical importance in the maintenance and regulation of the neurogenic niche .
Supported by FCT-Portugal.
O10
NEUROPROTECTIVE POTENTIAL OF NEUROPEPTIDE Y AND NPY 13-36 IN ISCHEMIC MODELS
Smialowska M.1, Jantas D.2, Kozniewska E.3, Przykaza L.3, Boguszewski P.4, Domin H.1
1 Department of Neurobiology and 2 Department of Experimental Neuroendocrinology, Institute of
Pharmacology, PAS, Krakow, Poland; 3 Department of Neurosurgery, Mossakowski Medical Research Centre
PAS, Warsaw, Poland
4 Laboratory of Limbic System, Nencki Institute of Experimental Biology PAS, Warsaw, Poland
Some studies indicate that NPY and Y receptor agonists may be neuroprotective against excitotoxic damage but
much less is known about their effect in ischemic neurodegeneration. In the present study we investigated the
protective potential of NPY in an ischemic in vitro model (oxygen-glucose deprivation; OGD). Since our
previous study showed that Y2 receptor agonist, NPY13-36, significantly diminished the brain damage after
middle cerebral artery occlusion (MCAO) in rats, in the present study we sought whether such protection may
also have functional significance for the improvement of motor functions.
In cortical cultures NPY was applied twice: immediately before the start and after the end, or once - 30 min or 1h
after the end of OGD. We found that both double and single application of NPY (0.5 or 1µM) significantly
decreased ischemic neurodegeneration by 16-26%.
In rats, transient focal cerebral ischemia was induced for 90 min by the MCAO. NPY 13-36 (10 µg/6 µl) or
vehicle (6 µl) was administered to the right lateral ventricle, 30 min after the start of occlusion. 72 h after
reperfusion, the behavioural tests and evaluation of brain damage were carried out. It was found that NPY 13-36
diminished the infarction volume by 58% in comparison to MCAO vehicle-treated rats. In conclusion, our
results indicate that NPY and NPY 13-36 may be not only neuroprotective against ischemic neuronal damage but
may also diminish postischemic functional deficits.
Supported by grant no N N401 091037, MS&HE, Poland.
24 Session 4
O11
INTERACTIONS OF NEUROPEPTIDE Y, CATECHOLAMINES AND ANGIOTENSIN AT THE
VASCULAR NEUROEFFECTOR JUNCTION
Thomas C. Westfall, Heather Macarthur, Mirnela Byku and Jessica Murray,
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St.
Louis, MO 63104
The sympathetic nervous system and renin-angiotensin system are both thought to contribute to the development
and maintenance of hypertension in experimental models such as the Spontaneously Hypertensive Rat (SHR).
We demonstrated that periarterial nerve stimulation (NS) increased the perfusion pressure (PP), NE overflow
and neuropeptide Y (NPY) overflow from the perfused mesenteric arterial beds of SHR and WKY at 4-6, 10-12
and 18-20 wk of age. The effects were greater in vessels obtained from SHR of all 3 ages compared with WKY.
Angiotensin II (Ang II) produces an enhancement of the NS-induced increase in the release of NPY and NE and
increase in PP in the mesenteric bed obtained from SHR and the effect was greater in beds obtained from SHR
compared to age matched controls. In contrast Ang (1-7) decreased NS-induced release of NE and NPY as well
as the increase in PP. This effect was greater in arteries obtained from SHR than WKY. A MAS receptor
antagonist attenuated the Ang (1-7) induced decrease in both NE and NPY release while an AT2 receptor
antagonist only affected the Ang (1-7) induced decrease in NE without affecting the decrease in NPY overflow.
Both an ACE inhibitor and an AT1 receptor blocker decreased the NS-stimulation induced overflow of NE and
NPY. Plasma Ang II levels are greater in the 10-12 wk SHR than WKY while plasma Ang (1-7) levels are lower
in the SHR than WKY. Taken together the opposing effects of Ang (1-7) and Ang II suggest that there is a
normal balance between these two hormones in modulating sympathetic neurotransmission and an imbalance
may contribute to the development and maintenance of hypertension.
Supported by: HL60260 GM and the American Heart Association
O12
ROLE OF PROSTANOIDS IN THE MODULATION BY ANGIOTENSIN-(1-7) OF NEUROPEPTIDE Y
AND NOREPINEPHRINE RELEASE AT SYMPATHETIC NEUROEFFECTOR JUNCIONS.
Murray J., Macarthur H., Westfall TC.
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St.
Louis, MO 63104.
Sympathetic neurotransmission is modulated by several mediators including nitric oxide, serotonin, and
prostaglandins, just to name a few. Prostacyclin (PGI2), Prostaglandin E2 (PGE2) and their analogues are
known to attenuate the evoked release of the sympathetic cotransmitters Norepinephrine (NE) and NPY from the
mesenteric arterial bed of the rat. Similar results were obtained from studies with the counterregulatory product
of the Renin-Angiotensin System, Angiotensin-(1-7) (Ang-(1-7)). It is not clear if Ang-(1-7) inhibits the evoked
release of NE and NPY from sympathetic neurons via direct activation of receptors on nerve terminals or
indirectly by promoting the production and release of another mediator, which in turn mediates the inhibitory
actions of Ang-(1-7). We examined the hypothesis that the Ang-(1-7) induced inhibition of sympathetic
neurotransmission is mediated by PGI2, a known inhibitor of sympathetic neurotransmission and mediator of the
vasodilator action of Ang-(1-7). Mesenteric arterial beds obtained from Wistar-Kyoto and Spontaneously
Hypertensive Rats were used as models of a sympathetic neuroeffector junction. The effect of Ang-(1-7) on the
evoked release of NPY and NE was determined before and after the administration of the COX inhibitor
Indomethacin and the PGI2 receptor blocker CAY10441 to inhibit PGI2 synthesis and binding, respectively.
Indomethacin reverses the decrease in perfusion pressure and the attenuation of the evoked release of NPY and
NE by Ang-(1-7), while CAY10441 has a partial effect. These data support the notion that modulation of
sympathetic neurotransmission by Ang-(1-7) is at least partially mediated by PGI2.
25 O13
DEVELOPMENT OF NEUROPEPTIDE Y-CONTAINING NEURONS IN THE AUTONOMIC GANGLIA
Masliukov P.M., Emanuilov A.I., Konovalov V.V. Yaroslavl State Medical Academy, Yaroslavl, Russia,
150000
Neuropeptide Y (NPY) has been associated with numerous physiological processes, including feeding, memory,
circadian rhythms, and regulation of blood pressure. A large number of adult mammalian autonomic neurons
express NPY. However, there are only few works concerning the development of NPY-containing neurons in
sympathetic and intramural ganglia.
In the current study, immunohistochemistry was used to label cells expressing NPY in the sympathetic superior
cervical ganglion, stellate ganglion, celiac ganglion and intramural ganglia of the heart and small intestine from
rats and cats of different ages, from newborn until 180-day-old. NPY-immunopositive neurons were found in all
studied ganglia from the moment of birth. We observed that the percentage of NPY-immunoreactive neurons
increased during the early postnatal development in sympathetic ganglia from the moment of birth until 20-30
days of animals’ life. In intramural cardiac and enteric ganglia, the number of NPY-positive neurons enlarged
during first 10 days in rats and 20 days in cats. In sympathetic ganglia, NPY-immunoreactive neurons were
strongly TH-positive and NO-negative in all age groups. NPY was partially colocalized with calbindin in
sympathetic and intramural ganglia from the moment of birth in both species.
Thus, the development of autonomic neurons is accompanied with the increasing of the proportion of neurons
containing NPY. Maturation of NPY-immunopositive neurons becomes complete in the autonomic ganglia of
rats and cats by their first month of life.
This work was supported by RFBR, grant of President of Russian Federation for young scientists and Russian
Federal Special-Purpose Program “Scientific and scientific-pedagogical personnel of innovative Russia” for
2009-2013 years.
Session 5
O14
NPY SYSTEMS IN EXTENDED AMYGDALA ARE RECRUITED DURING THE TRANSITION TO
ALCOHOL DEPENDENCE.
Gilpin, N.W. Department of Physiology, Louisiana State University Health Sciences Center, 1901 Perdido
Street, New Orleans, LA 70112
Neuropeptide Y (NPY) appears to be recruited during the transition to alcohol dependence in rats. NPY typically
does not affect alcohol drinking by genetically heterogeneous non-dependent rats, but reduces alcohol drinking
by rats exposed to chronic intermittent alcohol vapor (i.e., alcohol-dependent rats) or rats selectively bred for
high alcohol preference. Various recent lines of research point to the extended amygdala, in particular the central
amygdala (CeA) and bed nucleus of the stria terminalis (BNST), in mediating NPY modulation of alcoholrelated behaviors and alcohol effects on synaptic transmission. For example, chronic NPY treatment abolishes
the development of excessive alcohol drinking characteristic of the transition to alcohol dependence in alcohol
vapor-exposed rats, and the ability of NPY to suppress alcohol drinking by alcohol-dependent rats has been
localized to the CeA. Recent electrophysiological data show that NPY suppresses GABA release in both the
CeA and BNST via actions at pre-synaptic Y2 receptors (Y2Rs), and opposes alcohol effects on GABAergic
transmission via the same mechanism, suggesting a dual role for Y2Rs as heteroceptor and autoreceptor. Recent
behavioral data from our group confirm that NPY may affect anxiety-like and alcohol-drinking behaviors via
Y2Rs in a complex interplay between pre-synaptic release of NPY and GABA. Data will also be presented
regarding the recruitment of NPY systems in other regions of the extended amygdala (e.g., BNST) during the
transition to alcohol dependence. These data will be discussed in the context of within-systems and betweensystems neuroadaptations characteristic of the transition to alcohol dependence.
26 O15
NPY AGONISTS PROTECT AGAINST BINGE-LIKE ETHANOL DRINKING IN NON-DEPENDENT
MICE: EVIDENCE FOR A ROLE OF THE CENTRAL AMYGDALA
Thiele, T.E. Dept. of Psychology & the Bowles Center for Alcohol Studies, U. of North Carolina, Chapel Hill,
NC 27599-3270.
Binge drinking, defined as excessive ethanol intake over a short period of time that generates elevated blood
ethanol levels (80 mg/dL or greater), has become a serious health problem world-wide. Binge drinking has been
linked to heart disease, high blood pressure, and type 2 diabetes. Perhaps most alarming, longer-term binge
drinking has been implicated in the subsequent development of ethanol dependence. Over the last decade,
evidence has emerged suggesting that neuropeptide Y (NPY) receptor agonists protect against excessive
dependence-like ethanol drinking in rodents exposed to ethanol vapor without alter ethanol intake in nondependent animals. In the present set of experiments, we provide parallel evidence showing that NPY protects
against binge-like ethanol drinking but does not reduce non-binge-like ethanol intake. Follow-up studies showed
that a selective NPY type-1 receptor (Y1R) agonist and type-2 receptor (Y2R) antagonist blunt binge-like
ethanol drinking, implicating the Y1R and Y2R. More recently, we have found that binge-like ethanol
consumption caused a significant decrease of NPY and Y1R IR in the central nucleus of the amygdala (CeA),
effects that became more pronounced following repeated cycles of binge-like drinking. Interestingly, after 24
hours of abstinence following the last binge-like drinking session, NPY IR in the CeA continued to be blunted
relative to water drinking controls, but Y1R and Y2R IR were significantly elevated. Finally, we recently found
the centrally administered NPY failed to attenuate binge-like ethanol drinking in mice given CeA injection of
NPY conjugated to the neurotoxin saporin (NPY-SAP), indicating that intact NPY signaling in the CeA is
necessary for the protective effect of NPY on binge-like drinking. The present results suggest that binge-like
ethanol drinking in non-dependent mice triggers alterations within central NPY pathways analogous to what
occurs in ethanol-dependent rodents. Thus, in addition to treating excessive ethanol intake stemming from
dependence, Y1R agonists, and Y2R antagonists are potential attractive therapeutic targets for treating problem
binge drinking in the human population. (Supported by NIH grants AA017803, AA013573, AA015148, and
AA017818).
Session 7
O16
NPY AND ITS RECEPTORS AS NEW TARGETS IN NEUROBLASTOMA THERAPY
Congyi Lu, Magdalena Czarnecka, Anna Kuan-Celarier, Jason Tilan, Ewa Izycka-Swieszewska, Joshua Patrick
Earnest, Susanna Galli, and Joanna Kitlinska. Georgetown University, Washington DC
Neuroblastoma is a pediatric tumor developing from the precursors of sympathetic neurons. Due to this,
neuroblastoma cells synthesize and release high levels of NPY, as well as express its Y2 and Y5 receptors. Early
clinical studies indicated that high systemic levels of NPY correlate with poor prognosis in neuroblastoma
patients. In agreement with this, we have shown that NPY and its receptors create a functional autocrine loop in
neuroblastoma cells, essential for maintaining their proliferative potential. This proliferative effect is mediated
mainly via Y2 receptors, constitutively expressed in neuroblastoma. Strikingly, the same Y2 receptors mediate
the angiogenic effect exerted by endogenous NPY in neuroblastoma tissues. Consequently, blocking Y2
receptors significantly inhibits growth of neuroblastoma tumors in vivo, which is associated with markedly
reduced tumor cell proliferation, decreased vascularization and fibrosis of the tumor tissue. In contrast to
constitutively expressed Y2 receptors, expression of Y5 receptors in neuroblastoma cells is induced by their
known survival factor – brain-derived neurotrophic factor (BDNF). We have shown that such up-regulation of
Y5 receptors augments the pro-survival effect of BDNF in chemotherapy-treated neuroblastoma cells. This
effect seems to be mediated by cross-talk between Y5 and TrkB receptors, since blocking Y5Rs inhibits BDNFinduced TrkB phosphorylation and p44/42-MAPK activation. In line with this, expression of Y5Rs in human
neuroblastoma tissue correlates with expression of both BDNF and its receptor, TrkB, while Y5R protein is
detected preferentially in the aggressive, undifferentiated neuroblastoma cells. Moreover, expression of Y5Rs
and NPY is elevated in cell lines derived from neuroblastoma patients at relapse, as compared to those at
diagnosis. In summary, our findings demonstrate an essential role of NPY system in neuroblastoma growth and
progression and identify the peptide and its receptors as potential targets in therapy of the aggressive form of
these pediatric tumors. 27 O17
NPY AND ITS RECEPTORS, A NOVEL THERAPEUTIC TARGET FOR BREAST CANCER
Sulaiman Sheriff., T.A. Rizvi., Shagufta Khan., Miki Watanabe., Michael A Kennedy., Hassane Amlal.,
Balasuramaniam A. Department of Surgery, Hematology, Pathology, Internal Medicine, University of Cincinnati
Medical Center, OH, USA. Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA
We investigated the role of NPY Y5R in human breast carcinoma. Towards this goal, we determined the
expression profile of Y5R protein in human breast carcinoma and the metabolites involved in Y5R signaling in
BT-549 cell line using 1D 1H-NMR spectra. We analyzed Y5R protein expression in a total of 40 female breast
carcinoma patients with ages ranging from 37-66 years. Immunohistochemistry was performed on tumor tissue
sections using Y5R antibody. The Y5R labeling in breast tumor and non-tumor samples was localized to cellular
membrane as well as to cytoplasm. The intensity of Y5R staining is categorized to low (1+), moderate (2+), and
high (3+) groups. Among the 40 samples tested, 31 tumor parenchyma tissue sections show high intensity
staining (3+), 8 tumor parenchyma with moderate staining (2+), and 1 tissue section with no tumor parenchyma.
Normal breast parenchyma is observed in 11 tissue sections, of which 4 sections show low staining (1+) and 7
sections with moderate (2+) staining. Low intensity staining (1+) in stoma tissue is observed in 37 samples.
Low intense staining (1+) is also observed in endothelial and inflammatory cells in 11 tissue samples. No
staining is observed in myoepithelial and fat cells. In summary, Y5R staining is strong and uniform in tumor
areas. When both benign and malignant breast tissues are present in the same sample, the tumor areas stain with
higher intensity than benign areas in >90% of the samples studied. In BT-549 cell line, NPY decreased
intracellular lactate, succinate, myo-inositol, creatinine and increased acetate, glutamate and aspartate. NPY
treatment also decreased lactate and increased pyruvate levels in the medium. These results suggest that there is
upregulation of Y5R protein in human breast carcinoma and Y5R activation leads to increased utilization of
lactate and decreased consumption of pyruvate in breast cancer cells.
O18
RADIOLABELLED NPY PEPTIDES TO IMPROVE CANCER DIAGNOSIS BY POSITRON EMISSION
TOMOGRAPHY
Dumulon-Perreault V.,1 Ait Mohand, S., 1 Benard F., 2; Guérin, B. 1
1.Université de Sherbrooke, Département de médicine nucléaire et radiobiologie, Sherbrooke, Québec, Canada.
2. BC Cancer Research Centre, Vancouver, BC, Canada.
Neuropeptide Y1 receptors (NPY1R) have been shown to be significantly overexpressed on a large proportion of
breast cancer making these receptors prime candidates for receptor targeting with radiopharmaceutical for highly
specific tumor imaging by PET. We are currently developing radiolabelled peptides targeting NPY1R as tools to
improve cancer diagnostic by PET.
We will present a convenient approach to functionalize peptides targeting these receptors with NOTA, a chelator
for 64Cu- and 68Ga-labelling. Structural optimization of NOTA-peptides will be discussed by presenting results
of binding assays done on human breast cancer cells (MCF-7) overexpressing NPY1R. Biological evaluations of
the potent radiolabelled peptides using stability studies, biodistribution and PET imaging in human breast
cancer nude mouse xenografts will be also presented.
28 Session 8
O19
DESIGN OF NOVEL PYY ANALOGUES FOR THE TREATMENT OF OBESITY
Jensen, K. J. University of Copenhagen, Faculty of Science, Denmark
Here we report (1) substitution of the whole N-terminal segment of PYY3-36 with a non-native amphipathic αhelix to create novel two-helix analogs of PYY3-36 with potential for membrane-binding, and (2)
pharmacological and biophysical comparison of PYY3-36 itself with the new two-helix variants. These studies
including unprecedented measurement of the liposome binding of PYY3-36 and its two-helix analogs, using the
new and powerful biophysical method, single liposome curvature (SliC) assay. We believe that the findings
reported here are of significant interest for peptide medicinal chemistry.
A number of different amphipathic heptad repeat sequences were incorporated. The design resulted in a helixloop-helix structure, where the N-terminal helix originates from a heptad repeat sequence and the loop as well as
the C-terminal helix originates from the native PYY sequence. Affinity as well as functionality studies of the
two-helix analogs revealed that heptad repeat sequences with a net positive change were preferred. One of the
most promising analogues was further investigated by solution NMR techniques in the presence of phospholipid
micelles. In solution the peptide exits in an oligomeric state preventing its detailed structural analysis. In contrast
in presence of dodecylphosphocholine (DPC) micelles that mimic biological membranes, the peptide is
characterized by two well-defined α-helices. The single liposome curvature (SliC) assay revealed for the first
time that native PYY3-36 preferentially binds to liposomes with high curvature. In addition, one of the novel
two-helix PYY3-36 analogs bound with even higher preference to liposome. The improved biological activity
provides further supports for the hypothesis that some peptide hormones bind to the membrane prior to
activating the receptor.
O20
THE Y2 RECEPTOR IN INTERACTION WITH NPY – MOVING TOWARDS STRUCTURAL STUDIES BY
NMR
Schmidt P.,1 Witte K.,1 Thomas L.,1 Scheidt H.A.,1 Theisgen S., 1 Nietlispach D.,3 Beck-Sickinger A.G.,2 Huster
D.1
1 Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany
2 Institute of Biochemistry, University of Leipzig, Brüderstr. 34, 04103 Leipzig, Germany
3 Departement of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK
Although highly resolved crystal structures of G protein-coupled receptors have become available within the last
decade, the need for studying these molecules in interaction with their ligands in their natural environment,
where the molecules are rather dynamic, has been widely appreciated. NMR spectroscopy is an excellent method
to study structure and dynamics of membrane proteins in their native lipid environment.
Using solution as well as solid-state NMR measurements, we obtained spectra of uniformly 15N labelled Y2
receptor reconstituted into small and respectively large bicelle-like lipid structures where the dispersion of the
15N signals are comparable to native rhodopsin. To provide the required milligram quantities of labelled target
protein the Y2 receptor was expressed in Escherichia coli using high density fed-batch fermentation. The
structural integrity of the receptor molecules was achieved by in vitro refolding, determined using radioligand
binding assays and ligand affinity chromatography.
The receptor preparations will be used to investigate the structure of NPY in its bound state and the dynamics
upon ligand binding by using selectively 13C, 15N labelled ligand.
29 O21
COMPUTATIONAL DOCKING OF POTENTIAL THERAPEUTICS IN THE Y4-PANCREATIC
POLYPEPTIDE COMPARATIVE MODEL
Sliwoski, G., Dong, E.N., Meiler, J. Center for Structural Biology, Vanderbilt University, Nashville, TN, U.S.A.
Human Y4 receptor, a class A G-protein coupled receptor (GPCR), primarily mediates the action of pancreatic
polypeptide (PP) released from F-cells of pancreatic islets. Its role in regulating food intake and energy balance
suggests that increasing the activity of pancreatic polypeptide at the Y4 receptor would be a promising
therapeutic for the treatment of obesity. Structural studies of Y4 would provide a deeper understanding of the
interaction between PP and positive allosteric modulators that could act as such therapeutic compounds.
While there is no high resolution structure of the transmembrane region of Y4, comparative structural models of
GPCRs using the protein structure prediction software package ROSETTA is shown to accurately predict critical
residues for modulation. Comparative structural models of Y4 docked with bovine pancreatic peptide were
constructed using mammalian GPCR crystal structures as templates, demonstrating that four of the residues
important for PP interaction in trans-membrane domains 6 and 7 cluster in the same space (N6.55, D6.59, N7.32,
F7.35), while the remaining three functional residues in EL1 (Y2.64, D2.68, W2.70) line the top face of the
receptor.
