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Neuropeptides linking the control of appetite with reproductive function in
domestic animals
Clay A. LentsReproduction Research Unit, USMARC, ARS, USDA
EAAP, Copenhagen Denmark; August 25, 2014.
U.S. Meat Animal Research CenterReproduction Research Unit
Hypothalamus
Leptin
Insulin IGF-I Food Intake
-
Reproduction
+
Energy+/-
Growth
Barb et al., ICAFE 2008
Nutrients; FFA, Glucose
Brainstem
Liver
+/-
Common pathways of FI, growth & Reproduction
U.S. Meat Animal Research CenterReproduction Research Unit
Low LeptinNegative Positive
High Leptin
α-MSH AgRP
Increased Food Intake and Weight
Gain
Decreased Food Intake and Weight
Loss
α-MSH AgRP
+
-
-
-
+
+
-
+ -
NPY
+
-NPY
+
Reproduction- +
Barb et al., 2008 Reprod. Dom. Anim.
Interaction of appetite with reproduction
U.S. Meat Animal Research CenterReproduction Research Unit
Kisspeptin and reproductive function
Funes et al., 2003
GPR54 KO
Redmond et al., 2011
Mutations in Kiss or GPR54 lead to idopathichypogonadotropic hypogonadism.
Expression of Kiss is developmentally regulated and increased expression corresponds to increased frequency of LH pulses.
Kiss treatment restored LH secretion in underfed animals.
Nutrient restriction or perturbations in metabolism associated with decreased Kiss expression.
U.S. Meat Animal Research CenterReproduction Research Unit
Control
0
1
2
3
4
5
6
7
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180 0 30 60 90
LH
, ng/
mL
KISS110 g
0
1
2
3
4
5
6
7
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180 0 30 60 90
LH
, ng/
mL
KISS1 100 g
0
1
2
3
4
5
6
7
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180 0 30 60 90
Time, min
LH
, ng/
mL
ICV GnRH
ICV GnRH
ICV GnRH
Period 1 Period 2 Period 3
Kisspeptin stimulates LH and FSH secretion in prepubertal gilts
Lents et al., 2008, Reproduction
Control
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180 0 30 60 90
FSH
, ng/
mL
KISS110 g
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180 0 30 60 90
FSH
, ng/
mL
KISS1100 g
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180
30 60 90
Time, min
FSH
, ng/
mL
0
Period 1 Period 2 Period 3
ICVGnRH
ICVGnRH
ICVGnRH
LH FSH
U.S. Meat Animal Research CenterReproduction Research Unit
0102030405060708090
100
Kisspeptin only NKB only Kisspeptin/NKBcolocalized
Prop
ortio
n of
imm
unor
eact
ive
neur
ons,
%
Kisspeptin and neurokinin B (NKB) coexpressed in the ARC of gilts
Lents et al., USMARC, unpublished
Kiss NKB Merged
U.S. Meat Animal Research CenterReproduction Research Unit
Goodman et al, 2013; Navarro et al, 2013
GnRH
KNDy neurons
U.S. Meat Animal Research CenterReproduction Research Unit
POM
C m
RN
A
Hyp
otha
lam
us
Prepubertal gilt
6 mo-old
a
b P<0.01
3.5 mo
Lin et al. Biol. Reprod. 2001
Expression of POMC increases with age in the gilt
In general, activation of POMC neurons is associated with increased LH secretion – MSH
U.S. Meat Animal Research CenterReproduction Research Unit
Effects of MSH on LH secretion in gilts
Barb et al., 2004 J. Endocrinol.