Ligands identified in a high-throughput screen of small molecules have been docked to the Y4-PP comparative
model, predicting critical residues on both Y4 and PP for ligand interaction. The development of a computational
comparative structural model of the Y4-PP complex together with ligand docking studies using ROSETTA will
be combined with quantitative structure-activity relationship (QSAR) studies to allow for a novel approach to the
development of therapeutic agents for obesity.
O22
THE ANTEROGRADE TRANSPORT OF HUMAN NEUROPEPTIDE Y RECEPTORS IS REGULATED BY
SEQUENCE MOTIFS IN THE PROXIMAL C-TERMINUS
Moerl K., Walther C., Lotze J. and Beck-Sickinger A.G.
Institute of Biochemistry; Faculty of Biosciences, Pharmacy and Psychology; Leipzig University; Germany
Structural and functional dependence of G protein-coupled receptors (GPCR) on their subcellular localization
demands synthesis, folding and assembly of newly synthesized proteins in the endoplasmic reticulum, further
posttranslational modification and transport of properly folded receptors via the Golgi apparatus to the plasma
membrane. However, in comparison to the significant progress which has been made concerning ligand-binding
modes, receptor activation and regulation of subsequent internalization mechanisms, only little information is
available on molecular mechanisms involved in cell surface transport of newly synthesized GPCR. Experimental
results achieved over the past few years indicate that cell surface expression and membrane density of the
transmembrane receptors are controlled in a tightly regulated process. Both parameters have the potential to
regulate the magnitude of intracellular signaling cascades elicited upon extracellular ligand binding. Numerous
accessory proteins and chaperons capable to associate with GPCR via conserved sequence motifs and thus
assisting in proper receptor folding have been identified recently. Deletion and mutation studies of neuropeptide
Y receptors transfected in HEK293 cells allowed us to identify amino acid sequences located in the receptors C
termini which are critical for the anterograde transport of human neuropeptide Y receptors (hYR). For example
we identified a short sequence motif in the membrane proximal C terminus of hY2R which is critical for the
anterograde transport of hY2R. The functional relevance of this motif is sequence and position specific.
30 Session 9
O23
LEPTIN-NPY LINK IN THE ETIOLOGY OF DIABETES TYPE 1 AND 2, INDEPENDENT OF WEIGHT
AND APPETITE REGULATION
Kalra S., Kalra P. Departments of Neuroscience, Physiology and Functional Genomics , University of Florida ,
Gainesville, Florida, USA.
Subsequent to our discovery of hypothalamic arcuate nucleus -neuropeptide Y(ARC-NPY) as a physiological
appetite transducer and delineation of the appetite regulating network (ARN) during 1980-1990, we have now
identified the operation of two distinct new pathways, the energy expenditure network (EEN) and the fat accrual
network (FAN). These mediate glucose homeostasis under tight regulation by adipocyte leptin, independent of
ARN (Progress in Brain Res. 181:17, 2010 ; Gene Therapy 18:319, 2011). The ARC-NPY projections of the
EEN terminate in brown adipose tissue to regulate glucose disposal via thermogenic energy expenditure in
skeletal muscle and liver. The other descending pathway along the FAN restrains pancreatic secretion of insulin,
the peripheral hormone that promotes adipogenesis and body fat accrual. We have shown that breakdown in
leptin signaling due to insufficiency of leptin at target sites in the ARC-NPY evoked by either leptinopenia( e.g.
diabetes type 1, lipodystrophy or lipoatrophy due to HIV infection etc), or hyperleptinemia of obesity that
decreases leptin entry into the hypothalamus across the blood brain barrier, induces decreased energy
expenditure, hyperglycemia, hyperinsulinemia (due to a lack of restraint on pancreas).These cumulatively
upregulate adipogenesis and fat deposition culminating in insulin resistance and glucose intolerance, the
hallmark of diabetes type 2 pathology. Based on this new disclosure of a common etiology of leptin
insufficiency in the pathophysiology of both types of diabetes and experimental evidence showing lifetime
amelioration of diabetes by leptin alone, therapeutic clinical trials with leptin are underway to replace the
currently employed cumbersome insulin therapy.
O24
THE DIFFERENTIAL ROLE OF CENTRAL AND PERIPHERAL Y2 RECEPTORS IN REGULATING
GLUCOSE HOMEOSTASIS
Shi Y.C., Lin S., Castillo L., Lau J., Zhang L. Sainsbury A. & Herzog H.
Neuroscience Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst,
Sydney, NSW 2010, Australia
Neuropeptide Y (NPY) plays a critical role in regulating food intake and energy balance via activation of various
receptors including Y2. The Y2 receptor is widely distributed in central and peripheral tissues and is known to
be a key player in the regulation of energy homeostasis. Recently, emerging evidence has also shown that Y2
receptors are important regulators of glucose homeostasis. However, the specific role of Y2 receptors in the
central nervous system, notably on hypothalamic NPY-ergic neurons where they are known to be expressed – as
well as in peripheral tissues in the control of glucose homeostasis remains elusive. To this end, we generated
cell- and tissue-specific inducible knockout mice, NPYY2lox/lox and Mx1CreY2lox/lox, in which Y2 receptors
are ablated or knocked down either in NPY expressing neurons in the brain, by intracerebroventricular (ICV)
injection of doxycycline, or in peripheral tissues, by i.p. injection of polyinosinic-polycytidylic acid,
respectively. We demonstrate that specific deletion of brain Y2 receptors on NPY neurons has no effect on
glucose metabolism despite significantly increased adiposity and increased hepatic triglyceride levels. On the
other hand, on a chow diet, deletion of Y2 receptors predominantly in peripheral tissues leads to no alteration in
glucose tolerance. On a high fat diet however, in addition to significantly less weight gain than wild type
controls, mice with peripheral Y2 receptor deletion display significant improvements in i.p. glucose tolerance as
well as a more rapid restoration of serum glucose levels after i.p. administration of insulin. Moreover, expression
of several key enzymes in lipid metabolism in the skeletal muscle, the major tissue responsible for glucose
uptake, was altered by either hypothalamic or peripheral Y2 receptor deletion, consistent with the lipotoxicity
hypothesis that lipid abnormalities in muscle have profound effects on glucose homeostasis. Thus, these data
suggest that Y2 receptors in the brain and in peripheral tissues elicit differential effects on glucose regulation,
with lack of Y2 receptors in NPY expressing neurons having no effect on glucose tolerance, and lack of Y2
receptor in the periphery enhancing glucose tolerance and response to insulin, possibly due – at least in part – to
effects on fat metabolism in muscle. Supported by Australian NHMRC grant. 31 O25
DORSOMEDIAL HYPOTHALAMIC NPY AND GLUCOSE HOMEOSTASIS
Bi S. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Previous studies have suggested the importance of the dorsomedial hypothalamus (DMH) in glucose
homeostasis. The DMH contains both glucose-sensitive and glucose responsive neurons. Lesions of the DMH
resulted in deficits in feeding responses to both glucose and insulin challenges. Despite these observations, the
neural mechanisms through which the DMH acts to regulate glucose homeostasis remain undetermined. The
Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of obesity and non-insulin dependent
diabetes mellitus (NIDDM). Based on the evidence that NPY expression is elevated in the DMH of OLETF rats,
we have hypothesized that this elevation of DMH NPY contributes to hyperphagia, obesity and diabetes. To test
this hypothesis, we have generated recombinant vectors of adeno-associated virus (AAV)-mediated expression
of NPY or RNAi for knockdown of NPY expression and examined whether virus-mediated alterations in NPY
expression in the DMH affect food intake, body weight and glucose homeostasis in OLETF rats and intact
Sprague Dawley rats. We found that knockdown of NPY expression in the DMH ameliorated the hyperphagia,
obesity and diabetes of OLETF rats. Moreover, we found that NPY overexpression in the DMH of Sprague
Dawley rats resulted in increased food intake and body weight and impaired glucose tolerance, whereas
knockdown of NPY expression in the DMH decreased food intake and body weight and enhanced insulin
sensitivity. NPY knockdown ameliorated high-fat diet-induced hyperphagia and obesity and alleviated dietinduced alterations in glucose homeostasis. Together, our results not only demonstrate critical roles for DMH
NPY in body weight regulation, but also suggest a potential role for DMH NPY in glucose homeostasis.
Supported by: NIH R01DK074269 and R01DK087888
Session 10
O26
CHANGES IN SKIN INNERVATION IN A NEUROPATHIC PAIN MODEL
Ribeiro-da-Silva, A., Magnussen, C., Peleshok, J.C., St. Louis, M.
Dept. Pharmacology & Therapeutics, McGill University, Montreal, QC, Canada
Lesions of peripheral nerves can lead to neuropathic pain, a condition extremely difficult to treat. Our laboratory
has been studying changes both in the skin and spinal dorsal horn following the application of a chronic
constriction injury (CCI) to the sciatic nerve. In the thick skin of the ipsilateral hind paw of male SpragueDawley rats, we have observed that there was an initial loss of primary afferent innervation, detectable as early
as 3 days following application of the CCI. Specifically, the peptidergic nociceptive afferents, detected by
calcitonin gene-related peptide (CGRP) immunoreactivity (IR), recovered to sham levels by 4 weeks post-lesion
followed by a hyper-innervation that persisted to 1.5 years. The non-peptidergic C-fibres, detected by
immunoreactivity for the purinergic P2X3 receptor, provided most of the innervation of the epidermis in control
animals. Following lesion, there was a marked decrease in P2X3-immunoreactive fibre density in the upper
dermis that persisted to 4 weeks and slowly recovered such that by 16 weeks post-lesion, fibre density was still
significantly lower than in controls. The thick myelinated afferents, detected by NF200-IR, virtually disappeared
from the skin by day 3 post-lesion and recovered to about 1/4 of their sham density by 8 weeks. Sympathetic
fibres, detected by dopamine β-hydroxylase-IR, were mostly restricted to the lower dermis in control rats, where
they innervated blood vessels. Following CCI, the sympathetic fibres sprouted into the upper dermis at 2 weeks
post-lesion where they wrapped around CGRP-immunoreactive fibres and persisted in the new territory to 1.5
years post-lesion. We suggest that the sympathetic sprouting, as well as the excessive innervation by CGRPimmunoreactive fibres following lesion, seem to be mediated in part by nerve growth factor (NGF) as an
increase in proNGF-IR was also observed. In control animals, neuropeptide Y (NPY)-IR was restricted to
sympathetic fibres in the lowers dermis. Data on changes in NPY-IR after CCI will be presented at the meeting.
Our data suggests that the abnormal sensory-autonomic interactions that develop in the upper dermis following
CCI may contribute to the triggering of the mechanical allodynia experienced by these animals.
Supported by CIHR and Louise and Alan Edwards Foundation
32 O27
TONIC INHIBITON OF CHRONIC PAIN BY NEUROPEPTIDE Y.
Bradley Taylor
University of Kentucky, Medical centre, Lexington KY, USA
Dramatically up-regulated in the dorsal horn of the mammalian spinal cord following inflammation or nerve
injury, neuropeptide Y (NPY) is poised to regulate the transmission of sensory signals. We found that
doxycycline-induced conditional in vivo (Npytet/tet) knockdown of NPY produced rapid, reversible, and
repeatable increases in the intensity and duration of tactile and thermal hypersensitivity. Remarkably, when
allowed to resolve for several weeks, behavioral hypersensitivity could be dramatically reinstated with NPY
knockdown or intrathecal administration of Y1 or Y2 receptor antagonists. In addition, Y2 antagonism increased
dorsal horn expression of Fos and p-ERK. Taken together, these data establish spinal NPY receptor systems as
an endogenous braking mechanism that exerts a tonic, long-lasting, broad-spectrum inhibitory control of spinal
nociceptive transmission, thus impeding the transition from acute to chronic pain. NPY and its receptors appear
to be part of a mechanism whereby mammals naturally recover from the hyperalgesia associated with
inflammation or nerve injury.
Session 12
O28
YOU EAT WITH YOUR BRAIN -NPY AND ENERGY BALANCE
Colmers, William F. Department of Pharmacology, University of Alberta, 9-36 MSB, Edmonton, AB, Canada,
T6G 2H7
NPY was early recognized as having profound effects on behavior. One of the first observations with the peptide was its
effect on food intake. Careful mapping work by Glenn Stanley localized the orexigenic actions to a series of sites in the
hypothalamus, including the PVN and the perifornical hypothalamus. I became intrigued with the question of the sites and
mechanisms of NPY's actions in stimulating food intake as a means of investigating the circuitry underlying the control of
energy homeostasis. Fortunately, Eli Lilly and Don Gehlert (our 3rd Speaker) helped out at just that moment to make it
possible to do so. Together with Nina Pronchuk, we set out to identify the actions of NPY in the PVN using brain slices,
reasoning that any effects of the peptide on physiological properties of neurons might relate to its orexigenic effect- the
most prominent action of NPY described in PVN. Once we learned that NPY inhibited a GABA synaptic response in a
subclass of parvocellular neurons, a collaboration with Roger Cone from OHSU began. Michael Cowley (our next speaker)
came from Portland to learn brain slice from Nina, and they learned that melanocortin agonists enhanced the GABA
response in these cells. AgRP, the natural antagonist of melanocortins, blocked their effects in the slice. Next we looked at
the pharmacology of the NPY response, which was described by Stanley in 1992 as a"Y1 variant". Using tools developed
by Annette Beck-Sickinger and others, we learned that the NPY response in PVN had components related to Y1, Y5 and Y2
receptors, making the complexity of the response in vivo understandable. This has been borne out by the enormous work
needed to do triple and quadruple NPY receptor knockouts in order to find effects on feeding
With a greater understanding of the circuitry involved, it became important to learn if the responses to any of these signals
was sensitive to obesity. We first looked at NPY effects in Zucker fa/fa rats, and learned that in the animals that became
obese, the NPY responses declined modestly. We next got rats fat on a high-fat diet and again studied in PVN responses.
Again, the NPY responses declined a little in the obese rats, but the melanocortin responses declined sharply with excess
weight. Because the NPY system is on and remains on probably from birth, but the melanocortin system only turns on at
weaning, the plasticity of the melanocortin system may be the root cause of obesity.
Supported by Canadian MRC, CIHR and Alberta Heritage Foundation for Medical Research.
33 O29
NPY NEURONS IN THE DORSOMEDIAL HYPOTHALAMUS REGULATE ENERGY BALANCE AND
BLOOD PRESSURE
Cowley M.A.,Verma S., Simmonds S.E., Enrioi P.G., Draper S.J., Smith M.S., Grove K.L.
Monash Obesity & Diabetes Institute, Monash University, Clayton, Victoria, Australia, and
Oregon National Primate Research Center, Beaverton, Oregon, USA
NPY has actions in many parts of the brain, and has a well studied role in energy balance, especially in sites such
as the arcuate nucleus and the paraventricular nucleus of the hypothalamus. We have evaluated the effects of
leptin on dorsomedial hypothalamus (DMH) neurons using several experimental techniques. We have used an
NPY-GFP mouse that expresses Renilla-GFP in NPY neurons to record the electrical effects of leptin on NPY
neurons. We have also used in-vivo pharmacology studies, including telemetry to evaluate the effects of leptin
on food intake, body temperature, and cardiovascular parameters in lean and obese mice.
Here we report that NPY neurons in the DMH are activated by leptin. Leptin depolarizes DMH neurons, and
increases the frequency of action potentials they fire. This action in the DMH is in contrast to the actions of
leptin in the arcuate nucleus, were leptin inhibits NPY neurons.
In lean mice leptin microinjected into the DMH reduces appetite, however this action is lost in obese mice. In
lean and obese mice leptin microinjected into the DMH can increase sympathetic outflow to brown adipose
tissue. Surprisingly, we find that leptin increases sympathetic tone to elevate heart rate and blood pressure. We
also find that leptin levels linearly correlate with blood pressure, and blockade of leptin action in the DMH
normalizes blood pressure in obese hypertensive mice.
These data show that DMH neurons can regulate several critical physiological processes, including energy
balance and blood pressure.
Supported by: NIH, NCRR, NHMRC, National Heart Foundation of Australia.
O30
TRANSCRIPTOME OF THE HYPOTHALAMIC ARCUATE NUCLEUS OF THE RAT: A COMPARISON
OF MICROARRAY AND NEXT GENERATION RNA SEQUENCING.
Gehlert, D1, Calley, J2, Lu, Y3, Vrang, N4, Paulson, S4 and Morin, M1
1Diabesity Group, 2Translational Sciences Bioinformatics Group,and3 Global Statistical Science and Advanced
Analytics, Lilly Research Labs, Eli Lilly and Company Indianapolis IN USA, 4Gubra ApS ,Hørsholm,Denmark
Next generation RNA sequencing (RNA-seq) is quickly becoming an important tool to evaluate transcriptomes,
digitally quantify mRNA abundance and identify alternative splice sites and previously unknown single
nucleotide polymorphisms (SNPs). RNA-seq is also reported to be particularly suited to detecting mRNA
species encoding neuropeptides and G-protein coupled receptors (GPCRs) that have a low level of expression
and are under-represented on commercially available microarrays. In this experiment, we compared RNA-seq
and microarray analysis utilizing samples from the hypothalamic arcuate nucleus (Arc). The Arc contains a
variety of cell types expressing neuropeptides known to be involved in food intake including AgRP, NPY,
CART and POMC. This complex, yet well studied, phenotype made the Arc an ideal nucleus to validate the
utility of RNA-seq.
To accomplish this comparison, the arcuate nuclei from ad lib fed Sprague Dawley male rats were dissected by
laser capture microscopy (LCM) using a Veritas Microdissection system and total RNA isolated. One half of the
RNA from each sample was used for Affymatrix 230 2.0 microarray analysis and the other half was sequenced
on an Illumina HiSeq 2000 analyzer. The resulting data sets were compared and the limits of detection, linearity
and variability determined for each platform. Consistent with previously reported results, the microarray
detected over 8000 genes as present in the Arc of the rat, including NPY and CART. However, over 14000 genes
were detected utilizing RNA-seq, including many neuropeptides that were either not detected or not represented
on the Affymatrix chip. In summary, RNA-seq provided an improved method for determining the complete
transcriptional profile of the Arc nucleus.
34 Session 13
O31
NEUROPEPTIDES IN THE REGULATION OF FOOD INTAKE AND ENERGY METABOLISM.
Shioda S., Takenoya F., Kageyama H. Department of Anatomy, Showa University School of Medicine.
Shinagawa-ku, Tokyo 142-8555, Japan.
Novel neuropeptides of G-protein coupled receptor (GPCR) ligands are shown to be localized in brain and play a
range of physiological functions including feeding regulation and energy metabolism. I will describe the
distribution and localization of these GPCR neuropeptide ligands identified recently and to review their
involvement in neuronal networks, particularly feeding regulation and energy metabolism. This presentation
concerns some novel GPCR ligands of feeding and energy metabolism-regulating neuropeptides such as orexin,
ghrelin, galanin-like peptide (GALP) and neuropeptide W (NPW), such as those studied by our research group
and others, and neuronal interactions among these and other well known neuropeptides such as neuropeptide Y
(NPY) and alpha-melanocyte stimulating hormone (alpha-MSH) in the hypothalamus. Cross-talk among several
these neuropeptides-containing neurons in the hypothalamus plays a key role in determining feeding states as
well as feeding behavior and energy metabolism. I will show some structural and functional characteristics of
these very recently discovered neuropeptides and summarize the known interactions between these different kind
of feeding regulating neurons and leptin-targeting neurons in the hypothalamus. Moreover, I will present a new
strategy for analyzing the neural circuit of these feeding- and energy metabolism-regulating GPCR ligandscontaining neurons in orexin-containing neurons in brain by use of transgenic model mice. Finally, I will present
our very recent results of GALP and NPW which are involved in regulation of feeding as well as energy
homeostasis including body temperature, heart rate and so on. I will show our hot data of intranasal infusion of
GALP to decrease body weight and locomotor activity in animal models of ob/ob, db/db and high-fat diet (DIO)
mice. Research in this field will serve a very important role of clarifying neurologically-based causes for appetite
dysfunctions and diseases and it may help to establish and to lead new therapies for people who are suffering
from such conditions.
O32
FEEDING-RELATED PANCREATIC AND GASTROINTESTINAL HOROMONES INCULUDING NPY
FAMILY PEPTIDES DIRECTLY REGULATE VAGAL AFFERET NEURONS.
Yada T. and Iwasaki Y., Department of physiology, Jichi Medical University School of Medicine, Shimotsuke,
Tochigi, Japan
The effects of pancreatic and gastrointestinal hormones on the brain are crucial for biological homeostasis,
particularly energy homeostasis. Pancreatic polypeptide (PP) and peptide YY3-36 (PYY3-36), the members of
neuropeptide Y family, are released at the postprandial period and act on the feeding center in the hypothalamus
and medulla, thereby decreasing food intake. However, the action route and mechanism through which PP and
PYY3-36 act on the brain are not fully understood. The anorectic effects of PP and PYY3-36 are reportedly
abolished in vagotomized rodents, indicative of the involvement of the vagal afferent nerves. To investigate
direct action of PP and PYY3-36 on the vagal afferents, we examined the effects of PP and PYY3-36 on
cytosolic Ca2+ concentration ([Ca2+]i) in isolated nodose ganglion (NG) neurons of the vagal afferent nerves
using fura-2 microfluorometry. PP and PYY3-36 increased [Ca2+]i in NG neurons in a dose-dependent manner,
in which the potency of PP was approximately 2 log orders greater than that of PYY3-36. Moreover, the
majority of the PP-responsive NG neurons also responded to cholecystokinin-8, a hormone that is known to
induce satiety through activating vagal afferents. The present study demonstrates that PP and PYY3-36 directly
activate vagal afferent NG neurons and that PP has a greater potency than PYY3-36, and suggests that the potent
action of PP to stimulate cholecystokinin-8-responsive NG neurons is linked to the regulation of feeding.
35 O33
S-2367, A NOVEL NPY Y5 RECEPTOR ANTAGONIST BEING INVESTIGATED FOR THE TREATMENT
OF OBESITY
Hideo Yukioka Medicinal Research Laboratories, Shionogi Pharmaceutical Research Center, Shionogi & Co.,
Ltd. Toyonaka, Osaka, Japan
Neuropeptide Y (NPY) has been implicated as a key regulator of energy balance in the brain. Among NPY
receptors, NPY Y5 receptor is involved in the energy homeostasis. Therefore, an NPY Y5 receptor antagonist
may be effective in inducing negative energy balance, and consequently weight loss, by inhibiting NPY
signaling.
To verify the hypothesis, we synthesized a novel NPY Y5 receptor antagonist, S-2367, in our laboratory. S-2367
showed a high affinity and specificity for the cloned human NPY Y5 receptor with much lower affinity for other
NPY receptor subtypes. S-2367 antagonized the inhibitory effect of NPY on the forskolin-induced cyclic
adenosine mono phaosphate production in human NPY Y5 receptor-expressing cell. Pharmacokinetic analysis
showed sufficient oral availability and brain permeability of S-2367 in mice. After 8 week repeated
administration of S-2367, body weight gain, abdominal fat mass and hepatic steatosis were significantly
suppressed in diet-induced obese mice. S-2367, on the other hand, had no significant effect on body weight gain
in high-fat diet-induced obese NPY Y5 receptor deficient mice.
In 12 week treatment clinical study, S-2367 showed the good oral bioavailability, less frequent side effects and
significant weight loss in obese subjects. Further, to assess the long-term effectiveness of S-2367 in weight loss
and weight loss maintenance settings, two different studies were conducted under distinct diet conditions,
denoted as the Reduced Calorie Diet (RCD) and Low Calorie Diet (LCD) studies for one year. S-2367 was
found to induce the significant body weight reduction and suppression of weight regain, compared to placebo.
Now, a clinical study is going to examine the effect of S-2367 on body weight in Japanese patients.
O34
PANCREATIC POLYPEPTIDE AND BODY WEIGHT REGULATION
Akihiro Asakawa1, Ambikaipakan Balasubramaniam2, Akio Inui1
1Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and
Dental Sciences, Kagoshima 8908544, Japan, 2Department of Surgery, University of Cincinnati Medical Center,
Cincinnati, OH 45267, USA
Pancreatic polypeptide (PP) belongs to a family of peptides including neuropeptide Y and peptide YY. We
investigated the effects of PP and a novel agonist, BVD-74D, with high selectivity and affinity for Y4 receptor
on feeding and body weight. Peripherally administered PP decreased food intake in feeding studies performed
during the dark phase as well as during the light phase and gastric emptying rate in mice. PP-overexpressing
mice gained less weight with decreased food intake and fat mass. Vagotomy eliminated inhibitory effects on
feeding induced by PP. In the electrophysiologic experiments, intravenous administration of PP decreased
efferent activity of the gastric vagal nerve and afferent activity of the hepatic vagal nerve. PP decreased mRNA
expression of NPY, orexin and CRF and increased urocortin mRNA expression in the hypothalamus. PP also
decreased ghrelin mRNA expression in the stomach. Gene expression of hypothalamic orexin and gastric ghrelin
in PP-overexpressing mice was decreased. PP activated sympathetic nerves innervating the adrenal medulla,
brown adipose tissue and epididymal adipose tissue, and increased oxygen consumption. Repeated
administrations of PP decreased food intake and body weight gain, and ameliorated insulin resistance and
hyperlipidemia both in ob/ob obese mice and in FLS-ob/ob obese mice. Peripherally administered BVD-74D
decreased food intake and body weight gain in mice. BVD-74D also decreased food intake both in mice that
were fed with the high-fat diet and in FLS-ob/ob mice. These observations indicate that PP and Y4 receptor
would be promising targets for the treatment of obesity. 36 Session 14
O35
NPY IN EXPERIMENTAL AND HUMAN EPILEPTIC TISSUE
Gunther Sperk, Department of Pharmacology, University Innsbruck, Austria
Recurrent epileptic seizures in the rat enhance the expression of NPY and its mRNA in many brain areas
particularly in the hippocampus, cerebral cortex and the amygdala. In the hippocampus, the most prominent
expression of NPY is observed in mossy fibers and in GABAergic interneurons. At the same time, expression of
Y2 receptors is also increased whereas Y1 receptors are reduced. Similar changes in Y1 and Y2 receptors were
observed in the hippocampus of patients with temporal lobe epilepsy (TLE). In contrast to the rat, NPY
expression is not enhanced in mossy fibers in TLE. In the same tissue, surviving NPY interneurons show marked
axonal sprouting into areas innervated by mossy fibers, i.e. the dentate hilus, stratum lucidum, inner molecular
layer of the dentate gyrus. Stimulation of presynaptic Y2 receptors inhibits glutamate release, and exert an
anticonvulsant action in experimental models. In contrast activation of Y1 receptors results in proconvulsive
effects. Overall the findings findings suggest that changes in the NPY system induced by seizures represent
endogenous adaptive mechanisms aimed at counteracting hyperexcitability underlying epileptic activity. This
concept is strongly supported by evidence that genetically modified rats overexpressing the NPY gene are less
susceptible to seizures while deletion of NPY or Y2 receptor genes results in increased susceptibility to seizures.
Enhancing the concentration of NPY by local injection of viral vectors expressing the peptide gene exert potent
anticonvulsive actions.
Supported by the Austrian Research Funds (P12159, P19464, I644)
O36
NEUROPEPTIDE Y MODULATES EMOTIONAL MEMORY PROCESSES IN THE DORSAL
HIPPOCAMPUS
Tasan RO., Verma D., Herzog H. & Sperk G.
Institute of Pharmacology, Medical University Innsbruck, Innsbruck, Austria
NPY has attracted considerable attention in modulating emotional-affective behavior. NPY exerts a pronounced
anxiolytic effect most likely mediated by Y1 receptors, whereas stimulation of predominantly pre-synaptic Y2
receptors results in increased anxiety. The role of NPY in hippocampus dependent emotional learning processes,
however, remains still elusive. The current study aimed to investigate the role of Y2 receptors in 1) Pavlovian
context and 2) trace fear conditioning, two simple forms of hippocampus dependent associative learning and in
3) a fear context discrimination task, a more complex emotional learning paradigm that requires the presence of
adult-born neurons. Furthermore, the Barnes maze and object recognition tasks were employed to elucidate the
role of NPY in spatial and object identity memory.
While in delay fear conditioning Y2KO mice performed similar to WT controls, recall of a context and trace fear
memory was significantly increased in Y2KO mice. Temporal precision of memory recall was, however,
significantly impaired. Furthermore, Y2KO mice exhibited improved working memory during acquisition as
well as improved long-term memory in the Barnes maze test, a paradigm reflecting spatial learning. Context and
trace fear conditioning as well as spatial memory are predominantly mediated by the dorsal hippocampus.
Deletion of Y2 receptors in the dorsal hippocampus of conditional Y2 KO mice revealed improved short-term
memory in the Barnes maze and in the object location test. Local over-expression of the Y2 preferring agonist
NPY3-36 in the dorsal hippocampus by injecting a respective rAAV vector delayed the acquisition and reduced
the recall of trace fear memory while impairing the performance in the object-in-place and temporal order tasks.
In summary, our data indicate that Y2 receptors play an inhibitory role on the acquisition/expression of
hippocampus dependent fear memories. Moreover, Y2 receptors in the dorsal hippocampus are crucial for spatial
memory formation. These actions are probably mediated by inhibition of glutamate release in dorsal
hippocampal circuitries.
37 Session 15
O37
O38
NEW INSIGHTS TO THE REGULATION OF METABOLISM FROM A SYMPATHETIC NERVOUS
SYSTEM SPECIFIC NPY TRANSGENIC MOUSE MODEL
Savontaus E., Vähätalo L., Ailanen L., Ruohonen S.T. Department of Pharmacology, Drug Development and
Therapeutics, and Turku Center for Disease Modeling, University of Turku, Turku, Finland
Neuropeptide Y (NPY) is a co-transmitter in the sympathetic nerves (SNS) with increased release in prolonged
stress. To address the role of NPY colocalized with noradrenaline in the SNS and brain noradrenergic neurons,
we created a transgenic mouse (OE-NPYDBH) overexpressing NPY under the dopamine-beta-hydroxylase
promoter.
The OE-NPYDBH mice develop a gene-dose- and age-dependent metabolic phenotype that is most pronounced
in the homozygous male NPY-OEDBH mice. The primary finding is increased adipose tissue mass starting
from age 2 months. By the age of 7 months, male NPY-OEDBH mice become obese with increased body
weight. Liver triglyceride content is increased and histopathology shows hepatic steatosis. Serum triglyceride
and NEFA levels are significantly decreased and total cholesterol level increased. Brown adipose tissue has
increased lipid content and decreased UCP1 mRNA expression. Glucose and insulin tolerance are impaired and
fasting and glucose-stimulated serum insulin levels increased in obese NPY-OEDBH compared to wild-type
mice. Food intake, physical activity or energy expenditure by indirect calorimetry are not different between
genotypes. In addition to increased NPY, decreased noradrenergic tone and increased endocannibinoid levels
may contribute to the metabolic phenotype.
Thus, overexpression of NPY in the noradrenergic neurons leads to increased adiposity, impaired glucose
tolerance, hyperinsulinemia, hepatic steatosis and hypercholesterolemia, ie. traits of the metabolic syndrome.
This suggests that in addition to the hypothalamus, NPY promotes lipid storage and metabolic disturbances in
the brainstem and in the sympathoadrenal system.
38 O39
LONG-TERM MODULATION OF INGESTIVE AND SOCIAL BEHAVIOURS USING VIRAL VECTORS
EXPRESSING PYY IN THE SALIVARY GLAND
Zolotukhin S., Hurtado M.D., Acosta A., Dotson C.D., Brown A., Voutetakis A., Riveros P., Baum J.B. Dept of
Pediatrics, University of Florida, Gainesville, Fl 32610, USA.
Recently we have documented the presence of PYY3-36 in the saliva of mice and humans, as well as the
expression of Y receptors in the lingual epithelial cells, taste receptor cells (TCRs), and in cells of the
submandibular salivary gland (SMG). To characterize the functions of salivary PYY and oral Y receptors, we
have developed novel models of somatic transgenic mice expressing PYY in brain, liver, gut, or SMG. This was
achieved by targeted expression of the recombinant Adeno-associated virus vector encoding pre-pro-PYY
(rAAV-PYY). We show that centrally expressed PYY dramatically increases food intake (FI) and body weight
(BW) accumulation. On the other hand, ten-fold increase in systemic PYY followed hepatic transgene delivery
resulted in no net change of BW accumulation. Finally, local expression of PYY transgene in SMG reduced BW
accumulation in lean mice and in diet-induced obese mice. As an initial foray to characterize the mechanisms of
salivary PYY action, we have documented the involvement of salivary PYY in mediating lipid taste modality. In
germline PYY KO mouse model, animals displayed significantly blunted lipid sensing behavior which was
completely restored in mice treated with SMG-targeted rAAV-PYY. Interestingly, the chronic over-expression
of salivary PYY also produced a curious effect on the social interactions of mice - an apparent reduction in
aggressive behavior. This effect was tested in a study using a standard resident-intruder behavioral test. The
rAAV-PYY vector-treated mice showed a marked decrease in aggressive behavior. There was, however, no
difference in other behavioral parameters, such as social sniffing.
O40
NEUROPEPTIDE Y RECEPTOR SIGNALLING IN BONE TISSUE IS REQUIRED FOR NORMAL
GLUCOSE HOMEOSTASIS IN MICE
Paul A Baldock1, Nicola J Lee1, Daniela Sousa1,2, Ronaldo F Enriquez1, Amanda Sainsbury1, Herbert
Herzog1. 1. Neuroscience Research Program, Garvan Institute of Medical Research, Sydney, Australia, 2010. 2.
INEB, Institute of Biomedical Engineering, NEW Therapies Group, Porto, Portugal.
The skeleton has recently emerged as a potential player in the control of whole-body glucose metabolism;
however, the mechanism behind this regulatory axis has not been fully elucidated. Here we demonstrate that
neuropeptide Y (NPY) receptor signalling in osteoblasts is required for normal glucose metabolism, in addition
to bone formation. The osteoblastic NPY receptor (Y1R) was deleted from the osteoblast (OB Y1R KO). In
addition, the circulating Y1R ligand, peptide YY (PYY), was over expressed in osteoblasts. Mice were examined
for glucose homeostasis at 13-14 weeks.
Mice lacking Y1 receptors solely in cells of the osteoblastic lineage displayed a high bone mass phenotype due
to significantly increased bone formation rate mineral apposition rate and reduced bone resorption. Y1R KOOB
mice also displayed altered whole-body glucose metabolism. This was due to significantly decreased pancreatic
insulin content, pancreas weight, and decreased insulin secretion leading to elevated glucose levels and reduced
glucose tolerance, but with no effect on insulin induced glucose clearance.
Furthermore, increased activity of Y1R signalling induced by over-expression of its ligand, PYY in osteoblastic
cells, in PYY TGOB mice also led to impaired glucose tolerance. However, insulin secretion was elevated and
insulin sensitivity was impaired.
These data reveal a novel mechanism by which NPY receptor signalling in bone tissue is involved in the control
of glucose and energy homeostasis. Maintenance of Y1R signalling in osteoblasts appears crucial to preservation
of optimal glucose homeostasis.
39 Session 16
O41
HUMAN ENDOCARDIAL ENDOTHELIAL CELLS HYPERTROPHY AND RELEASE OF ENDOTHELIN1: ROLE OF NEUROPEPTIDE Y AND ANGIOTENSIN II.
Jacques D.1, Magder S.2, D’Orléans-Juste P.3 and Bkaily G.1.1Department of Anatomy and Cell Biology,
3Department of Pharmacology, Faculty of medicine, University of Sherbrooke, Sherbrooke, Québec, Canada,
J1H 5N4. 2Critical Care Division, Royal Victoria Hospital, McGill University Health Centre, McGill University,
Montreal, Quebec, Canada, H3A 1A1.
Endocardial endothelial cells (EECs) line the cardiac cavities and constitute a barrier between the circulating
blood and the adjacent cardiomyocytes. In addition, EECs produce and release various cardioactive factors such
as Neuropeptide Y (NPY) and Angiotensin II (AngII). Theses peptides are implicated in various cardiac
pathologies including hypertrophy of cardiomyocytes. However, it is not known whether EECs volume is also
increased and whether this modulates excitation-secretion coupling of these cells. Using real 3D confocal
microscopy imaging, immunofluorescence and various molecular biology techniques, we found that NPY and
AngII induce an increase in the volume of human EECs. This increase in cell volume is accompanied by an
increase in hypertrophic markers such as the DNA/protein ratio and ANP mRNA. We also found that AngIIinduced hypertrophy of EECs is primarily mediated via AT1 receptor activation whereas the NPY-induced
hypertrophy is mediated via activation of the Y1, Y2 and Y5 receptors. In addition, NPY and AngII induce the
release of Endothelin-1 from normal and hypertrophic human EECs. Thus, similar to cardiomyocytes, cardiac
hypertrophy is associated with EECs hypertrophy and modulates the secretory capacity of these cells. Increased
secretion may in turn contribute to the cardiac remodelling that occurs in hypertrophy and heart failure.
Supported by the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of
Quebec (HSFQ).
O42
VECTOR-INDUCED OVEREXPRESSION OF NPY REDUCES PARKINSONIAN SYMPTOMS IN MICE.
Christiansen S.H.1, Jensen M.1, Gøtzsche C.R.1, Hansen P.H.1,2, Wörtwein G.1,2, Weikop P.1,2, Woldbye D.P.D.1
1Department of Neuroscience and Pharmacology, Univ. of Copenhagen, Denmark; 2Laboratory of
Neuropsychiatry, Psychiatric Center Copenhagen, Denmark
Current treatment of Parkinson’s disease (PD) provides only symptomatic relief over a limited time span and
does not prevent the progressive loss of dopamine neurons. Consequently, there is still great need to develop
novel treatments for PD. NPY has been shown to modulate dopaminergic neurotransmission and to exert
neuroprotective effects and could consequently be a novel target in PD. Using microdialysis, we showed that
NPY infusion into the striatum of adult mice results in increased extracellular dopamine levels that was
accompanied by reduced Parkinsonian-like symptoms as revealed by haloperidol-induced catalepsy.
In recent years, gene therapy has shown promising potential as a future treatment for PD, however, no previous
studies have explored NPY-related transgenes for this purpose. Using recombinant adeno-associated virus
(rAAV) encoding the NPY transgene, we induced overexpression of NPY in the striatum and tested potential
effects on Parkinsonian symptoms in adult mice. As hoped for, rAAV-NPY-treatment caused a significant
decrease in haloperidol-induced catalepsy and also reversed amphetamine-induced rotational behaviour in 6OHDA-lesioned mice. These data indicate that gene therapy with viral vectors inducing overexpression of NPY
in the striatum deserves attention as a potential future therapy in PD.
40 O43
THE BETA-ARRESTIN-2-MEDIATED PATHWAY IS NOT REQUIRED FOR PANCREATIC
POLYPEPTIDE (PP)-MEDIATED REDUCTION IN FOOD INTAKE AND BODY WEIGHT
Ning Lee, Jun Zhu, John Lehrach, Camelia Chiriac, Yuying Zhang, Jiyi Fu, Mary Jane Cullen, Suzanne Rooney,
Ving Lee, Yanting Huang, Chongqing Sun, Jian Chen, Keith Miller, Claudio Mapelli, William Ewing, Wen-Pin
Yang, Liang Schweizer, Anthony Azzara, Mary Ann Pelleymounter.
Metabolic Disease Drug Discovery, Bristol-Myers Squibb Company, Pennington, NJ, USA
β-arrestins (β-arrestin-1 and β-arrestin-2), originally identified as scaffold proteins involved in desensitization of
G protein-coupled receptors (GPCRs), are receiving greater attention with regard to their involvement in
activating signaling pathways independent of G protein. The interactions between β-arrestins and NPY receptors
(Y1, Y2, Y4 and Y5) have been demonstrated via in vitro assays; however, the functional significance of this
interaction is not clear. In this study, we have used siRNA directed against β-arrestins to show that, knocking
down of β-arrestin-1 and/or β-arrestin-2 in a NPY Y4-overexpressed CHO stable line has no impact on
pancreatic polypeptide (PP)- induced Y4 receptor signalings, including cAMP, and ERK activation. Using βarrestin-2 KO mice, Y4 agonist-induced colon contractile responses were conducted and shown no difference
from observed in wild-type mice. We have also shown that, peripheral injection of PP produces a similar
reduction of acute food intake in both normal lean mice and β-arrestin-2 KO mice. In addition, after a 4-day subchronic treatment with long acting pegylated PP, similar levels of food intake and body weight reduction were
observed in both wild-type mice and β-arrestin-2 KO mice. These data suggest that the β-arrestin-2-mediated
pathway, downstream of Y4 activation, is not required for PP-mediated regulation of food intake and body
weight.
Session 17
O44
PYY MEDIATES GPR119 ACTIVITY IN THE INTESTINE.
Helen Cox,1 Iain Tough,1 Anne-Marie Woolston,1 Lei Zhang2, Amy Nguyen2, Amanda Sainsbury2 and Herbert
Herzog2. 1King’s College London, Wolfson CARD, Guy’s Campus, London SE1 1UL, UK, 2Neuroscience
Program, Garvan Institute of Medical Research, 384 Victoria Street, NSW 2010, Sydney, Australia.
Peptide YY (PYY) is released with glucagon-like peptide (GLP) following food intake and regulates intestinal
function and glucose homeostasis, but the mechanisms underpinning these processes are not well understood.
Enteroendocrine L-cells contain PYY, GLP-1 and GLP-2 and express the acylethanolamine receptor, GPR119
whereas the surrounding epithelia express Y1 receptors but not GPR119 or peptides. In functional assays utilising
intact mucosae from human and mouse colon we showed that GPR119 activation inhibited epithelial electrolyte
secretion in a glucose-sensitive manner [1]. Endogenous PYY selectively mediated these effects since PYY-/- mice
showed no GPR119 response, but these mucosal effects were observed in NPY-/- mice.
Importantly, GPR119 responses in wild type mouse tissue and healthy human colon were abolished by Y1 receptor
antagonist BIBO3304 but were not enhanced by dipeptidyl peptidase 4 blockade, indicating that PYY processing
to PYY(3-36) was not important. In addition, Gpr119 agonism reduced glycaemic excursions after oral glucose
delivery to WT mice, but not PYY-/- mice. Taken together, these data demonstrate a previously unrecognised
paracrine functional role of PYY in mediating intestinal GPR119 activity and an associated function in controlling
glucose tolerance, in parallel with the better known GLP-1 effects.
1. Cox HM et al. (2010) Cell Metab. 11, 532-542.
This work was funded by The Wellcome Trust.
41 O45
O46
BILE ACID INDUCED SECRETION OF PYY; PATHOPHYSIOLOGICAL IMPLICATIONS.