Time 0 = ICV treatment
U.S. Meat Animal Research CenterReproduction Research Unit
Time after ICV injection (hrs)
Feed
Inta
ke (k
g)
**
**
**
12 240
1
2
3
4
5
Saline NDP-MSH
12 24 12 24
D298 N298D298N
* P < 0.05
Cumulative food intake of pigs after ICV injection of NDP-MSH
Barb et al., 2010 Physiol. Genomics
U.S. Meat Animal Research CenterReproduction Research Unit
Summary of differentially expressed (DE) genes (q<0.07) in hypothalamus, liver and adipose tissues of pigs 24 hr after
ICV injection of NDP-MSH
Barb et al., 2010 Physiol. Genomics
278 249 5,066
Fat = reduced leptin mRNA Hypothalamus = increased NPY mRNA
U.S. Meat Animal Research CenterReproduction Research Unit
-3 -2 -1 0 1 2 3 402460
1
2
Hours from ICV treatment
GH
ng/
ml
LH
ng/
ml 100 μg NPY
ICV Injection OVX prepuberal gilt
100 μg NPY
Barb et al., Reprod. 2006
***
*
*
*** *
*
*
*** *
*
**
NPY inhibits LH secretion in OVX prepubertal gilts
U.S. Meat Animal Research CenterReproduction Research Unit
• Early weaned heifers
• Diet: high concentrate• Low gain (n=12): 0.5 kg/d• High gain (n=12): 1.0 kg/d
• Slaughtered at 9 mo of age
Nutrition and neuroendocrine function of heifers
Alves et al. 2014
U.S. Meat Animal Research CenterReproduction Research Unit
(n = 6)
(n = 5)
*P ≤ 0.05
**P ≤ 0.01***P ≤ 0.001
Cardoso et al. 2014
Nutrition and neuroendocrine function of heifers
U.S. Meat Animal Research CenterReproduction Research Unit
Nutrition and neuroendocrine function of heifers
• NPY axons contacting Kiss cells are evident
• Much fewer than NPY to GnRH contacts
Amstalden et al. 2014, Front. Endocrinol.
Kiss NPY Syn
U.S. Meat Animal Research CenterReproduction Research Unit
Alves et al. 2014
NPYGnRH
# co
ntac
t/cel
l
GnRH neuronal soma and proximal dendrites in close
apposition of NPY fibers
Perc
ent
GnRH neurons highly innervated by NPY fibers
Nutrition and neuroendocrine function of heifers
U.S. Meat Animal Research CenterReproduction Research Unit
PeripheralCirculation
Leptin
POMC
-MSH
Food Intake
Growth
NPY
Reproduction
GnRH
KISS
Fat (leptin)Energy
+/?+
+
+
++
+ -/?
-
+/-
-
-
Hypothalamus
-
Adapted from Hausman et al., 2012, Biochimie
U.S. Meat Animal Research CenterReproduction Research Unit
Gonadotropin-inhibitory hormone
• Gonadotropin-inhibitory hormone (GnIH)• Hypothalamic neuropeptide first identified in Japanese quail • Inhibits the gonadotropic axis of birds
• Suppresses gonadal development and sexual behavior
• GnIH receptor expression in the pituitary of chicken decreases with sexual maturation