Christoph Beglinger, Anne C. Gerspach, Robert E. Steinert
Department of Biomedicine and Division of Gastroenterology, University Hospital Basel, Basel, Switzerland
Gut hormones peptide YY and GLP-1 play integral roles in appetite control and energy homeostasis. Eneteroendocrine cells
can be stimulated by nutrients and bile acids.. Bile acids (BAs) are amphipathic molecules that facilitate the uptake of lipids,
and their levels fluctuate in the intestine as well as in the blood circulation depending on food intake. The G protein-coupled
receptor TGR5 is expressed in PYY-secreting cell lines and in primary L cells from mice; pharmacological and genetic
gain- and loss-of-function models suggest a physiological role for TGR5 activation on PYY secretion in rodents. In vivo
TGR5 signaling was associated with improved postprandial glucose tolerance in diet-induced obese mice, an the
improvement was associated with postprandial PYY release and insulin secretion. In contrast, TGR5_/_ mice exhibited
reduced glucose tolerance. We therefore hypothesized that intraduodenal perfusion of TGR5 agonists stimulate the secretion
of PYY with respective changes in the glucose metabolism of healthy humans. Aim: To study the effect of BAs and OA on
PYY and GLP-1 release in healthy human subjects.
Methods: The study was conducted as a randomized, double-blind, placebo-controlled, parallel-designed trial, including
twelve healthy male volunteers. Subjects received intraduodenal perfusions of i) a bile acid (chenodeoxycholic acid), ii) a
fatty acid (pos. control) or iii) OA and iv vehicle (placebo, neg. control). The different test solutions were perfused for 180
minutes at a rate of 2.0 mL/min. After one hour subjects performed an oral glucose tolerance test (oGTT, 75 g glucose in
300 mL tab water). Blood samples were collected for hormone and glucose analysis.
Results: BAs induced a significant secretion of PYY and GLP-1 within the first 60 minutes compared to placebo (P =
0.05); however no the oGTT was more potent to stimulate hormone release. Plasma insulin and glucose were not affected
by BAs within the first 60 minutes. Further analyses, including plasma CCK and serum bile acids.
Summary: BAs can modulate the secretion of PYY and GLP-1. However, the results of the oGTT indicate a minor role for
BAs in the regulation of gut peptides with little effect on glucose homeostasis in humans. The attenuated insulin release
after the oGTT infers a peptide independent effect of BAs. The results are in agreement with studies performed in
patients after bariatric surgery.
Perspectives: Our results in healthy subjects do not support an important role for bile acids in regulating PYY release and
postprandial glucose metabolism. A major limitation of the hypothesis is the fact that the evidence is based on animal
studies and cell models.
42 Session 18
O47
GPR83 – A NOVEL REGULATOR OF SYSTEMIC ENERGY METABOLISM CONTROL
Müller TD, Müller A, Habegger K, Yi C-X, Heppner K, Trivedi C, Bielohuby M, Abplanalp W, Piechowski L,
Stemmer K, Lehti M, Holland J, Hembree J, Bhardwaj N, Raver C, Ottaway N, Bartley E, Krishna R, Sah R,
Perez-Tilve D, Biedlingmaier M, DiMarchi R, Krude H, Pfluger PT, Kleinau G, Biebermann H, Tschöp MH
Helmholtz Centre Munich, Munich, Germany
GPR83 is an orphan G-protein coupled receptor awaiting physiological characterization. GPR83 is widely
distributed in the brain including hypothalamic nuclei governing energy balance. Similar to ghrelin action,
hypothalamic expression patterns of GPR83 are tightly regulated in response to nutrient availability and are
decreased in the state of chronic high-fat diet exposure. In the arcuate nucleus, GPR83 is co-localized with the
ghrelin receptor (GHSR1a) and in vitro analyses show that GPR83 modulates GHSR1a activity through direct
and physical interaction. In line with its ability to modulate GHSR1a activity, ghrelin’s effect on food intake and
adipogenesis is enhanced in GPR83 KO compared to WT mice. In summary, our data indicate that
GPR83/GHSR1a dimerization modulates ghrelin action in response to nutrient availability, thus identifying
GPR83 as a novel regulator of systemic energy metabolism control.
O48
DIRECT INFLUENCES OF GASTRO-INTESTINAL PEPTIDES ON BRAIN NETWORKS AS REVEALED
BY NEUROIMAGING IN HUMANS
Schloegl H, Kabisch S, Horstmann A, Lohmann G, Lepsien J, Pleger B, Villringer A, Stumvoll M, University of
Leipzig, Germany
The glucagon-like peptide-1 (GLP-1) analog exenatide is used in clinical practice to enhance insulin secretion in
type-2-diabetes. In some, but not all patients a decrease in energy intake and a reduction in body weight can be
found, independent of the insulinotropic effects and occurrence of nausea. In animal studies the hypothalamus
was identified as a GLP-1 binding site.
To investigate the central nervous effects of exenatide in humans we performed functional MRI and obtained
data of 22 male obese (BMI 30 - 46 kg/m²) during IV exenatide administration in a cross-over, placebocontrolled study. Inside the MRI scanner subjects rated food pictures for tastiness and thereafter consumed an ad
libitum meal where energy intake was assessed.
Subjects were classified according to the drug effects on energy intake (% kcal reduction compared to placebo):
>10 % = “responders”, n=11, mean -375 kcal, -24 % and <10% = “non-responders”, n=11, mean +122 kcal, +10
%. For fMRI analysis we used a parameter-free method called 'eigenvector centrality mapping' (ECM), that
attributes a centrality value to each brain voxel (volumetric pixel) reflecting its degree of connectedness with
other voxels. Only "responders" showed a significantly higher centrality/connectivity of the hypothalamus in the
exenatide condition compared to placebo (z-value 3.88, maximum at Talairach coordinates -2, -9, -3), in "nonresponders" there were no significant effects in this region.
We demonstrate for the first time that peripherally administered exenatide exerts its effects on energy intake in
humans via the hypothalamus, and suggest that absence of anorectic effects of the drug administration is related
to an absent hypothalamic response.
Supported by Eli Lilly and Company
43 O49
ROLE OF GI HORMONES IN THE TREATMENT SUCCESS OF BARIATRIC SURGERY
TA Lutz1,3, M Büter2,3. 1Inst. Vet. Physiol., Uni. Zurich, Switzerland; 2Clin Visc Transplant Surg, Uni. Hospital
Zurich, 3Center Integr. Human Physiol., Uni. Zurich; Switzerland.
Roux-en-Y gastric bypass (RYGB) surgery is one of the most effective anti-obesity therapies. The body weight reducing
effect of RYGB seems to depend on a variety of factors such as lower spontaneous eating, decreases in meal size and lack
of a decrease in energy expenditure (EE) that often is associated with body weight loss; mechanical restriction and caloric
malabsorption seem to play only minor roles. Further, RYGB also changes food preferences in that intake of high caloric
food items seems to be reduced (e.g., high fat and high sugar solutions or intake of a high fat versus low fat diet).
The first publication indicating that some of the changes induced by bypass surgery may be related to humoral factors was
the study published by Atkinson and colleagues (Atkinson, AJP 1982). They showed that recipient rats ate less when they
received postprandial plasma from RYGB rather than from sham rats. More recent studies elaborated in more detail that the
enhanced synthesis and release of gastrointestinal (GI) hormones like glucagon-like peptide-1 (GLP-1), peptide YY (PYY),
amylin and perhaps others seem to contribute to the eating inhibitory and weight reducing effect of RYGB. In our own
laboratory, we have shown that antagonizing the action of endogenous GLP-1 or cholecystokinin (CCK) with exendin-9 or
devazepide, respectively, increased eating in RYGB rats. The effect was seen in male and female animals. In addition, a test
meal induced a much stronger activation of the caudal brainstem (where CCK, GLP-1, PYY and amylin act to reduce
eating) in female RYGB than in sham rats; the effect was even more pronounced when ovariectomized females received
physiological estradiol replacement.
Reduction of body weight by dieting typically leads to a compensatory decrease in EE. This reaction is markedly attenuated
by RYGB; in other words, RYGB rats expend more energy than body weight matched control animals. Unlike the effect of
RYGB on eating, it is unclear whether the changes of EE are also mediated by altered release or action of GI hormones.
Together, these data indicate that RYGB surgery alters the physiology of weight control and affects both food intake and
energy expenditure; decreased eating and body weight after RYGB are not counteracted by a decrease in EE. RYGB leads
to the enhanced release of satiation hormones. The typical meal pattern with a reduction in average meal size after RYGB is
consistent with these findings; smaller meal sizes are only partly compensated by an increase in meal frequency
O50
HIGH DENSITIES OF PEPTIDE YY AND SEROTONIN CELLS IN THE COLON OF PATIRNTS
LYMPHOCYTIC COLITIS
M. El-Salhy, D. Gundersen, J. G. Hatlebakk3and T . Hausken, Section for Gastroenterology, Department of
Medicine, Stord Helse-Fonna hospital, Department of Research, Helse-Fonna, Haugesund,
section for Gastroenterology, Institute of Medicine, University of Bergen, Norway
Objective: The endocrine cell density has been reported to be increased in the colon of patients with lymphocytic
colitis, as revealed by chromogranin A. The aim of this study was to investigate which endocrine cell type is
affected in these patients.
Methods: 57 patients with LC and 27 controls were included in the study. Biopsies from the right and left colon
were obtained from both patients and controls during colonoscopy. The biopsies were immunostained by
Avidin-biotin-complex method for serotonin, PYY, PP, entroglucagon and somatostatin cells. The cell densities
were quantified by computerized image analysis.
Results: Serotonin and PYY cell densities were increased in the colon of LC patients. This increase was found
in both the right and left colon. PP-, entroglucagon- and somatostatin -immunoreactive cells were few to make a
reliable quantification.
Conclusion: It has been speculated that the high density of serotonin cells in LC patients are caused by the
interaction between the immune cells, occurring in the epithelium and submucosa in LC patients, and serotonin
cells, and that the increase in PYY density is secondary to the increase in serotonin cell density. LC and postinfectious IBS (PI-IBS) show striking similarity. The question as whether LC and PI-IBS are the same disorder
is raised.
44 P01
MOOD AND MEMORY-ASSOCIATED BEHAVIORS IN NEUROPEPTIDE Y5 KNOCKOUT MICE.
Ito, M., Dumont Y., Quirion R.
Douglas Mental Health University Institute, Dept. Psychiatry, McGill University, Montreal QC, Canada.
The Y5 receptor subtype has been recognized and often called the “feeding receptor”. More recent data let to
suggest that in addition to Y1 and Y2 subtypes, Y5 receptors can be involved in mood-related behaviors
(Morales eat al., Brain Res. 1314:194-205, 2010). The impact of lacking Y5 receptors have been investigated
with regards to feeding status, but have not been evaluated yet for mood and memory associated behaviors.
In the present study, using a battery of behavioral test to assess anxiety and depression-like paradigms, as well as
memory function, we have evaluated the potential behavioral deficits induced in mice devoid of Y5 receptors.
Those paradigms were evaluated using the open field (OF), elevated plus maze (EPM), forced swim test (FST),
social interaction test (SI), object recognition test (ORT) and Morris water maze (MWM) in Y5 knockout (KO)
mice and wild type (WT) animals. In behavioral test associated to anxiety related behaviors (OF, EPM and SI),
time spend in the central area as well as grooming and rearing events evaluated in OF were similar in Y5 KO
and WT mice. Similar results were observed for time and number of entry in open arms as compared to closed
arm in EPM. Additionally, Y5 KO mice spent similar amount of time and number of entry in the stranger
chamber and empty chamber as compared to WT animals. All together these data suggest that compare to WT.
Y5 KO mice display similar anxiety-like phenotype. In the FST, a test that is commonly used to evaluate
depression-like paradigm, the Y5 KO mice had similar immobility time as compared to WT mice, against,
suggesting that Y5 KO mice have similar phenotype as compare to WT animals with regards to depressive-like
behaviors. . Furthermore, no memory dysfunction was observed in Y5 KO mice, as compared to WT, as
addressed in the MWM and OR tests. On the other hand, male Y5 KO mice have higher body weight and
female Y5 KO mice gave birth to higher number of pups per litter. All together these data suggest that under
basal conditions Y5 receptors do not appears to play significant roles in anxiety and depression-like behaviors
and memory function. Hence, the impact of Y5 receptor on fertility and it’s role on stress induced mood
disorders remain to be investigated in more details.
P02
THE SELECTIVE NEUROPEPTIDE Y Y5 AGONIST [CPP1–7,NPY19–23,ALA31,AIB32,GLN34]HPP
DIFFERENTLY MODULATES EMOTIONAL PROCESSES AND BODY WEIGHT IN THE RAT
Morales-Medina JC, Dominguez-Lopez S, Gobbi G, Beck-Sickinger AG and Quirion R
Douglas Mental Health University Institute, McGill University, Montréal, QC, Canada, H4H 1R3
The neuropeptide Y (NPY) has been suggested to act as a major regulator of emotional processes and body
weight. The full spectrum of biological effects of this peptide is mediated by at least four classes of receptors
known as the Y1, Y2, Y4, and Y5 subtypes. However, the respective contribution of each of these receptor
subtypes, especially the Y5 subtype, in emotional processes is still mostly unknown. In the present study, we
investigated the effect of long term administration of a selective Y5 agonist [cPP1-7, NPY19-23, Ala31, Aib32,
Gln34]hPP on emotional processes and body weight using two rat models of emotional dysfunctions, the
corticosterone (CORT)-induced anxiety model as well as the olfactory bulbectomized (OBX) model of
depression and anxiety in Wistar and Sprague-Dawley rats, respectively. The administration of the Y5 agonist
reversed the high levels of locomotion, rearing and grooming in the open field test and the impaired social
activity induced by OBX, while increased the percentage of entries and time in the open arm of the elevated plus
maze in CORT-treated rats. Furthermore, this Y5 agonist increased body weight in both strains of naïve, control
rats. These data further demonstrate that Y5 receptors are not only involved in the control of body weight but
also mediate anxiolytic- and antidepressant-like effects under challenged conditions. Thus, the
pharmacotherapeutic administration of a Y5 agonist could be considered as a potentially novel strategy to
alleviate some forms of anxiety and depression in humans.
Supported by: the Canadian Institutes of Health Research (RQ) and by the German National Science Foundation
45 P03
CHRONIC ADMINISTRATION OF THE Y2 RECEPTOR ANTAGONIST JNJ-31020028 INDUCED ANTIDEPRESSANT LIKE-BEHAVIORS IN OLFACTORY BULBECTOMIZED RAT
Morales-Medina J.C.1, Dumont Y.1, Bonaventure P.2, Quirion R.1
1 Douglas Mental Health University Institute, Dept. Psychiatry, McGill University, Montreal QC, Canada. 2
Janssen Research & Development, L.L.C. San Diego, CA, USA
Recent studies from our group have shown that BIIE0246, a Y2 receptor antagonist, has antidepressant effect in
olfactory bulbectomized (OBX) rat (Morales-Medina et al., Neuropharmacology 62, 200-208, 2012). However,
this molecule has several inconvenient for useful therapeutic applications including: poor solubility, do not cross
the brain blood barrier and slow dissociation. Alternatively, the novel and brain penetrant Y2 receptor
antagonist, JNJ31020028 is opening new avenue to investigate the implication of Y2 receptors in mood
disorders. Although initial studies with this compound revealed on effects under basal conditions, a more
detailed investigation let to suggest that under challenging conditions it may have some beneficial effects on
mood associated behaviors (Shoblock et al., Psychopharmacol 208:265-277, 2010; Cippitelli et al., Alcohol,
45:567-576, 2011).
In the present study, we evaluated the effect of subchronic intracerebroventricular (ICV) administration of
JNJ31020028 in a battery of behavioral tests in an animal model that mimic several deficits observed in the
human condition, OBX rat. Chronic administration of JNJ31020028 induced a decrease of immobility time in
the forced swim test in OBX while having no effect in control animals. Additionally, it decreased number of
grooming events in OBX animals, but had no effects on some other behavioral deficits observed in this animals
such as rearing and hyperlocomotion. Furthermore, JNJ31020028 had no effect on behavior tests that are
commonly used to evaluate anxiety, namely the elevated plus maze and social interaction in both OBX and
control animals. These data further demonstrate the potential therapeutic potential of Y2 receptor antagonist in
the treatment of mood disorders.
Supported by: the Canadian Institutes of Health Research (RQ).
P04
THE ROLE OF NEUROPEPTIDE Y Y1 AND Y2 RECEPTORS IN THE LATERAL SEPTAL REGULATION
OF ANXIETY IN THREE ANIMAL MODELS
Trent, N.L., Menard, J.L. Department of Psychology and Center for Neuroscience Studies, Queen’s University,
Kingston, Ontario, Canada, K7L3N6.
Neuropeptide Y (NPY) is one of the most abundant peptides in mammalian brain and NPY binding sites are
highly expressed in the lateral septum (LS), an area extensively implicated in anxiety regulation.
Intracerebroventricular (i.c.v.) infusions of NPY are known to produce anxiolytic-like effects, but less is known
about the role of the LS in NPY-induced anxiolysis. Therefore, our goal was to characterize the contribution of
NPY and its receptor subtypes to the lateral septal regulation of anxiety using three animal models: the elevated
plus maze (EPM), novelty-induced suppression of feeding (NISF), and shock-probe burying (SPB) tests.
In our first study, infusions of NPY (1.5μg) into the LS decreased rats’ anxiety in the NISF and SPB tests, but
not the EPM (Experiment 1). Pre-treatment infusions of the Y1 antagonist BIBO3304 (0.30μg) blocked NPYinduced anxiolysis in the NISF test, but not in the SPB test (Experiment 2). In a subsequent study, we found that
infusions of the Y2 agonist NPY13-36 (0.75μg) into the LS decreased anxiety in the EPM, but not the NISF or
SPB tests (Experiment 1). Further, NPY13-36-induced anxiolysis in the elevated plus-maze test was blocked by
pre-treatment infusions of the NPY Y2 antagonist BIIE0246 (1μg; Experiment 2).
In summary, distinct NPY receptors differentially contribute to NPY-mediated anxiolysis in a test specific
manner, with the Y1 receptor mediating anxiolysis in the NISF test, and the Y2 receptor mediating anxiolysis in
the EPM. It has yet to be determined which receptor subtype is mediating NPY-induced anxiolysis observed in
the SPB test, therefore; further investigation into the role of the Y4 and Y5 subtypes is necessary.
46 P05
NPY ACCUMULATION IN DP4/CD26-DEFICIENT CONGENIC RATS.
Canneva F., Distler J., Meyer S., Dobner J. and von Hörsten S. Department of Experimental Therapy, FAU
Erlangen-Nürnberg, Erlangen, Germany 91054.
Dipeptidyl peptidase 4 (DP4/CD26) is a catalytic enzyme active on insulinotropic hormones, neuropeptides, and
chemokines, with the highest affinity for NPY, whose role as an anxiolytic peptide is well documented.
A blunted stress-responsive phenotype has already been described in congenic DP4-deficient (DP4mut) rats,
both on a Fisher and Dark Agouti (DA) genetic background.
Here we have investigated whether NPY metabolism was indeed altered in DP4mut rats.
Interstingly we found that NPY levels were upregulated (doubled) in the CSF of DP4mut rats as compared to wt
littermates, thus being nicely associated with the anxiolytic/stress-protective-like phenotype of these animals.
Additionally, in an attempt to differentiate genetic and epigenetic/environmental components of such
phenomenon, the stress-protective phenotype was investigated in animals raised by a foster mother of identical
or opposite genotype. As expected, DP4mut rats showed a blunted response to stress, in the non habituted
version of the Hot Plate test (reduced stress-induced analgesia), as well as increased levels of NPY in the CSF.
More interestingly, the behavioral display of DAwt rats raised by a DP4mut foster mother was similar to
DP4mut rats, and significantly reduced as compared to DAwt animals raised by a DAwt mother. Accordingly,
NPY levels in the CSF of these animals were found increased, similarly to DP4mut rats.
Our data indicate that DP4-deficiency, in rats, influences NPY accumulation in the CNS, thus suggesting a
possible molecular correlate for the stress-protective phenotype displayed. Moreover, we suggest that perinatal
experiences might also have long-lasting effects on NPY metabolism, possibly through modulation of DP4 or
other proteases' activity.
P06
NPY MEDIATES INJURY-INDUCED ADULT NEUROREGENERATION
Jia C., Hayoz S., Hegg CC. Pharmacology & Toxicology, Michigan State University, East Lansing, USA, 48824
NPY induces cell proliferation in the olfactory epithelium (OE), a region where adult neurogenesis occurs both
continuously and following damage. We tested the hypothesis that NPY has a role in injury-induced adult
neuroregeneration in the mouse OE. We found that NPY is predominately expressed in non-neuronal, IP3R3+
microvillous cells that are in the vicinity of progenitor cells. As damage to the OE releases ATP, we used ATP to
mimic injury. Intranasal ATP administration significantly increased NPY protein levels and number of NPY+
cells. ATP significantly increased NPY release as measured by immunoassays. Furthermore, ATP-induced NPY
release was significantly blocked by IP3 receptor inhibitor 2-APB, NPY release was evoked following uncaging
“caged IP3”, and ATP-induced NPY release was impaired in IP3R3-deficient mice. These data indicate that
injury stimulates the release of NPY in an IP3R3-dependent process. Next, we examined whether NPY release is
involved in injury-induced neuroregeneration. ATP significantly increased progenitor cell proliferation and
neuroregeneration, measured by BrdU incorporation and proliferating cell nuclear antigen expression, via p44/42
ERK activation. Intranasal instillation of NPY Y1 receptor antagonist BIBP3226 following ATP treatment
significantly inhibited p44/42 ERK activation and BrdU incorporation. This suggests that ATP indirectly
activates p44/42 ERK in the OE via ATP-induced NPY release and subsequent activation of Y1 receptors in
progenitor cells to promote proliferation and neuroregeneration. Collectively, these data indicate that injury
initiates neuroregeneration via NPY up-regulation, NPY release, and Y1 receptor mediated-p44/42 ERK
activation. Thus, the NPY system is a pharmacological target to promote regeneration of damaged neurons.
Support: NIHDC006897
47 P07
NEUROPROTECTIVE ROLE OF NPY IN RETINAL CELLS
Santos-Carvalho, A.1,2; Álvaro, A. R.3; Ambrósio, A. F.1,4; Cavadas, C.1,2
1CNC-Center for Neuroscience and Cell Biology, Univ.of Coimbra; 2Faculty of Pharmacy, Univ. of Coimbra;
3Department of Biology and Environment, Univ.of Trás-os-Montes and Alto Douro; 4Centre of Ophthalmology
and Vision Sciences, IBILI, Faculty of Medicine, Univ.of Coimbra, PORTUGAL.
It has been claimed that glutamate excitotoxicity has a role in retinal degenerative diseases. Neuropeptide Y
(NPY) has neuroprotective properties against excitotoxicity in hippocampus and MDMA-induced toxicity in the
retina. The main objective was to investigate the potential protective role of NPY against glutamate toxicity in
cultured rat retinal neurons, unraveling the NPY receptors and intracellular mechanisms involved.
Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500
µM) for 24h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death by
TUNEL and Caspase-3 assays. The NPY receptors involvement was studied using receptor selective agonists
and antagonists. The cell types were identified by immunocytochemistry.
Pre-treatment of cells with NPY (100 nM) inhibited glutamate-induced necrotic cell death (PI-positive cells) and
apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells). NPY was able to protect cultured
neurons, but had no protective effect in the integrity of the glial cells. NPY Y2, Y4 and Y5 receptors activation
inhibited necrotic cell death, but apoptotic cell death triggered by glutamate was only prevented by the activation
of Y5 receptor. The neuroprotective effect of NPY against glutamate cell death was inhibited by H89 and
SB258035, inhibitors of protein kinase A and p38K respectively.
NPY receptors might be potential neuroprotective targets in retinal degenerative diseases.
Supported by FCT (PTDC/SAU-NEU/73119/2006 & SFRH/BD/45311/2008)
P08
POLYMORPHISMS IN NEUROPEPTIDE Y SIGNALLING GENE ARE ASSOCIATED WITH THE RISK
OF MYOCARDIAL INFARCTION IN PATIENTS WITH ATHEROSCLEROSIS
Nima Aghili, Joseph M. Devaney, Mingyao Li, Muredach Reilly, Daniel Rader, Zofia Zukowska, Mary Susan
Burnett, Stephen Epstein, Medstar Health Research Institute, Washington DC, DC, USA
Background: Dipeptidyl peptidase IV (DPP-IV), enzyme that cleaves neuropeptide Y, has proinflammatory
actions that can contribute to atherosclerosis progression. Previously we showed that DPP-IV may also lead to
atherosclerotic plaque vulnerability in part by promoting neoangiogenesis within the plaque. These
characteristics of DPP-IV may predispose patients with coronary artery disease (CAD) to plaque rupture and
thus to myocardial infarction. The goal of this investigation was to determine whether genetic alterations in
DPP-IV predispose to plaque vulnerability and myocardial infarction (MI).
Methods: Between Aug 1, 2004, and March 31, 2007, blood samples of patients with angiographically
documented CAD were banked at Washington Hospital Center. 875 patients of European ancestry with
angiographic CAD were divided into those with MI (n=421) and those without (n=454).
A genome-wide association study was performed using the Affymetrix 6.0 chip to identify loci that predispose to
MI. In the current study we assessed the association of single nucleotide polymorphisms (SNPs) in the DPP-IV
gene region and risk of MI in patients with CAD. For genotyped SNPs, association was tested by logistic
regression with the assumption of additive genetic effects and the significance level of 0.05.
Results: Average patients’ age at diagnosis of CAD was 46.8 years for MI group and, by design, was higher
(50.8) in the non MI group. We tested the association of DPP-IV SNPs with risk of MI in patients with CAD.
From 25 SNPs in the DPP-IV and promoter region, we identified 6 SNPs that were significantly related to
angiographic CAD with MI, but not to angiographic CAD without MI (odds ratio: 1.22-1.34, p<0.05). The
association of Identified SNPs to the risk of MI was not attenuated after adjustment for traditional risk factors.
Conclusions: Polymorphisms in dipeptidyl peptidase IV gene, in patients with known CAD, may increase the
risk of MI.
48 P09
PRENATAL STRESS SUPPRESSES NEUROPEPTIDE Y LEVELS IN ADULT OFFSPRING
Nima Aghili, Roberta Lassances-Soares, Subeena Sood, Lee O Alderman, Amir Najafi, Hajra Nashin, Sunny S
Jhamnani, Lijun Li, Zofia Zukowska, Stephen E Epstein, Mary Susan Burnett. Medstar Health Research
Institute, Washington DC, USA
Background: There is strong evidence that early life environment may alter the hypothalamic-pituitary-adrenal
axis and sympathetic nervous system. Neuropeptide Y (NPY), a sympathetic neurotransmitter also present in
endothelial, immune cells and megakaryocytes/platelets is increased in response to psychosocial and physical
stressors and associated with atherosclerosis. Here we examine the effect of prenatal stress on NPY levels in a
murine model of atherosclerosis.
Methods: Female ApoE -/- mice on a lard-containing diet were divided into stressed (n=12) and non-stressed
(n=13) cages. Animals in the stressed group were subjected to cold stress for the last 17 days of gestation. At 16
weeks, both prenatally stressed (PNS, n=40) and non-prenatally stressed (NPNS, n=40) offspring were divided
into adult stress (AS, n=40) and non adult stress (NAS, n=40) groups. AS mice were stressed for 4 weeks. Urine
corticosteroid and platelet-rich and platelet poor plasma (PRP and PPP) NPY-ir levels were assessed.
Expression levels of NPY were measured in unfractionated bone marrow and peripheral blood mononuclear cells
(PBMCs) using real time PCR.
Results: At 6 weeks, urine corticosterone was lower in PNS mice (males, p= 0.006, females, p= 0.001). At 20
weeks, PRP NPY was lower in the PNS mice (p<0.001) while the NPY level was not different in PPP. Among
mice with AS, previous PNS further blunted the PRP NPY level (p=0.003). NPY gene expression was lower in
PBMCs and bone marrow of PNS mice (PBMCs, p=0.04; bone marrow, PNS, p=0.01.)
Conclusion: In this atherosclerotic mouse model, prenatal stress leads to a decrease in PBMC and bone marrowderived as well as platelet NPY, as measured in PRP. The latter is more evident when the prenatally stressed
mice are subject to adult stress. The influence of these changes in the predisposition to atherosclerosis and in
production of vulnerable plaque-like phenotype awaits further study.
P10
PROTEOME ANALYSIS OF HUMAN Y1-RECEPTOR EXPRESSING BREAST CANCER CELLS
Kostelnik K.†, Baumann S. ‡, Mörl K. †, von Bergen M. ‡, Beck-Sickinger A.G. †
† Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany
‡ Department of Metabolomics, Helmholtz Centre for Environmental Research-UFZ, 04318 Leipzig, Germany
Breast cancer is one of the most frequently diagnosed cancers in women with an estimated number of deaths of
39,510 in the US for 2012 [1]. In 2001, Reubi et al. observed a switch of the neuropeptide Y (NPY) receptor
subtype expression from Y2 to Y1 during breast neoplasm [2]. This discovery enabled the targeting of tumor and
metastasis formation by using labeled NPY-derived Y1-receptor ligands [3]. However, it is still unknown
whether this shift of the receptor subtype plays a functional role in tumor development or if it is just a side effect.
To understand the effects of NPY on the breast tumor on a system-wide scale the proteomic-based technique
SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) followed by tandem mass spectrometry
(MS/MS) has been applied. Human breast carcinoma-derived cell lines were used as an in vitro system to
investigate NPY-mediated changes in the proteome and therefore conclude possible cell signaling regulations.
The labeling of the amino acids lysine and arginine in breast cancer cells with the 13C-isotope and subsequent
treatment with NPY allowed the quantitative analysis of differences at the protein level in comparison to a nonlabeled, non-treated control. In the SILAC experiment specific breast cancer-related proteins indicated an
expression regulation after NPY-treatment. Hence, these proteins have been further investigated by using
antibody-based techniques.
[1] Cancer Facts & Figures 2012, American Cancer Society®. [2] Reubi et al. (2001) Cancer Research 61, 46364641. [3] Khan and Zwanziger et al. (2010) Angew. Chem. Int. Ed. 49, 1155-1158.
49 P11
CARBABORANE CONTAINING NEUROPEPTIDE Y ANALOGS FOR BREAST CANCER THERAPY
Ahrens, V.M. #, Frank, R. ‡, Hey-Hawkins, E. ‡, Beck-Sickinger, A. G. #
#Universität Leipzig, Institute of Biochemistry, Germany
‡Universität Leipzig, Institute of Inorganic Chemistry, Germany
The human Y1-receptor subtype is over-expressed in more than 90% of breast cancer patients and in 100% of
breast cancer derived metastases, whereas healthy breast tissue expresses receptors of the Y2 type. Peptides
which selectively bind to and activate receptors over-expressed in the membrane of cancer cells are a promising
tool for tumor diagnosis and therapy. [F7, P34]-NPY displays high affinity to the human Y1-receptor subtype; in
contrast, [Ahx(5-24)]-NPY specifically binds at human Y2-receptors. These peptides allow selective targeting of
only one Y-receptor subtype while the other remains unaffected.
Boron Neutron Capture Therapy (BNCT) is a binary therapy approach making use of nontoxic 10B, which is
able to capture nontoxic thermal neutrons. The neutron capture results in an excited state 11B. 11B decomposes
to form highly cytotoxic 4He particles (alpha particles) with a short radiation range of ca. 10 µm inside the cell.
Using these effects, BNCT can be applied in tumor therapy.
We describe the combination of both therapeutic approaches. Ortho-carbaboranyl propionic acid was
synthesized and linked to the ε-amino group of Nα-Fmoc protected L-lysine. The carbaborane-modified amino
acid was incorporated into NPY, [F7, P34]-NPY, and [Ahx(5-24)]-NPY. Binding studies and IP accumulation
assays confirmed nanomolar affinity and activity of the modified analogs despite of the large carbaborane
cluster. Internalization studies revealed excellent and receptor subtype specific uptake of the conjugates into
respective cells [1]. First boron uptake studies showed promising uptake of carbaborane-peptides into Y1receptor expressing cells.
[1] Ahrens et al. (2011) J. Med. Chem. 54(7):236
P12
POSTENDOCYTIC FATE OF THE HUMAN Y2-RECEPTOR-LIGAND-SYSTEM
Babilon S., Ahrens V., Mörl K., Beck-Sickinger A.G. Institute of Biochemistry, Universität Leipzig,
Brüderstraße 34, 04103 Leipzig, Germany.
Ligand-induced endocytosis and subcellular trafficking is known to contribute to the physiological regulation of
G protein-coupled receptors (GPCR). Consequentially, detailed knowledge about receptor desensitization and
resensitization processes tremendously enlarges the scope of clinical intervention when GPCRs are used as drug
targets. The human neuropeptide Y2 receptor (hY2R) was found to be a promising target to treat distinct types of
tumors, as well as obesity, epilepsy, and ischemic diseases [1]. While it is known that the hY2R rapidly
internalizes upon agonist stimulation in an arrestin3-dependent manner [2], not much is known about the fate of
this receptor-ligand-system subsequent to endocytosis.
To investigate the postendocytic pathways of the hY2R a stable HEK293 cell line was established for in vitro
studies, expressing the hY2R fused to the enhanced yellow fluorescent protein (EYFP) on its carboxy terminus.
By stimulating the receptor with carboxytetramethylrhodamine-modified neuropeptide Y (NPY), subcellular
trafficking was studied using live cell fluorescence microscopy. Those studies revealed that receptor and ligand
do co-internalize and can be found in the same intracellular compartment immediately after endocytosis.
Additional experiments comprised co-localization studies with different subcellular markers to further
characterize trafficking pathways of the receptor-ligand system. Moreover, receptor recycling and degradation
assays were performed, allowing quantitative studies of the hY2R fate.
In conclusion, new insights into the postendocytic hY2R-NPY-system could be obtained which augment the
scope of the hY2R in medical application.
[1]
Walther, C. et al. J Pep Sci (2011) [2]
Walther, C. et al. J Biol Chem (2010)
50 P13
SWITCHING Y-RECEPTOR SELECTIVITY OF REDUCED-SIZE NPY ANALOGS
Hofmann, S.#, Frank, R.‡, Hey-Hawkins, E.‡, Beck-Sickinger, A. G.#
#Universität Leipzig, Institute of Biochemistry, Brüderstr. 34, 04103 Leipzig, Germany
‡Universität Leipzig, Institute of Inorganic Chemistry, Johannisallee 29, 04103 Leipzig, Germany
Neuropeptide Y (NPY) is known to be the naturally potent agonist of the neuropeptide Y receptor 1, 2 and 5
(Y1R, Y2R, Y5R) whereas the pancreatic polypeptide (PP) mainly stimulates the neuropeptide 4 receptor (Y4R).
Apart from the Y receptor body distribution and their physiological functions, especially the Y1R and the Y4R
were shown to be promising pharmaceutical targets. The Y1R is particularly over-expressed on primary and
disseminated mammacarcinoma cells and can be utilized as peptide shuttle to selectively address breast cancer tissue for both
diagnosis and therapy. The Y4R plays a crucial role in food intake and is a key player in obesity signaling.
Despite their distinct physiological and pathophysiological functions, both receptors show a similar ligandbinding mode which exacerbates the development of highly selective NPY analogs.
Due to increased synthesis costs and metabolic stability problems, reduced-size NPY analogs with modified
amino acid residues are of great interest. Based on the previously developed first Y1R selective NPY agonist
[Pro30,Nle31,Bpa32,Leu34]-NPY (28-36) we could now discover a key to selectively switch between Y1R- and
Y4R-activity. Herein, the single incorporation of either an acyl chain or an ortho-carbaboranyl-Nε-modified Llysine
(K-Nε(Cpa)) on a crucial position could be shown to selectively modulate the activation potency.
By performing receptor activation studies with stably transfected Y receptor-positive COS-7 cells, we could
confirm the desired selectivity switch between Y1R and Y4R. EC50 values in range of the
[Pro30,Nle31,Bpa32,Leu34]-NPY
(28-36) lead structure could be determined.
P14
IMPROVED PANCREATIC POLYPEPTIDE-BASED ANALOGS FOR OBESITY TREATMENT
Mäde V., Bellmann-Sickert K., Beck-Sickinger A. G. Institute of Biochemistry, Faculty of Biosciences,
Pharmacy and Psychology, Universität Leipzig, Brüderstr. 34, 04103 Leipzig, Germany.
Pancreatic polypeptide (PP) is an appetite-inhibiting peptide hormone [1] exerting its effects via the G proteincoupled Y4-receptor. Thus, appetite as a fundamental cause of obesity could be regulated by mimics of this
satiety-inducing hormone [2].
Peptide therapeutics exhibit a lot of advantages like high selectivity, non-toxic degradation and predictable in
vivo-behavior [3]. The application of peptides as drugs, however, is limited by fast proteolytic degradation and
body clearance. This issue can be addressed by peptide engineering such as conjugation to polyethylene glycol
(PEG) or lipids [4,5].
In this work, we designed analogs of PP based on distinct core peptides [6], which are modified by either
PEGylation or lipidization. Peptides were synthesized by solid-phase peptide synthesis (SPPS) and analyzed by
reversed-phase HPLC and MALDI-mass spectrometry. Conformational analysis by circular dichroism
spectroscopy revealed no crucial disturbances by the modifications. Signal transduction and binding experiments
were performed for evaluating compound functionality and affinity. Moreover, in vitro stability tests of the most
potent peptide conjugates were carried out in order to investigate their bioavailability.
In conclusion, we designed promising, chemically stabilized conjugates based on human PP which might be
potential anti-obesity drugs.
[1] R. L. Batterham et al., J Clin Endocrinol Metab 2003, 88:3989-3992. [2] M. S. Huda et al., Obes Rev 2006,
7:163-182. [3] P. Vlieghe et al., Drug Discov Today 2010, 15:40-56. [4] K. Bellmann-Sickert et al., Trends
Pharm Sci 2010, 31:434-441. [5] K. Bellmann-Sickert et al., J Med Chem 2011, 54:2658-67.
[6] T. W. Schwartz, 2005, PCT/EP2005/002982.
51 P15
LONG CHAIN LIPIDATION OF Y4 RECEPTOR AGONISTS PROLONGS THEIR ACTIVITY
Jolly N., Mäde V., Elling C., Little P., Beck-Sickinger A., Schwartz T. & Cox H.
Wolfson CARD, King’s College London, UK.
The potential of pancreatic polypeptide (PP) as an anti-obesity agent is limited by its short half-life and so
development of longer-acting analogues is essential. We have developed lipidated Y4 agonists to gain increased
stability and longevity of response compared to an existing Y4 agonist (TM30339). Here we investigated the
anti-secretory effects of Y4 agonism on human (h) colonic epithelial (Col-24) monolayers that constitutively
express the Y4 receptor (Cox et al., 2001, BJP 132, 345 -353), which were mounted in Ussing chambers and
changes in short-circuit current (Isc) recorded.
Responses to single additions of TM30339 and hPP were transient in Col-24; reducing Isc with a peak response
within 10 min. Modification of TM30339 with a short chain fatty acid did not alter the longevity of the Isc
response. In contrast, the addition of a long chain fatty acid (E-PAM) resulted in a prolonged response, with
peak responses achieved at 45 minutes. Importantly, the potency of these peptides was not altered after chemical
modification, indicated by equipotent EC50 values. Basolateral addition of maximally effective concentrations
of hPP significantly attenuated the response to a second application of hPP in a concentration-dependent manner.
Interestingly, pre-treatment with low and high concentrations of the E-PAM modified peptide significantly
diminished all subsequent Y4 signals, suggesting rapid and sustained internalisation of the receptor.
In summary, our data provides the first functional evidence that long chain lipidated Y4 receptor agonists have
prolonged effects in comparison to their non-lipidated predecessors, and have the potential to act as anti-obesity
treatments.
Supported by the EU 7th Framework Programme, Grant Agreement: No. 223057 (GIPIO)
P16
FROM CHIMERIC RECEPTORS TO SINGLE RESIDUES: CHARACTERISATION OF NEUROPEPTIDE Y
RECEPTOR SELECTIVITY
Pedragosa Badia X., Stichel J., Beck-Sickinger A. G.
Institute of Biochemistry. Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, D-04103
Leipzig, Germany.
The NPY system is a multireceptor/multiligand system that consists of four GPCRs in humans: Y1R, Y2R, Y4R
and Y5R. This family of receptors is activated by three different agonists (pancreatic polypeptide (PP),
neuropeptide Y (NPY) and peptide YY (PYY)) with different potencies for each receptor. This family of
receptors and peptides plays a role in several physiological processes such as memory retention, regulation of
hunger and satiety, which makes the system interesting for pharmacological studies.
One of the reported receptor-ligand interactions involves the position Asp6.59. This amino acid forms a subtype
selective ionic interaction with an arginine on the peptide side. On Y2R/Y5R, Asp6.59 interacts with Arg33,
whereas on Y1R/Y4R, Asp6.59 interacts with Arg35 of the peptide.
In order to clarify the evolution of receptor-ligand interactions, we used a chimeric approach combining
segments of Y1R and Y4R. These two receptors share a high sequence homology which makes them suitable for
the design of chimeric receptors. We expressed chimeric YFP fused receptors in HEK-293 cells and tested
expression by fluorescence microscopy. We also tested affinity by saturation binding and signal transduction
assays. By using this approach, we could elucidate that TM2 plays a major role in the activation of the receptor
by NPY. By combining TM2 with the 3rd extracellular loop, we could slightly improve the activation of the
receptor with NPY. In a subsequent mutagenesis approach, we could show three residues that play a major role
in the Y4R.
52 P17
INVESTIGATION OF THE HUMAN Y2 RECEPTOR LIGAND-BINDING POCKET
Pruner J.1), Xu B.1), Fällmar H., Boukharta L.2), Keränen H.2), Gutiérrez-de-Terán H.3), Lundell I.1), Mohell
N.1), Åqvist J.2), Larhammar D.1) 1) Department of Neuroscience and 2) Department of Cell and Molecular
Biology, Uppsala University, Uppsala, Sweden, and 3) Fundación Pública Galega de Medicina Xenómica,
Hospital Clínico Universitario de Santiago, Santiago de Compostela, Spain.
The NPY-family receptor subtypes Y1 and Y2 arose from a common ancestor in early vertebrate evolution
approximately 500 million years ago. Today they display only about 30% overall sequence identity, yet bind the
two peptides NPY and PYY with similar affinities. The receptors have evolved interesting differences regarding
their interactions with NPY and PYY. Deepened knowledge about these differences will facilitate design of
subtype-selective compounds for pharmacotherapy.
We have docked the conserved carboxyterminal tetrapeptide RQRY-amide to a 3D model of human Y2 based on
the crystal structure of the human CXCR4 receptor as well as our previous mutagenesis studies of Y2. The
RQRY-amide-Y2 complex is now being validated by mutagenesis of residues that were predicted to have or not
to have a role in binding. Preliminary tests of E5.24L resulted in more than 10-fold reduced affinity of PYY3-36,
thereby indicating involvement of the extracellular loop 2 (EL2) in ligand binding. EL2 has been postulated to be
involved in ligand recognition of other members of the rhodopsin superfamily of G protein-coupled receptors.
Furthermore, we are presently exploring the role of several other positions in the binding pocket. Mutation
T2.61I abolished binding and F7.35A resulted in a minimum of 10-fold affinity loss. This result is consistent
with the model since the side chain of F7.35 appeared to be close to the hydrophobic R35 in the peptide ligand.