• Seasonal control of reproductive function
U.S. Meat Animal Research CenterReproduction Research Unit
• Gonadotropin-inhibitory hormone (GnIH)• 3 peptides in birds
• GnIH-RP-1, GnIH, GnIH-RP-2
• RFamide-related peptide (RFRP)• Mammalian homolog of GnIH• 2 peptides; RFRP-1 and RFRP-3
• Functions in mammals• Nociception, stress, prolactin secretion, energy
balance, and reproduction
Mammalian GnIH homolog; RFamide-related peptides
U.S. Meat Animal Research CenterReproduction Research Unit
Prepro-proteins encoding RFamide-related peptides
Sheep MEIISLKRFILLMLATSSLLTSNIFCTDESRIPSLYSKKNYDKYSEPRGDLGWEKERSLT 60Cow MEIISLKRFILLMLATSSLLTSNIFCTDESRMPNLYSKKNYDKYSEPRGDLGWEKERSLT 60 Pig -------------------LTSNIFCTDELVLSSLHSKKNYDTYSEPRRDSKWEKQRSLN 41 Human MEIISSKLFILLTLATSSLLTSNIFCADELVMSNLHSKENYDKYSEPRGYPKGE--RSLN 58 Rat MEIISSKRFILLTLATSSFLTSNTLCSDELMMPHFHSKEGYGKYYQLRGIPKGVKERSVT 60 * ***** **** * ** ** * * * ** Sheep FEEVKDWGPK--IKMNTPAVNKMPPSAANLPLRFGRNMEEERSTRVMAHLPLRLGKNRED 118 Cow FEEVKDWAPK--IKMNKPVVNKMPPSAANLPLRFGRNMEEERSTRAMAHLPLRLGKNRED 118 Pig FEELKDWGPKNVIKMSTPVVNKMPPSAANLPLRFGRTKEEESSPGATAGLPLRFGRNTED 118 Human FEELKDWGPKNVIKMSTPAVNKMPHSFANLPLRFGRNVQEERSAGATANLPLRSGRNMEV 120 Rat FQELKDWGAKKDIKMSPAPANKVPHSAANLPLRFGRNIEDRRSPRARA--------NMEA 112 * * *** * *** ** * * ********* * * * * Sheep SLSRRVPNLPQRFGRTIAAKSITKTLSNLLQQSMHSPSTNGLLYSMTCRPQEIQNPGQKN 180 Cow SLSRWVPNLPQRFGRTTTAKSITKTLSNLLQQSMHSPSTNGLLYSMACQPQEIQNPGQKN 180 Pig SMSRPVPNLPQRFGRTI-ARSITKALGALLQQSTRSPSAKGLLYSITR------------ 165 Human SLVRRVPNLPQRFGRTTTAKSVCRMLSDLCQGSMHSPCANDLFYSMTCQHQEIQNPDQKQ 180 Rat GTMSHFPSLPQRFGRTT-ARRITKTLAGLPQKSLHSLASSELLYAMTRQHQEIQSPGQEQ 171 * ******** * * * * * * * * ****
RFRP-1
RFRP-2
RFRP-3
* = amino acids completely conserved Blue letters = basic residues (potential for cleavage)Red letters = cleavage site with amide-donor
Lents et al., unpublished
U.S. Meat Animal Research CenterReproduction Research Unit
RFRP DAPI Merged
ParaventricularNucleus
Posteriorhypothalamus
VentromedialNucleus
50 m
RFRP immunoreactivity in the hypothalamus: prepubertal gilt
3V = third ventricle
Lents et al., unpublished
U.S. Meat Animal Research CenterReproduction Research Unit
• RFRP Immunoreactive fibers in the ewe• Neurosecretory zone of the median eminence
• Potential to regulate pituitary function• Preoptic area, septum, and diagonal band of Broca
• Potential to regulate GnRH secretion
• Implicates RFRP in the nutritional regulation of reproduction.