P3.29A resulted in about six to seven fold affinity loss compared to wild-type Y2. To validate the model, we
have pharmacologically characterized E2.69A and R4.64A and experimentally confirmed that they are not
involved in the binding, as predicted in our model. These results bring us further towards modeling of the entire
peptide.
Supported by: The Swedish Research Council.
P18
MODELING, DOCKING AND MUTAGENESIS OF THE HUMAN NPY/PYY RECEPTOR Y2 IDENTIFIES
RESIDUES INVOLVED IN PEPTIDE BINDING AND RECEPTOR STABILITY
Xu B.1, Fällmar H.1, Boukharta L.2, Gutiérrez-de-Terán H.3 , Pruner J.1, Lundell I.1, Mohell N.1, Åqvist J.2,
Larhammar D.1 1) Dept of Neuroscience and 2) Dept of Cell and Molecular Biology, Uppsala University,
Uppsala, Sweden. 3) Fundación Pública Galega de Medicina Xenómica, Hospital Clínico Universitario de
Santiago, Santiago de Compostela, Spain
We have docked both full human NPY (hNPY) and the conserved carboxy-terminal dipeptide RY-amide of
hNPY to a 3D model of human Y2 based on the crystal structure the human adenosine A2A receptor. hNPY-Y2
and RY-amide-Y2 complexes obtained by docking guided mutagenesis of six residues. The residues T2.61,
Q3.32 and H7.39 seem to form a hydrogen bond network, possibly involved in ligand binding and receptor
stability. The residues Y3.30, Y5.38 and L6.51 are possibly involved in peptide binding by forming a
hydrophobic binding pocket. Nine mutants were generated for T2.61, Q3.32 and H7.39 including one reciprocal
exchange mutant of Q3.32 and H7.39. Eight mutants were generated for Y3.30, Y5.38 and L6.51 including three
double mutants. Binding studies were performed using the radioligand 125I-pPYY and two Y2 specific ligands,
peptide hPYY3-36 and the antagonist BIIE0246. Binding data indicate that all mutated positions apart from
Y3.30 are involved in peptide binding. The binding data also showed the differences between peptide and
antagonist binding. 125I-pPYY lost binding to five of eight mutants generated from Q3.32 and H7.39, cellular
expression showed that these mutations affected the expression level or the cell membrane expression, which
suggests that Q3.32 and H7.39 also contribute to receptor stability. For the remaining three mutants and T2.61A
mutant, a functional inositol phosphate (IP) assay was performed by co-transfecting the mutants with a chimeric
G protein. We found that the change in potency of hPYY3-36 for these mutants corresponds to the change in
affinity. The binding results are in good agreement with the modeling and docking based on the CxC chemokine
receptor type 4 (CxCR4) which thereby adds further support for our human Y2 model.
Supported by: The Swedish Research Council (Sweden) and SAF2001-30104 (MICINN, Spain). 53 P19
ALTERED NPY SYSTEM ACTIVITY THROUGHOUT BONE REGENERATION
Alves C.J., Ribas J., Neto E., Ferreira S., Summavielle T., Lamghari M.
INEB- Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal
Nervous system is known to play an important regulatory role on bone metabolism. One of the neural arms now
accepted to be involved in bone remodeling is the NPY neuronal pathway, however, its relevance in bone
regeneration after injury requires further understanding. In this line of thought, this study aimed to analyze the
profile of NPY system (NPY, Y1R and Y2R) within the bone microenvironment throughout bone repair, and to
determine whether the feedback loop between bone and the nervous system targets central and peripheral NPY
neuronal pathway. C57/BL6 mice were submitted to bilateral femoral non-critical bone defect and 3 assessment
time points were defined: 1, 3 and 7 post-surgery days. At these time points, femurs, dorsal root ganglia (DRG)
and hypothalamic region were collected. Sham-operated and non-operated mice were used as controls. The data
showed increased NPY expression levels in bone tissue of femur-injured mice that was limited to the early
stages of bone repair, the Y1R expression also increased, but not in so early stages. The analysis of bone reinnervation process (GAP43) showed an inverse correlation trend with bone NPY expression. The NPY central
response revealed an increase of NPY and Y2R in the hypothalamus at the initial phases of bone repair with an
unchanged expression of Y1R, while in the DRGs the Y1R expression decreased throughout time and Y2R
levels remained unaffected. Overall, these results indicate that NPY system was targeted during the early steps of
bone regeneration, suggesting a possible role in bone repair. Furthermore, it might be linked to processes known
to occur in the first stages of bone regeneration, such as inflammation and revascularization.
P20
NPY CLEAVED BY DIPEPTIDYL-PEPTIDASE IV (DPPIV) INDUCES PRE-ADIPOCYTES
DIFFERENTIATION
Joana Rosmaninho-Salgado, Vera Cortez, Maria Alexandra Gonçalves, Magda Santana, Patrícia Marques, Marta
Estrada, Cláudia Cavadas
CNC- Center for Neurosciences and Cell Biology; Faculty of Pharmacy, Univ. Coimbra, Portugal
Adipocytes proliferation and differentiation control adipose tissue homeostasis and are associated with endocrine
and structural dysfunctions of adipose tissue in obesity. NPY is present on adipose tissue and it iscleaved by the
dipeptidyl-peptidase IV (rDPPIV) producing the Y2 and Y5 receptor agonist. Moreover, the NPY receptores
involved in adipogenesis induced by NPY is still controversial. The present work has two main aims: 1) to study
the role of NPY and its cleavage by DPPIV on adipogeneis; and 2) to study the intracellular pathways involved
in adipogenesis induced by NPY. It was used a murine preadipocyte cell line, 3T3-L1. Adipogenesis was
evaluated by quantifying lipid accumulation using the Oil red-O assay and also by analysing PPAR-γ expression
using the Western blotting. Our results show that DPPIV (25 ng/ml) induced an increase on lipid accumulation
and on PPAR-γ expression that was inhibited in the presence of the antibody anti-NPY (NPY 05, 6 μg/ml).In the
presence of NPY Y2 receptor antagonist (BIIE0246, 1 μM) or NPY Y5 antagonist (L-152,804, 1 μM) a decrease
on lipid accumulation and PPAR-γ expression was observed. Furthermore, NPY (100 nM), NPY Y2 (NPY3-36,
100 nM) or NPY Y5 (NPY19-23(GLY1, Ser3, Gln4, Thr6, Ala31, Aib32, Gln34)PP, 100 nM) agonists induced
an increase on adipogenesis. Furthermore we obserced that that MAPK, PKA, PKC and PI3K were involved on
adipogenesis induced by NPY. In conclusion, NPY cleaved by DPPIV induces NPY Y2 and NPY Y5 receptor
activation being responsible for adipogenesis.
This work was supported by FCT (SFRH/BPD/31547/2006, SFRH/BD/44664/2008, PTDC/SAUFCF/102415/2008),
54 P21
CENTRAL ROLE OF NPY IN THE REGULATION OF SYMPATHETIC TONE AND ENERGY
EXPENDITURE
Shu Lin, Yanchuan Shi, Lesley Castillo, Jackie Lau, A. Sainsbury and Herbert Herzog
Neuroscience Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst,
Sydney, NSW 2010, Australia
Neuropepetide Y (NPY) is best known for its powerful stimulation of food intake. However, long term elevation
of hypothalamic NPY also results in decreased energy expenditure, but the mechanism behind this is unclear.
We demonstrate that arcuate nucelus specific re-introduction of NPY into otherwise NPY deficient NPY-/- adult
mice results in accelerated body weight gain associated with hyperphagia and increased adiposity. Importantly,
enhanced adiposity is accompanied by marked reduction in energy expenditure due to decreased oxygen
consumption, physical activity and body temperature. The mechanism behind this control is decreased
sympathetic nervous system activity, as evidenced by decreased tyrosine hydroxylase (TH) expression in key
nuclei of the brain, decreased serum catecholamine levels and downregulated ß3 adrenergic receptor expression
in white adipose tissue (WAT) and brown adipose tissue (BAT). Similarly, arcuate only NPY overexpression
also decreases thermogenesis by downregulating UCP1 and PGC1α in the BAT and/or muscle, two well-known
sympathetic-innervated tissues, via signalling through Y1 receptors. Taken together, our data demonstrate a
potent inhibitory role of hypothalamic NPY on energy expenditure by interacting with the sympathetic nervous
system, thereby reducing thermogenesis and lipid oxidation. This also highlights the critical central regulation of
peripheral fat accretion by the NPY system.
P22
LIPID METABOLISM IN SKELETAL MUSCLE IS CONTROLLED BY ARCUATE NEUROPEPTIDE Y
Yan-Chuan Shi, Shu Lin, Lesley Castillo, Hui Zhang, Jackie Lau, Amanda Sainsbury & Herbert Herzog
Neuroscience Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst,
Sydney, NSW 2010, Australia
Hypothalamic neuropeptide Y (NPY) is best known for its strong stimulatory effect on appetite, but little is
know about its influence on peripheral metabolism. Here we show that arcuate nucleus (Arc) specific reintroduction of NPY into otherwise NPY deficient (NPY-/-) adult mice (ArcNPY) markedly impairs glucose
tolerance without altering serum insulin levels. In order to investigate the underlying mechanisms for this, we
focused on factors influencing glucose uptake and utilization in skeletal muscle. Compared to the effect of
overproducing NPY in the Arc of wild type (WT) mice (WT-NPY), Arc only re-introduction of NPY in NPY-/mice significantly increased muscle triglyceride content and acetyl-CoA carboxylase (ACC) expression level,
suggesting enhanced muscle lipogenesis. Moreover, decreased expression of carnitine palmitoyltransferase-1
(CPT-1) and mitochondrial respiratory chain complexes in the muscle of ArcNPY versus WT-NPY mice
indicates decreased capacity for transporting fatty acids into mitochondria for oxidation as well as reduced
mitochondrial oxidative capacity. Consistent with this, mitochondrial biogenesis appeared to be reduced in the
muscle of ArcNPY mice, as evidenced by downregulation of muscle PGC1α expression. Taken together, these
data demonstrate that in addition to its well known role in stimulating food intake, Arc NPY regulates glucose
homeostasis, at least partially via effects on muscle lipogenesis and lipid oxidation, thereby decreasing insulin
sensitivity in the muscle when chronically elevated.
55 P23
Y1 AND Y5 RECEPTORS ARE BOTH CRITICAL FOR THE REGULATION OF FOOD INTAKE AND
ENERGY HOMEOSTASIS IN MICE
Nguyen AD, Mitchell NF, Lin S, Macia L, Yulyaningsih E, Baldock PA, Enriquez RF, Zhang L, Shi YC,
Zolotukhin S, Herzog H and Sainsbury A. Neuroscience Research Program, Garvan Institute of Medical
Research, Sydney, Australia.
Neuropeptide Y (NPY) acting in the hypothalamus is one of the most powerful orexigenic agents known. Of the
five known Y receptors, hypothalamic Y1 and Y5 have been most strongly implicated in mediating hyperphagic
effects of NPY. However, knockout of individual Y1 or Y5 receptors induces late onset obesity and Y5 receptor
knockout also induces hyperphagia. These findings suggest redundancies between the functions of Y1 and Y5.
Thus, in order to investigate the role of Y1 and Y5 receptors in the control of food intake and energy
homeostasis, we generated germline and adult-onset hypothalamus-specific Y1Y5 receptor double knockout
mice. This study for the first time conclusively demonstrates that food intake in mice requires the co-ordinated
action of both the Y1 and Y5 receptors, since the simultaneous ablation of both receptors in germline Y1Y5
receptor double knockout (Y1Y5-/-) mice leads to reductions in spontaneous and fasting-induced food intake as
well as corresponding delays in weight regain after fasting. This hypophagic effect is at least partially mediated
in the hypothalamus, as mice with adult-onset hypothalamus-specific Y1Y5 receptor dual ablation also exhibited
reduced spontaneous and fasting-induced food intake, especially when placed on a high fat diet. Despite
hypophagia, Y1Y5 deficient mice exhibited increased body weight and/or increased adiposity, possibly due to
compensatory responses to gene deletion such as the decrease in energy expenditure observed in Y1Y5-/- mice.
Taken together, these data reveal the critical role of the Y1 and Y5 receptors in the physiological regulation of
food intake in mice.
Supported by: NHMRC
P24
NORADRENERGIC NEURON-ONLY EXPRESSION OF NPY EXACERBATS STRESS-INDUCED FAT
GAIN IN CHOW-FED MICE
Lei Zhang, I-Chieh J Lee, Rondaldo F Enriquez, Shu Lin, Paul Baldock, Errika Savontaus, Herbert Herzog
Neuroscience Research Program, Garvan Institute of Medical Research, 384 Victoria ST, Darlinghurst NSW
2010 Australia
Neuropeptide Y (NPY) is a major regulator of energy homeostasis that is strongly influenced by stress.
Additionally, NPY regulates stress responses mainly via the sympathetic nervous system (SNS). To investigate
the specific role of NPY in the noradrenergic neurons in the regulation of energy homeostasis, we generated a
transgenic mouse model that expresses NPY selectively only in the SNS and brain noradrenergic system
utilizing the dopamin-β-hydroxylase gene promotor to drive NPY expression in otherwise NPY deficient mice
(symNPY). We examined body composition and energy metabolism in chow-fed symNPY and control mice.
Mice underwent either minimal handling (non-stress) or a chronic stress protocol invovling 1-hr cold-stress 3
times per week for 10 weeks. At non-stressful condition, symNPY mice were not different from WT regarding to
adiposity or energy metabolism. Interestingly, 10-week chronic cold-stress led to a trend to increasing adiposity
in WT, that was more pronounced and significant in symNPY mice. A decreased lipid oxidation and/or increased
lipogenesis is likely an important contributor to the greater increase in adiposity by stress in symNPY compared
to WT, since symNPY but not WT showed an increased respiratory exchange ratio during the dark phase in
response to chronic cold- stress. Food intake, energy expenditure or physical activity were not significantly
altered by stress in symNPY or WT mice. Taken together, these results demonstrate that NPY expressed
specifically in the noradrenergic neurons has significant interaction with stress and plays a critical role in
regulating adiposity and lipid metabolism in stressful conditions.
56 P25
INVOLVEMENT OF PERIPHERAL Y1 AND Y2 RECEPTORS IN THE METABOLIC PHENOTYPE OF
TRANSGENIC MICE OVEREXPRESSING NPY IN THE SYMPATHETIC NERVOUS SYSTEM
Vähätalo LH, Ailanen L, Ruohonen ST, Savontaus E. Department of Pharmacology, Drug Development and
Therapeutics, FI-20014 University of Turku, Finland
We have previously constructed a transgenic mouse model overexpressing NPY (OE-NPY) in the sympathetic
nervous system and brain noradrenergic neurons. Homozygous OE-NPY show increased body weight, adiposity,
impaired glucose tolerance, insulin sensitivity and hepatosteatosis without changes in food intake or energy
expenditure. We hypothesized that the metabolic phenotype in OE-NPY mice is caused by increased NPY
release from the sympathetic nerves activating peripheral Y1 and Y2 receptors, and aimed to test if chronic Y1
or Y2 antagonist treatment attenuated the development of the metabolic syndrome in these mice.
20-week-old male OE-NPY and WT mice (n=11-13/group) were administered daily for 31 days with Y1 (BIBO3304, 1 mg/kg, ip), Y2 (BIIE-0246, 1,3 mg/kg, ip) receptor antagonist or vehicle. Body composition was
measured with quantitative NMR (Echo-MRI700) at days 0, 14 and 31. Body weight was measured twice and
food intake of group-housed mice once a week. Liver, white and brown adipose tissue mass, blood glucose,
serum free fatty acid (FFA) and insulin levels were measured after sacrifice.
Y2 antagonist significantly decreased fat gain (fat-%) in OE-NPY and WT mice during the first 14 days. From
that point on fat gain however increased in antagonist-treated groups and no difference in fat mass between Y2
antagonist and vehicle treated groups was observed in the end. Y1 receptor antagonist had no significant effect
on fat-%. Body weight, serum insulin, white adipose tissue, liver or interscapular brown adipose tissue weight
were not changed between treatment groups. However, significant difference between genotypes was maintained
in these parameters in all treatment groups. Blood glucose or serum FFAs did not differ between genotypes or
treatments.
In conclusion, the metabolic phenotype of OE-NPY mice was not attenuated by peripheral Y1 and Y2 receptor
antagonists. However, initial fat reduction followed by fat increse in Y2 antagonist treated mice revealed a
possible activation of compensatory mechanisms as a response to chronic antagonist treatment. Thus it cannot be
concluded that peripheral Y1 and Y2 receptors are not involved in the metabolic phenotype of OE-NPY mice.
P26
HEPATIC FATTY ACID AND CHOLESTEROL METABOLISM IN NPY OVEREXPRESSING MICE
Ailanen L, Vähätalo L, Ruohonen ST, Gylling H, Miettinen TA and Savontaus E. Department of Pharmacology,
Drug Development and Therapeutics, University of Turku, Turku, Finland.
Our group has previously published a mouse model overexpressing neuropeptide Y (NPY) in noradrenergic
neurons (OE-NPY). The transgenic mice show symptoms of the metabolic syndrome (MS), including
hepatosteatosis and late-on-set hypercholesterolemia, which were studied in this work. Gene expression of
hepatic fatty acid (FA) and cholesterol metabolism were studied by microarray and quantitative real-time PCR
(qPCR) analysis, and serum cholesterol metabolism and absorption markers by high performance liquid
chromatography (HPLC). H&E- and Oil Red O staining were made to confirm the hepatosteatosis.
There were 39 genes and 13 biological pathways differently regulated by OE-NPY in microarray analysis. They
refer to decreased hepatic β-oxidation and increased anti-inflammatory actions of NPY. Lipogenic enzyme FAS
expression was increased and oxidative enzymes ACOX1 and CRAT expression were decreased in OE-NPY
mice. There was no difference between genotypes in the expression of lipogenic enzyme ACC, oxidative
enzyme CPT1, FA uptake increasing enzyme ACSL4 or gluconeogenic enzyme PCK1. Serum cholesterol
absorption markers campesterol and sitosterol were significantly increased and the synthesis/absorption ratio
decreased in OE-NPY female mice. Squalene, a precursor of cholesterol, was decreased in OE-NPY male mice.
The results suggest that hepatic lipogenesis is increased and β-oxidation decreased in OE-NPY mice, which may
explain the hepatosteatosis. NPY overexpression may cause hypercholesterolemia by increasing intestinal
cholesterol absorption rather than increasing cholesterol synthesis, which is the opposite to what is commonly
seen in the MS in humans.
Supported by: Academy of Finland; Novo Nordisk Fondet.
57 P27
ROLE OF ENDOGENOUS PEPTITE YY IN GASTROINTESTINAL AND FEEDING RESPONSES TO
STRESS
Sarah Forbes, Herbert Herzog, Helen Cox. King’s College London, Wolfson Centre for Age-Related Diseases,
Guy's Campus, London, SE1 1UL, United Kingdom.
Neuropeptide Y (NPY) is critical for maintaining numerous stress-responses. Although there is little evidence to
implicate peptide YY (PYY) in stress, the possibility is raised as it has important roles in gastrointestinal (GI)
motility and feeding like NPY and acts through the same Y receptors. The aim of this study was to determine GI
transit rates, food intake and corticosterone levels after an acute restraint (AR; 30 min) or novel environment
(NE) stress in NPY-/-, PYY-/- and WT mice and establish the role of PYY(3-36) and CRF mechanisms. Both
upper gastrointestinal transit (UGIT), determined after intra-gastric administration of a charcoal marker, and
corticosterone levels were established under basal or AR conditions. During AR faecal pellets were counted and
after AR feeding was monitored. Faecal pellet output (FPO) and food intake were established after 15 min and 4
hr in a NE. Compared to WT mice both genders of NPY-/- mice had significantly slower UGIT after AR, whilst
male PYY-/- mice had significantly faster UGIT under basal and AR conditions, which was inhibited by
exogenous PYY(3-36). NPY and PYY-/- mice displayed higher FPO and less food intake relative to WT
counterparts in a NE and during/after AR, however only NPY-/- displayed an increase in corticosterone levels.
CRF1 promoted colonic motility in NPY-/- mice and interacted with NPY and PYY to inhibit food intake during
stress. CRF2 inhibited colonic motility in all mice and potently suppressed food intake independently of NPY
and PYY. Thus whilst endogenous NPY is important for stress resilience, PYY possesses a significant role in
inhibiting UGIT under normal and stressed conditions. Both peptides protect against stress-induced colonic
motility with CRF2.
P28
DIFFERENTIAL EFFECTS OF GPR119 AGONISTS IN WILD TYPE AND PEPTIDE YY KNOCKOUT
MOUSE AND HUMAN COLON MUCOSAE.
Iain R. Tough, Herbert Herzog and Helen M. Cox. Wolfson CARD, King's College London, London UK.
GPR119 is a recently deorphanised receptor located on enteroendocrine cells and its stimulation causes release
of the anorexigenic hormone, peptide YY (PYY) and the incretin, glucagon-like peptide 1 making the receptor
an attractive target for novel diabesity therapies. We demonstrated previously that GPR119-mediated responses
inhibited short-circuit current (Isc) in mouse or human colon mucosae mounted in Ussing chambers, using the
small molecule agonist, PSN632408 or the endogenous agonist, oleoylethanolamide (OEA) (Cox et al., 2010,
Cell Metab., 11, 532-42). The responses were attributed to the paracrine effects of PYY on epithelial Y1
receptors. Here we investigated the relative potencies of PSN632408 and OEA with two further GPR119
agonists, N-oleoyldopamine (OLDA) and AR231453 in descending colon mucosae from adult wild type (WT) or
PYY knockout (-/-) mice and from clinical specimens.
Antisecretory responses to each agonist were observed in WT mouse and human colon mucosae. In mouse
tissues, concentration-response curves had the following relative order of potencies: AR231453 >> OLDA >
OEA = PSN632408. In terms of relative efficacy, OED, OLDA and PSN632408 exhibited similar maximal
responses; however maximally effective AR231453 responses were ~50% of PSN632408. AR231453 and
OLDA were unaffected by neuronal blockade. While AR231453 was blocked by a Y1 receptor antagonist and
not observed in PYY-/- mucosa (reported previously with PSN632408 and OEA), OLDA was not Y1 receptoror PYY-dependent. As seen previously with PSN632408, AR231453 responses were glucose sensitive. In
conclusion, OLDA is a non-selective GPR119 agonist in colonic mucosa, whereas AR231453 may be a partial
GPR119 agonist.
Supported by The Wellcome Trust
58 P29
THE NPY SYSTEM: A NOVEL PHYSIOLOGICAL DOMAIN
Maria Daniela Hurtado, Alicia Brown, Shawn Dotson, Herbert Herzog, Paola Riveros and Sergei Zolotukhin
Pediatrics Department, University of Florida, Gainesville, Fl 32610.
We have recently reported the presence of PYY in saliva and the expression of this peptide and its receptor,
Y2R, in lingual epithelial and taste receptor cells (TRC). In this report we extend our finding to more NPY
system members and characterize their expression in murine lingual epithelium, TRC and salivary glands. We
show how morphologically different layers of the keratinized stratified epithelium express YRs in a very
distinctive yet overlapping manner, in a cascade fashion switching from Y1R/Y2R+ progenitor cells in the basal
layer, to Y1R+ cells in the prickle cell layer, to Y1R/Y5R+ cells in the granular layer, to Y5R+ cells in the
keratinocytes. Y4R is shown to be expressed in the subepithelium, specifically in somatosensory neurons
innervating basal layer. In taste buds of the circumvallate papillae, Y4R was shown to be expressed in nerve
fibers innervating TRC. A significant population of TRC was positive for Y1R, Y2R, Y4R, or Y5R. A fraction
of TRC expressing Y receptors also expressed Neural Cell Adhesion Molecule suggesting their possible role in
the gustatory signal transduction.
It has been demonstrated that NPY, another member of the family, is also present in saliva and is expressed in
TRC modulating taste sensitivity to sweet modality. To characterize the functions of salivary PYY, we tested
whether it modulates taste perception as well. Using behavioral studies in PYY KO model, we show that PYY
modulates bitter taste sensitivity and lipid sensing. Taken together these experiments characterize, for the first
time, the expression of all NPY family members in the oral cavity suggesting multiple and diverse functions in
proliferation/differentiation and in taste perception.
P30
SALIVARY PYY: A NOVEL PHYSIOLOGICAL ROLE
Maria Daniela Hurtado, Andres Acosta, Michael La Sala, David Duncan, C. Shawn Dotson, Herbert Herzog,
Bruce J. Baum, and Sergei Zolotukhin
Pediatrics Department, University of Florida, Gainesville, Fl 32610.
Peptide YY (PYY) is a hormone released postprandially into the bloodstream from gastrointestinal L-endocrine
cells to induce satiety. We have previously demonstrated that PYY is present in murine and human saliva. In
mice, salivary PYY derives from plasma and is also synthesized in taste receptor cells of lingual taste buds. In
this report we show that the acute one-time augmentation of salivary PYY resulted in food intake (FI) reduction
as demonstrated in feeding behavioral studies. Because of the sustained 24-hr anorectic effect, we tested whether
repeated once-a-day treatment over an extended period of time would also affect FI and body weight (BW)
accumulation when mice were fed high fat (HF) diet ad libitum. Indeed, DIO mice treated with daily PYY oral
spray consumed significantly less HF food on a daily basis resulting in retarded BW accumulation. This effect is
mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial and taste receptor
cells.
To fully characterize the physiological role of salivary PYY, we provided a sustained supply of exogenous PYY
in saliva using viral vector-mediated gene delivery targeting salivary glands of PYY knock-out (KO) mice and
DIO WT mice. We demonstrated that long term increase of salivary PYY resulted in a significant long-term
reduction in food intake (FI) and body weight (BW) in DIO mice and in a retarded BW accumulation in the PYY
KO model. Thus we provide evidence for new physiological role of the previously characterized gut peptide
PYY suggesting a potential simple and efficient alternative therapeutic approach for the treatment of obesity.
59 P31
SALIVARY PYY ACTIVATES CNS NUCLEI INVOLVED IN FEEDING BEHAVIOR AND TASTE
PERCEPTION
Maria Daniela Hurtado, Andres Acosta, Oleg Gorbatyuk, Valeriy G. Sergeyev, Cedrick D. Dotson, and Sergei
Zolotukhin.
Pediatrics Department, University of Florida, Gainesville, Fl 32610.
Peptide YY (PYY) is a hormone released postprandially into the bloodstream from intestinal L-endocrine cells
and induces satiation upon interaction with its cognate receptor, Y2R. Recently we have demonstrated the
presence of PYY in human and murine saliva, and the expression of Y2R receptors in lingual epithelium and
taste receptor cells. Importantly, we have shown that augmentation of salivary PYY through genetic and/or
pharmacological approaches reduces food intake (FI) and body weight (BW) in mice. In this report we describe
the activation of brainstem and hypothalamic nuclei in response to acute pharmacological augmentation of
salivary PYY in mice at doses that reliably reduce FI and BW. In fasted mice, hypothalamic satiety centers were
activated significantly in response to orally applied PYY, similar to the fed control group and the peripherally
injected PYY group. Activated areas in the nucleus of the solitary tract (NST), identified by c-Fos reactivity,
were markedly different for the rostral and caudal NST, in response to systemic vs. local oral PYY treatment.
This distinctive pattern suggests that PYY’s impact on FI and BW may be mediated, at least in part, by the
modulation of taste responsiveness. This pattern of neuronal activation was corroborated by behavioral studies
assaying induction of taste aversion by salivary PYY. Orally applied PYY, while inducing a strong anorexigenic
response, did not induce conditioned taste aversion as the circulating form does. Taken together these data
provide support for the existence of a metabolic circuit associated with Y2R-positive cells in the oral cavity and
extending through brainstem nuclei into hypothalamic satiety centers.
P32
A TRIPEPTIDE SWITCH REGION TO INDUCE AGONISM AND INVERSE AGONISM AT THE
GHRELIN RECEPTOR
Els S.*, Schild E.*, Peterson P.S.#, Chollet C.*, Holst B.#, Schwartz T.W.#, Beck-Sickinger A.G.*
*Institute of Biochemistry, Universität Leipzig, Germany
#Department of Neuroscience and Pharmacology, Laboratory for Molecular Pharmacology, The Panum Institute,
University of Copenhagen, Denmark
The ghrelin receptor is a GPCR possessing an unique constitutive activity. The signaling of the receptor is
controlled by its endogenous ligand ghrelin and considerably contributes to the regulation of appetite, food
intake and energy homeostasis. Thus, reducing the constitutive activity can be an approach to decrease body
weight and to develop an anti-obesity drug. Inverse agonists are able to reduce basal signaling of a receptor.
Holst et al. introduced variants of substance P acting as inverse agonists. The hexapeptide KwFwLL-NH2
significantly reduces the constitutive activity[1].
We describe the design of highly potent ligands for the ghrelin receptor. Small changes in the switch region wFw- of the hexapeptide KwFwLL-NH2 could easily swap the peptide behavior from inverse agonism to
agonism, indicating the importance of this sequence. In activity assays, introduction of β-(3-benzothienyl)-Dalanine, 3,3-diphenyl-D-alanine and 1-naphthyl-D-alanine at position 2 resulted in highly potent and efficient
inverse agonists, whereas the substitution of tryptophan of position 4 with 1-naphthyl-D-alanine and 2-naphthylD-alanine induces agonism. Thereby, modification of only few atoms can decide between agonism and inverse
agonism. Only minor changes in the peptide sequence can have a major impact on biological actions.
Furthermore, the peptide with highest potency and affinity, K-(D-1-Nal)-FwLL-NH2, was tested in acute food
intake studies, revealing an almost 5-fold decrease of acute food intake compared to vehicles.
[1] Holst et al., J. Biol. Chem., 282, 15799 (2007)
60 P33
THE EFFECT OF NOVEL GHRELIN RECEPTOR AGONISTS ON CELL GROWTH
Tolhurst R., Els S., Beck-Sickinger A., Holst B., Schwartz T., Cox, H.
Wolfson CARD, KCL, London, SE1 1UL, UK.
Ghrelin receptor 1a (GHSR1a) mediates the effects of ghrelin, a major hunger-inducing peptide that is produced
mainly in the stomach. GHSR1a has high constitutive activity, being able to increase IP3 levels and activate both
serum and CREB response-element reporter assays in cells in the absence of ghrelin. Ghrelin treatment increases
activation of these signaling pathways and novel agonists, such as wFw-Isn-NH2 (a partial agonist) and
KwFwLL (an inverse agonist) have been used to further study GHSR1a signaling, with an aim to develop antiobesity therapies without side effects (Sivertsen et al., 2011, JBC, 286, 20845–20860).
Ghrelin can increase the growth of different types of cancer cell lines, via mitogen activated protein kinase
(MAPK) activation (Chopin et al., 2011, MCE, 340, 65-69). Therefore, we aimed to use two different cell lines,
1) HEK-293 (which have endogenous levels of GHSR1a and ghrelin) and 2) HEK-GHSR1a (HEK-293 cells
stably transfected with GHSR1a) in a sulphorhodamine B growth assay, to study the effects of novel GHSR1a
agonists on growth.
HEK-GHSR1a cells showed increased growth when treated with serum, compared to HEK-293 cells (but not
when cells were grown in the absence of serum). We found that ghrelin increased growth in a concentration
dependent manner in both cell lines and this was MAPK mediated. Surprisingly, we found that the inverse
agonist increased growth in both cell lines, but the partial agonist only increased growth in HEK-293 cells.
Furthermore, a novel GHSR1a antagonist also increased growth in both cell lines. This demonstrates a different
ligand pharmacology (in terms of growth) from their original classification as determined by IP3 turnover.
Supported by: EU 7th Framework Programme, Grant Agreement: no.223057 (GIPIO).
P34
NEUROPEPTIDE Y RECEPTORS AND NEUROPEPTIDE Y-INDUCED SECRETION ARE DIFFERENT IN
LEFT COMPARED TO RIGHT VENTRICULAR HUMAN ENDOCARDIAL ENDOTHELIAL CELLS.
Ahmarani L.1, Perreault C.1, Bkaily G.1, Magder S.2, D’Orléans-Juste P.3 and Jacques D.1.1Department of
Anatomy and Cell Biology, 3Department of Pharmacology, Faculty of medicine, University of Sherbrooke,
Sherbrooke, Québec, Canada, J1H 5N4. 2Critical Care Division, Royal Victoria Hospital, McGill University
Health Centre, McGill University, Montreal, Quebec, Canada, H3A 1A1.
The aim of the study was to test the hypothesis that excitation-secretion coupling of left ventricular human
endocardial endothelial cells (EECs) could be different from that of right ventricular human EECs. Using
immunofluorescence coupled to 3D confocal microscopy and ELISA, our results show that stimulation of
secretion by neuropeptide Y (NPY) induced the release of ET-1 from both right and left ventricular EECs.
However, the secretory capacity of right ventricular EECs is higher than that of left ventricular EECs.
Furthermore, our results show that the effect of NPY was probably mediated via Y2 receptors activation in right
ventricular EECs and via Y5 receptor activation in left ventricular EECs. This secretory process seems to also
implicate ET-1 receptors activation only in right ventricular EECs. In conclusion, our results suggest that
differences in excitation-secretion coupling may exist between right and left ventricular EECs and this may
contribute to the functional differences between right and left ventricular muscle. Supported by the Canadian
Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Quebec (HSFQ).
61 P35
EXAMINATION OF BVD15 AS A TEMPLATE FOR NEW Y1 RECEPTOR IMAGING AGENTS.
Oscar Lui1, Simon Mountford1, Lei Zhang2, Herbert Herzog2, Philip Thompson1
1Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, VIC 3052 Australia,
2Neuroscience Program, Garvan Institute of Medical Research, Sydney, Australia
Radiolabelled peptides are an important class of radiopharmaceuticals for imaging and therapy of tumours. They
can be designed upon endogenous peptide hormones making them an attractive target.
Neuropeptide Y (NPY) is a 36 amino acid peptide that exhibits a wide range of central and peripheral activities.
These actions are mediated by a number of G-protein coupled receptor subtypes. In particular, Y1 receptors are
overexpressed in human breast carcinomas. They also have important functional roles in breast tumour growth
and metastasis. Y1 receptors therefore can potentially be exploited for the detection of such tumours and also for
the delivery of radiotherapy.
This project aims to develop highly selective Y1-receptor peptide ligands suitable for imaging and/or delivery of
radiotherapy with improved in vivo properties over native NPY and other pancreatic polypeptide family
members.
Herein we report the synthesis of 16 truncated NPY analogues. These peptides are based on the previously
reported BVD15 scaffold [1]. Different measures to improve Y1 affinity and plasma metabolic stability were
investigated. Extending from the previously reported [Lys(DOTA)4]BVD15 analogue [2], it was found that the
Lysine4 is capable of tolerating various modifications, including prosthetic groups and other bifunctional
chelators. Substitution of Lysine4 for its mimics 2,3-diaminopropionic acid and ornithine can further improve
Y1 receptor affinity of the analogues. Modifications at the N-terminal Isoleucine however resulted in dramatic
reduction of Y1 affinity.
1/ Leban, J. et al., (1995). J. Med. Chem. 38, 1150-1157. 2/ Guérin, B. et al., (2010). Bioorg. Med. Chem. Lett.
20, 950-953.
P36
EXPRESION OF PEPTIDE YY, PANCREATIC POLYPEPTIDE, Y1 AND Y4 RECEPTORS IN HUMAN SKIN AND
RECONSTRUCTED HUMAN EPIDERMIS.
Duranton, A1, Dumont, Y2, Bastianetto, S2, Quirion R2, Breton, L1.
1) L’Oréal Recherche, Clichy, France. 2) Douglas Mental Health University Institute, McGill University, Dept. Psychiatry,
Montreal QC, Canada.
The presence of NPY as well as peptide YY (PYY) and pancreatic polypeptide (PP) has not yet been evaluated in human
epidermis. Using antibodies directed against NPY, PYY and PP revealed that PYY and PP-like imunoreactive (ir) materials
were present in epidermal cells, but no fluorescent signal detected for NPY. Antibodies directed against human Y1, Y2, Y4
and Y5 receptor subtypes demonstrated that Y1 and Y4 receptors are present in all layers of the epidermis. Fluorescent
labelling seen with Y1 and Y4 antibodies is found in cells that are also positively labelled with cytokeratin (CK) 1, 10, 14
and 15 as well as filaggrin in the epidermis, demonstrating that Y1 and Y4 receptors are expressed in basal stem cells,
suprabasal and granular layers of the epidermis. Additionally, CK15 immunoreactive materials are also observed in cells
that were immunoreactive to Y1 and Y4 receptor antibodies.
The SkinEthic™ reconstructed human epidermis (RHE) model consists of normal, human-derived keratinocytes, which
have been cultured to form a fully differentiated three-dimensional epidermis (Bernard et al.Exp Dermatol. 11:59-74, 2002).
Normal human keratinocytes that were cultures for 3 days showed one to two cell layer that were immunoreactive to CK1
and CK14 but not to CK10 and fillagrin. At day 7, cells in suprabasal layer is positively labelled with CK10 antibody while
CK14 immunoreactive cells are mostly located in the basal layer and fully differentiated human skin epidermis is obtained
at day 14 and 17, demonstrating that SkinEthic™ fully expressed markers of keratinocytes as seen in normal human
epidermis. As also seen in normal human epidermis PP like-ir, Y1 like-ir and Y4 like-ir is observed in SkinEthic™ that
were cultured for 3, 7, 14 and 17 days. Immunoreactive materials are observed in all layers of the SkinEthic™ as also
shown for normal human epidermis. However, and rather surprising, we were not able to detect PYY immunoreactive signal
in SkinEthic™ that were cultured for 3, 7, 14 and 17 days. These data contrast with the expression of PYY-like-ir detected
in normal human epidermis. Hence, SkinEthic™ represents a useful model to investigate the role and functions of PP, Y1
and Y4 receptors in keratinocytes.
62 P37
EFFECT OF AGING AND LONG-TERM CALORIC RESTRICTION ON NEUROPEPTIDE Y RECEPTOR
SUBTYPES IN THE RAT BRAIN
Veyrat-Durebex C.1, Gaudreau P.1, Dumont Y.2, Ferland G.3, Quirion R.2
1) Laboratory of Neuroendocrinology of Aging, University of Montreal, Montreal, Quebec, Canada, 2) Douglas Institute,
McGill University, Montreal, Quebec, Canada, and 3) Department of Nutrition, University of Montreal, Montreal, Quebec,
Canada.
Neuropeptide Y (NPY) mediates its physiological effects in the brain through multiple receptor subtypes. They are involved
in memory functions (Rangani RJ et al. Peptides. 2012;33:317-318), mood disorders and stress responses (Morales-Medina
et a.l, Brain Res. 2010;1314:194-205), as well as feeding behavior and energy homeostasis (Pjetri et al. Genes Brain Behav.
2012;11:105-112). Their dysregulation may play a role in aging processes (Akimoto and Miyasaka Geriatr Gerontol Int.
2012;10:S107-S119). Moreover, it has been proposed that hypothalamic NPY neurons contribute to downstream
physiological benefits in calorie-restricted rats (Minor et al., Aging Cell. 2011;10:483-492). We aimed to assess the effect
of aging and caloric restriction (CR) on brain NPY receptor subtypes, by quantitative receptor autoradiography. Eightmonth-old male Sprague Dawley rats were fed ad-libitum (AL) or submitted to a 12-month CR. They were compared to 3month-old AL rats. Labeling of Y1, Y5 and Y2 receptors was performed as previously described using radiolabeled ligands
in the presence of selective antagonists. Aging was associated with a decrease of Y1 (cingulate cortex, CA1), Y2 (CA1) and
Y5 (CA2), and an increase of Y2 (lateral septum) receptor densities. CR resulted in a decrease of Y1 (CA3, dentate gyrus)
and an increase of Y2 (CA2) and Y5 (CA2) receptor densities. Some changes were also observed in hypothalamic nuclei of
old AL and RC rats. Altogether these results suggest that long-term CR may contribute to healthy brain aging in regulating
NPY receptor subtypes involved in cognition and food intake.
This research was supported by the Quebec Network for Research on Aging/Fonds de Recherche du Québec-Santé (FRSQ))
and the Canadian Institutes of Health Research. CVD was recipient of a postdoctoral studentship from FRSQ.
P38
NPW INDUCED HYPOPHAGIA IS MEDIATED THROUGH STRESS RESPONSE.
Takenoya F1,2)., Kageyama H1)., Ogawa T1)., Shioda S1). 1) : Department of Anatomy, Showa University School
of Medicine. Shinagawa-ku, Tokyo 142-8555, Japan. 2) :Department of Exercise and Sports Physiology, Hoshi
University School of Pharmacy and Pharmaceutical Science, Tokyo 142-8501, Japan
Neuropeptide W (NPW) is identified as an endogenous ligand for GPR7 (NPBWR1) and GPR8 (NPBWR2) which belong
to the orphan G protein-coupled receptor (GPCR) family. Both NPW and NPBWR1 are widely distributed in the central
nervous system (CNS) in mammals. NPW is affected by leptin, while it inhibits NPY and activates POMC neurons,
followed by inducing the suppression of food intake. Moreover, NPW is involved in the regulation of the hypothalamuspituitary-adrenal cortex (HPA) axis. As the intracerebroventricular (i.c.v.) administration of NPW decreases nocturnal
feeding behavior in the dark phase, we undertook to elucidate the underlying mechanism with the central effect of NPW on
diet-induced obese (DIO) mice and to know whether the HPA axis is involved in this process. The i.c.v. infusion of NPW
was performed 2 h before the dark phase, then the alterations to food and water intake and locomotor activity were
observed. In addition, immunostaining of brain tissue sections in these animals was performed using anti-CRH, antivasopressin, anti-oxytocin and anti-c-Fos antibodies. NPW infusion did not affect the food and water intake and locomotor
activity in the DIO mice, but a decrease of food and water intake was demonstrated in standard diet-fed normal mice. NPWinduced hypophagic response in the sedentary mice was suppressed in animals that were permitted to undertake voluntary
exercise on a running wheel. These results imply that the failure of the HPA axis feedback system and the attenuation of
stress by the voluntary exercise lead to a reduction in NPW induced hypophagia. In the dark phase, NPW i.c.v. infusion
induced an increased c-Fos expression in the supraoptic (SON) and paraventricular nucleus (PVN) of the sedentary mice
compared to control animals that received a saline injection. A lot of CRH-positive neurons but not vasopressin- or
oxytocin-positive neurons in the PVN showed a significant expression of c-Fos in response to NPW administration. On the
other hand, voluntary running attenuated the level of NPW-induced c-Fos expression in the sedentary mice as compared
with the animals that received a saline injection. These findings suggest that NPW activates CRH-positive neurons and the
anorexigenic effects induced by NPW are influenced by the stress response.
63 P39
NEUROPEPTIDE Y MODULATES FEAR, ANXIETY AND DEPRESSION-LIKE BEHAVIOR IN
DISTINCT NUCLEI OF THE AMYGDALA
Tasan RO., Verma D., Herzog H. & Sperk G.
Institute of Pharmacology, Medical University Innsbruck, Innsbruck, Austria
Fear and anxiety are integrated in the amygdaloid nuclei and involve the interplay of the amygdala with various
other brain areas. Neuropeptides play a critical role in regulating these processes. Neuropeptide Y (NPY) is
highly expressed in limbic brain areas, including the amygdala. Depending on the receptor subtypes involved
(Y1, Y2 or Y4), NPY has different, in part opposing effects on anxiety, fear and depression-related behaviors.
We combined site-specific deletion of NPY receptors and locally restricted over-expression of NPY receptor
subtype selective ligands with behavioral analysis to elucidate the contribution of the individual receptor
subtypes in the modulation of emotional behavior. In Pavlovian fear conditioning NPY KO mice display a
dramatically accelerated acquisition of conditioned fear while fear extinction was impaired. Interestingly this
phenotpye was only reproduced in mice lacking both, the Y1 and the Y2 receptor. In Y1 single KO mice
acquisition was moderately faster while fear extinction was delayed. Deletion of NPY and in particular of Y2
receptors resulted also in a generalization of cued as well as also context fear. Local over-expression of NPY by
an rAAV vector in the basolateral amygdala delayed the acquisition and facilitated the extinction of fear, both in
WT and NPYKO mice, emphasizing the crucial role of this area in NPY mediated fear acquisition and
extinction. On the other hand, deletion of Y2 receptors in the central amygdala resulted in an increased
expression and delayed extinction of conditioned fear, while there was no change in fear acquisition.
Taken together, our data demonstrate that NPY delays acquisition and reduces expression of conditioned fear
whereas it promotes fear extinction. Both, Y1 and Y2 receptors are involved in these processes. Y1 receptors in
the basolateral amygdala are modulating the acquisition and extinction of fear while Y2 receptors in the central
amygdala are preferentially inhibiting the expression but facilitating the extinction of learned fear. Furthermore,
Y2 receptors are crucially involved in the discrimination of fear related stimuli.
P40
NEUROPEPTIDE Y AS A MEDIATOR OF STRESS INDUCED EFFECTS ON CANCER INITIATION AND
PROGRESSION.
Jason Tilan, Ewa Izycka-Swieszewska, Joshua Patrick Earnest, Susanna Galli, Asim Shabir, Congyi Lu,
Magdalena Czarnecka, Olga Rodriguez, Chris Albanese, and Joanna Kitlinska.Georgetown University,
Washington DC
Epidemiological and experimental studies suggest that psychosocial stress contributes to cancer development and
progression. Yet, the mechanism of this phenomenon is not clear. The goal of our study was to test whether neuropeptide Y
(NPY), which is up-regulated in chronic stress and acts as an angiogenic and growth factor, mediates this process. To this
end, wild type (WT) and NPY knockout (NPY KO) mice were treated with a chemical carcinogen, 7,12
dimethylbenz[a]anthracene (DMBA). To isolate NPY-dependent effects of stress, DMBA-treated WT and NPY KO mice
were subjected to chronic stress (daily cold exposure) for two weeks in the following groups: 1) Control; 2) Early stress
during DMBA administration to establish its effect on tumor initiation; 3) Late stress applied when tumors were detectable
to determine its effect on their progression. DMBA induced various neoplastic changes: lymphoma/leukemia (Leu), uterine
hemangiomas and angiosarcomas, folliculomas and mammary tumors. Under basal conditions, lack of NPY resulted in
reduced incidence of Leu and a protective effect on mammary tumor formation in NPY KO mice suggesting its role in
development of these malignancies. Early stress increased incidence of uterine angiosarcomas in an NPY-dependent
manner. In contrast, a stress-induced increase in frequency of Leu and ovarian folliculoma was observed only in NPY KO
mice suggesting that this effect may be mediated by catecholamines (CA), which are excessively released in the absence of
an inhibitor of their secretion, NPY. Surprisingly, early stress had a protective effect on mammary tumor formation that was
observed in both WT and NPY KO mice suggesting an NPY-independent mechanism. Late stress accelerated the
progression of Leu in an NPY-independent manner. Similar phenomenon was observed in uterine angiosarcomas induced in
WT mice, while late stress in NPY KO mice caused their regression. This, again, may be explained by a growth inhibitory
effect of CAs excessively released in stressed NPY KO mice. Lastly, in mammary tumors, stress increased tumor
vascularization and proliferation in NPY-dependent manner. Our data demonstrate potent and differential effects of NPY
and other stress mediators on tumor development and progression and lay a foundation for further, more comprehensive and
mechanistic studies. This, in turn, may open new therapeutic and preventative avenues for cancer patients, as well as
identify populations at risk. 64 P41
NEUROPEPTIDE Y RECEPTOR TYPE 5 – INTERACTIONS WITH G PROTEIN-COUPLED AND
TYROSINE KINASE RECEPTORS AND THEIR FUNCTIONAL CONSEQUENCES.
Magdalena Czarnecka, Congyi Lu, Zofia Zukowska, Joanna Kitlinska; Department of Biochemistry and
Molecular & Cellular Biology, Georgetown University, Washington DC
Neuropeptide Y (NPY) functions are mediated by class A GPCRs. While Y1 and Y2 receptors (Rs) are well characterized,
functions of the Y5Rs are not fully elucidated. This enigmatic R is usually co-expressed with other NPY Rs at very low
levels, often in a temporary, inducible manner. Its structure strikes with a presence of a large intracellular loop 3, classified
as an intrinsically disordered region (IDR) which could account for the R’s signaling diversity. Therefore we hypothesized
that Y5Rs play a regulatory role in the NPY system.
Indeed, we have shown that co-expression of Y5Rs with Y1Rs in CHO-K1 cells, sensitizes them to NPY and triggers the
mitogenic response to the peptide at its picomolar concentrations, significantly below known Kds for single Rs. The
immunoprecipitation experiments revealed presence of Y1/Y5R heterodimers. Our data indicate also that in addition to their
direct and functional interactions with other NPY Rs, Y5Rs are involved in cross-talk with tyrosine kinase Rs for brainderived neurotrophic factor (BDNF) – TrkB. In neuroblastoma (NB) cells constitutively expressing only Y2Rs, BDNF
additionally induced expression of Y5Rs. Y5R antagonists significantly reduced BDNF-driven TrkB phosphorylation and
p44/42 MAPK activation. Moreover, blocking Y5Rs significantly decreased BDNF-incited NB cell survival and resistance
to chemotherapy, while combining Y5R and TrkB antagonists completely abolished pro-survival effect of BDNF.
In summary, Y5R is a potent modulatory R acting via interactions with other GPCRs, as well as tyrosine kinase Rs. These
unique features of Y5Rs might stem from the dynamic capacities embedded in the intrinsically disorder region of ICL-3.
Such interactions of the Y5Rs enhance functions of NPY mediated by its other Rs and enable cross-talk of NPY system
with other pathways. Understanding the mechanisms and functional consequences of Y5Rs actions may open new
therapeutic opportunities for NPY-associated disorders.
P42
ANCIENT GRANDEUR OF THE NPY SYSTEM CONFIRMED BY DISTANTLY RELATED
VERTEBRATES.
Larhammar D. and Bergqvist C. Department of Neuroscience, Uppsala University, Uppsala, Sweden.
Previous analyses of the evolution of the NPY-family peptides and their receptors led to the conclusion that the
ancestral vertebrates probably had a single NPY-like peptide and three ancestral receptors, namely Y1, Y2 and
Y5. Two tetraplodizations, i.e., genome doublings (called 2R), quadrupled this set. Of the resulting four
peptides, two remain today as NPY and PYY. The receptor trio’s quadruplication should have resulted in 12
receptors, but only 7 were deduced to have remained in the ancestor of the jawed vertebrates. This was supported
by the discovery of all seven receptor genes in the elephant shark genome. Other vertebrate lineages, in contrast,
seem to have lost genes: frogs have lost Y6 (pseudogene), amniotes (mammals, reptiles and birds) have lost Y8,
mammals have lost also Y7, and several orders of mammals lack Y6. In the sequenced teleost fish genomes,
neither Y1, Y5 or Y6 were identified, although we have subsequently identified Y1 in zebrafish. On the other
hand, some teleosts have a duplicate of Y8 (Y8b) and a duplicate of Y2 (Y2-2). Recently, the genome
assemblies were reported for two basally radiating fishes, namely the coelacanth Latimeria chalumnae, an early
branch on the sarcopterygian (flesh-finned) fish lineage otherwise dominated by the tetrapods (amphibians,
birds, reptiles and mammals), and the spotted gar Lepisosteus oculatus, which branched off early in the
actinopterygian (ray-finned) fish lineage, before the teleost ancestor underwent the third tetraploidization (3R).
We have found that both of these early vertebrate branches possess all seven deduced ancestral NPY receptors,
thereby confirming the duplication scenario reported previously. Thus, mammals have lost more NPY receptors
than any other vertebrate lineage.
Supported by: The Swedish Research Council.
65 P43
HIGH THROUGHPUT DISCOVERY OF NEUROPEPTIDE Y Y2 RECEPTOR ANTAGONISTS
Brothers, S.P. and Wahlestedt C.
Center for Therpaeutic Innovation, Department of Psychiatry and Behavioral Sciences, University of Miami
Miller School of Medicine
The neuropeptide Y Y2 receptor (Y2R) has been implicated in a number of human diseases such as obesity,
mood disorders and alcoholism and its contributions to each of these diseases could be better resolved by use of
selective, chemical probes that are publicly available. To discover potential Y2R antagonists, the NIH Molecular
Libraries Small Molecule Repository (MLSMR) library was screened for Y2R antagonist activity in a live whole
cell cAMP biosensor assay.
We discovered five potent and selective Y2R antagonists. These five antagonists, SF-11, SF-21, SF-22, SF-31
and SF-41 (with IC50 values of 199 ± 10, 440 ± 30, 750 ± 30, 1200 ± 100 and 4400 ± 40 nM respectively), were
also found to bind with high affinity to the Y2R and completely lack NPY-Y1 receptor (Y1R) binding or
activation. These compounds belong to four chemical scaffolds that are structurally distinct from previously
reported Y2R antagonists such as BIIE0246 and JNJ-5207787. Additionally, these compounds had better brain
penetration and selectivity profiles compared to BIIE0246, the most widely used Y2R antagonist.
We conclude that these newly discovered compounds have the potential to be developed into useful probes to be
used in Y2R related research.
This work was supported by the National Institutes of Health grants R21NS056950-01S1 and U54MH084512.
P44
IDENTIFICATION OF EXTRINSIC SOURCES OF NEUROPEPTIDE Y INPUT TO THE RAT
BASOLATERAL AMYGDALOID COMPLEX AND REGULATION BY CONDITIONED CONTEXTUAL
FEAR.
Janice H. Urban, Mary R. De Joseph, and Randy J. Leitermann.
Depart. Physiol. & Biophysics, Rosalind Franklin Univ. of Medicine and Science, North Chicago, IL 60064
Neuropeptide Y (NPY) acts within the basolateral amygdaloid complex (BLA) to suppress anxiety and fear responses, and
provide resilience to stressful challenges. Identifying NPYergic input to the BLA is crucial to further understanding the
regulation of BLA activity and anxiety, as well as providing novel targeting sites for managing anxiety. We have recently
found that NPY-containing fibers in the BLA are not exclusively from interneurons, suggesting additional NPY inputs to
the BLA. To identify these extra-BLA sources, these studies used Fluorogold (FG) retrograde tract-tracing coupled with
immunohistochemistry (IHC) for NPY. Injections of FG into the BLA of rats produced back-filled cells containing NPYimmunoreactivity in the amygdalostriatal transition area (AStr) and stria terminalis. These regions contained dual-labeled
cells regardless of the anterior-posterior location of the injection, suggesting these projections innervate a large portion of
the BLA. To identify which source of NPY is responsive to fear and anxiety provoking stimuli, rats were subjected to
conditioned contextual fear. Amidated (releasable) NPY immunoreactivity in the BLA and NPY mRNA levels, in regions
with known projections to the BLA, were assessed using IHC and in situ hybridization coupled with emulsion
autoradiography, respectively. Conditioned rats displayed lower amidated NPY-immunoreactive fiber intensities in the BLA
than controls shortly after testing fear responses, suggesting an increased NPY release in response to the fearful
environment and/or removal from the environment. NPY mRNA levels in the AStr were increased in conditioned rats 1hr
after testing and returned to baseline within 5hr, while mRNA levels in the BLA and stria terminalis were not changed at
any time points examined. Together, these results suggest that the biosynthetic capacity of AStr-containing NPY projections
to the BLA are increased in response to conditioned fear and may participate in the regulation of anxiety and fear responses
to conditioned stimuli. Overall, our studies suggest that in addition to local-circuit BLA neurons, a number of limbic regions
associated with the stress circuitry provide NPY input to the BLA. Ultimately, these projections provide exciting novel
targets to consider when assessing NPY regulation of BLA function. While little is known regarding the function of the
AStr, future studies will further characterize NPY-containing AStr projections to the BLA and determine their role in
modulating stress responses. 66 List
Nima Aghili
Medstar Health Research Institute
Washington DC, USA
Lena Ahmaranie
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, Canada
Verena Ahrens
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Liisa Ailanen
University of Turku
Turku, Finland
Johny Al Koury
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, Canada
Cecilia Alves
Univiversity of Porto,
Biomedical Institute
Porto, Portugal
Akihiro Asakawa
Kagoshima University
Grad. school of Medical and Dental Sci.
Kagoshima, Japan
Levon Avedanian
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, Canada
Stephanie Babilon
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Paul Baldock
Garvan Institute of Medical Research
Sydney, NSW, Australia
Annette Beck-Sickinger
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Christoph Belinger
University Hospital
Basel, Switzerland
Sheng Bi
Johns Hopkins University
School of Medicine
Baltimore, MD, USA
Ghassan Bkaily
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, Canada
67 Pascal Bonaventure
Janssen Research & Development
San Diego, CA, USA
Lionel Breton
L’Oréal Recherche
Clichy, France
Shaun Brothers
University of Miami
Miami, FL, USA
Fabio Canneva
University of Erlangen-Nuernberg
Erlangen, Germany
Claudia Cavadas
University of Coimbra
Center Neuroscience and Cell Biology
Coimbra, Portugal
William F. Colmers
University of Alberta
Department of Pharmacology
Edmonton, AB, Canada
Robert Corringham
Michael Cowley
Monash University
Monash Obesity & Diabetes Institute
Clayton, Victoria, Australia
San Diego, CA, USA
Helen Cox
King’s College London
Wolfson CARD, Guy’s Campus
London, UK
Elizabeth Dong
Vanderbilt University
Center for Structural Biology
Nashville, TN, USA
Yvan Dumont
Douglas Mental Health Univ Institute
McGill University
Montreal, QC, Canada
Albert Duranton
L’Oréal Recherche
Clichy, France
Sylvia Els
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Magdy El-Salhy
University of Bergen
Stord Helse-Fonna Hospital
Stord, Norway
68 Sarah Forbes
King’s College London
Wolfson CARD, Guy’s Campus
London, UK
Pierrette Gaudreau
University of Montreal
Lab of Neuroendocrinology of Aging
Montreal, QC, Canada
Donald Gehlert
Lilly Research Laboratories
Indianapolis, IN, USA
Nicholas Gilpin
Louisiana State University
Health Science Center
New Orleans, LA, USA
William Gray
National Institute for Neuroscience
and Mental Health Research
Cardiff, UK
Brigitte Guerin
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, CA
Silveira Heika
University of Alberta
Edmonton, AB, Canada
Herbert Herzog
Garvan Institute of Medical Research
Sydney, NSW, Australia
Sven Hofmann
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Peter Holzer
Medical University of Graz
Graz, Styria, Austria
Xinyan Huang
Lundbeck Research USA.
Paramus NJ, USA
Maria Daniela Hurtado
University of Florida
Pediatrics Department
Gainesville, FL, USA
Akio Inui
Kagoshima University
Grad. School of Medical and Dental Sci.
Kagoshima, Japan
Masanobu Ito
Douglas Mental Health Univ Institute
McGill University
Montreal, QC, Canada
69 Danielle Jacques
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, Canada
Knud Jensen
University of Copenhagen
Faculty of Sciences
Copenhagen, Denmark
Cuihong Jia
Michigan State University
Dept. of Pharmacology & Toxicology
East Lansing, MI, USA
Javjeet Jolly
King’s College London
Wolfson CARD, Guy’s Campus
London, UK
Pushpa Kalra
University of Florida
Department of Physiology
Gainesville, FL, USA
Satya Kalra
University of Florida
Department of Neuroscience.
Gainesville, FL, USA
Joanna Kitlinska
Georgetown University
Washington, DC USA
Katja Kostelnik
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Meriem Lamghari
University of Porto
Instituto de Engecharia Biomédica
Porto, Portugal
Anja Landsmann
Gastro Intestinal Peptide In Obesity
Leipzig University
Leipzig, Germany
Dan Larhammar
Uppsala University
Department of Neuroscience
Uppsala, Sweden
Ning Lee
Bristol-Myers-Squibb
Pennington, NJ, USA
Shu Lin
Garvan Institute of Medical Research
Sydney, Australia
Thomas Lutz
University of Zurich
Institute of Veterinary Physiology
Zurich, Switzerland
70 Veronika Mäde
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Alex Magder
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC. Canada
Joao O. Malva
University of Coimbra
Center for Neuroscience & Cell Biology
Coimbra, Portugal
Petr Masliukov
Yaroslavl State Medical Academy
Yaroslavl, Russia
Michael Meaney
Douglas Mental Health Univ. Instute
McGill University
Montreal, QC, Canada
Janet Menard
Queen’s University
Department of Psychology
Kingston, ON, Canada
Karin Moerl
Leipzig University
Institute of Biochemistry
Leipzig, Germany
Julio Cesar Morales-Medina
Douglas Mental Health Univ. Institute
McGill University
Montreal, QC. Canada
Timo Müller
Helmholtz Centre Munich
Munich, Germany
Jessica Murray
Saint Louis University
School of Medicine
St-Louis, MO, USA
Amy Nguyen
Garvan Institute of Medical Research
Sydney, NSW, Australia
Torben Osterlund
Zealand Pharma A/S
Glostrup, Denmark
Johan Frederik Paulsson
Novo Nordisk A/S
Maloev, Denmark
Xavier Pedragosa Badia
Leipzig University
Institute of Biochemistry
Leipzig, Germany
71 Claudine Perreault
University of Sherbrooke
Faculty of Medicine
Sherbrooke, QC, Canada
Jasna Pruner
Uppsala University
Department of Neuroscience
Uppsala, Sweden
Rémi Quirion
Douglas Mental Health Univ. Institute
McGill University
Montreal, QC, Canada
Alfredo Ribeiro-da-Silva
McGill University
Dept. Pharmacology & Therapeutics
Montreal, QC, Canada
Joana Rosmaninho-Salgado
University of Coimbra
Center for Neuroscience & Cell Biology
Coimbra, Portugal
Renu Sah
University of Cincinnati
Dept Psychiatyr & Behav. Neurosci.
Cincinnati, OH, USA
Ana Santos-Carvalho
David Sarruf
Novo Nordisk A/S.
Maloev, Denmark
University of Coimbra
Center for Neuroscience & Cell Biology
Coimbra, Portugal
Eriika Savontaus
University of Turku
Dept Pharmacol, Drug Dev & Therap
Turku, Finland
Peter Schmidt
Leipzig University
Institute Medical Physics & Biophysics
Leipzig, Germany
Thue Schwartz
Sulaiman Sheriff
University of Cincinnati Medical Center
Cincinnati, OH, USA
Copenhagen, Denmark
Yan Chuan Shi
Garvan Institute of Medical Research
Sydney, NWS, Australia
Seiji Shioda
Showa University
School of Medicine
Tokyo, Japan
72 Gregory Sliwoski
Vanderbilt University
Center for Structural Biology
Nashville, TN, USA
Maria Smialowska
Instute of Pharmacology
Polish Academy of Sciences
Krakow, Poland
Günther Sperk
Medical University Innsbruck
Innsbruck, Austria
Scott Sternson
Howard Hughes Medical Institute
Janelia Farm Research Campus
Ashburn VA, USA
Michael Stumvoll
University of Leipzig
Leipzig, Germany
Fumiko Takenoya
Hoshi University
School Pharmacy & Pharmaceutical Sci.
Tokyo, Japan
Ramon O. Tasan
Medical University of Innsbruck.
Innsbruck, Tirol, Austria
Bradley Taylor
University of Kentucky
Department of Physiology
Lexington, KY, USA
Mira Thakur
Douglas Mental Health Univ. Institute
McGill University
Montreal, QC, Canada
Tood Thiele
University of North Carolina
Dept Psycho & Bowles Ctr Alcohol Studies
Chapel Hill, NC, USA
Neil Thompson
PolyPeptide Laboratories A/S
Hillerod, Denmark
Iain Tough
Kings’s College London
Wolfson CARD, Guy’s Campus
London, UK
Robert Tolhurst
Kings’s College London
Wolfson CARD, Guy’s Campus
London, UK
Nathalie Trent
Queen’s University
Dept Psychol and Ctr Neurosci Studies
Kingston, ON, Canada
73 Mohit Trikha
San Diego, CA, USA
Janice Urban
Rosalind Franklin University
Medicine and Science
North Chicago, IL, USA
Laura Vahatalo
University of Turku
Dept Pharmacol, Drug Dev & Therap
Turku, Finland
Thomas C. Westfall
Saint Louis University
School of Medicine
St. Louis, MO, USA
David PD Woldbye
University of Copenhagen
Dept. Neuroscience & Pharmacology
Copenhagen, Denmark
Brigitte S. Wulff
Novo Nordisk A/S
Maloev, Denmark
Bo Xu
Uppsala University
Department of Neuroscience
Uppsala, Sweden
Toshihiko Yada
Jichi Medical University
School of Medicine
Tochigi, Japan
Hideo Yukioka
Shionogi Pharmaceutical
Research Center
Toyonaka, Japan
Lei Zhang
Garvan Institute of Medical Research
Sydney, NSW, Australia
Sergei Zolotukhin
University of Florida
Department of Pediatrics
Gainesville, FL, USA
74