Smith et al., 2008
Clarke et al., 2006
Mammalian GnIH homolog; RFamide-related peptides
Clarke, 2014
Projections of RFRP neurons to appetite regulating cells in the ovine hypothalamus
Clarke et al., 2012
U.S. Meat Animal Research CenterReproduction Research Unit
0.00.51.01.52.02.53.03.5
-240
-210
-180
-150
-120 -9
0-6
0-3
0 0 30 60 90 120
150
180
210
240
LH, n
g/m
l
SalineID 216
0.00.51.01.52.02.53.03.5
-240
-210
-180
-150
-120 -9
0-6
0-3
0 0 30 60 90 120
150
180
210
240
LH, n
g/m
l
RFRP-310 μg
ID 206
0.00.51.01.52.02.53.03.5
-240
-210
-180
-150
-120 -9
0-6
0-3
0 0 30 60 90 120
150
180
210
240
LH, n
g/m
l
0.00.51.01.52.02.53.03.5
-240
-210
-180
-150
-120 -9
0-6
0-3
0 0 30 60 90 120
150
180
210
240
LH, n
g/m
l
RFRP-350 μg
ID 202
RFRP-3100 μgID 208
ICV ICV
ICVICV
Time, min Time, min
ICV injection of RFRP3 to pre-pubertal ovariectomized gilts
Heidorn et al., Nat. ASAS 2010
U.S. Meat Animal Research CenterReproduction Research Unit
*
0.00.51.01.52.02.53.03.54.04.5
Saline 10 µg 50 µg 100 µg
Mea
n G
H, n
g/m
l
Pre -ICV Post -ICV
RFRP-3
a
b
0.0
0.5
1.0
1.5
2.0
Saline 10 g 50 g 100 g
Mea
n LH
, ng/
ml
RFRP-3
Pre-ICV
Post-ICV
LH GH
Central (ICV) injection of RFRP3 to pre-pubertal ovariectomized gilts
*P < 0.01a,bP < 0.04
Heidorn et al., Nat. ASAS 2010
U.S. Meat Animal Research CenterReproduction Research Unit
0.0
0.5
1.0
1.5
2.0
2.5
Saline 450 g 850 g0.0
0.5
1.0
1.5
2.0
2.5
Saline 450 g 850 g
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Saline 450 g 850 g0.0
0.5
1.0
1.5
2.0
2.5
Saline 450 g 850 g
RFRP-3 RFRP-3
Bas
al L
H, n
g/m
lN
umbe
r of L
H P
ulse
s
Mea
n LH
, ng/
ml
Puls
e am
plitu
de, n
g/m
l
2 h before treatment2 h during treatment2 h after treatment
RFRP-3 RFRP-3
Peripheral (IV) injection of RFRP3 to pre-pubertal ovariectomized gilts
Heidorn et al., Nat. ASAS 2010
U.S. Meat Animal Research CenterReproduction Research Unit
Saline SB75Low RFRP2
(0.01 mg/kg BW) SB75 SB75High RFRP2
(0.1 mg/kg BW)
RFRP-2 does not affect LH secretion in pre-pubertal ovariectomized gilts
Thorson et al., Endo 2014
U.S. Meat Animal Research CenterReproduction Research Unit
GnRHR antagonist suppressed LH secretion in pre-pubertal ovariectomized gilts
2 h before SB-75 2 h after SB-75
Thorson et al., Endo 2014
U.S. Meat Animal Research CenterReproduction Research Unit
Intravenous Injection of RF9 & GnRHPre-pubertal Ovary Intact Gilts
0
1
2
3
-120 0 120 2400
1
2
3
-120 0 120 240
80901
0
3
6
9
240 3600
3
6
9
240 360
Saline RF9 GnRHGnRH
78801
0
1
2
3
-120 0 120 2400
3
6
9
240 3600
1
2
3
-120 0 120 2400
3
6
9
240 360
Saline RF9 GnRHGnRH
LH
, ng/
mL
Treatment Time, Minutes
0.5 mg RF9/kg BW; 100 µg GnRHThorson et al., Endo 2013
U.S. Meat Animal Research CenterReproduction Research Unit
GWAS for Age at Puberty in Gilts
SNP Window, bp
Chr Start End Rank
Genetic variance
%Bootstrap P-value
Candidate gene Function Reference
12 14,910,939 15,006,310 1 9.7332 0.001 GH1 Somatic growth7 75,121,635 75,460,294 2 7.1362 0.003 PRKD1 BMI
Puberty, swineGraff et al., Hum Mol Gen. 2013Nonneman et al., Anim. Gen. 2013
9 142,232,369 142,338,853 4 1.4283 0.004 SMYD2 Puberty, swine Tart et al., Anim. Gen. 20131 121,626,075 121,978,687 21 0.4196 0.006 RORA Menarche Demerath et al., Hum. Mol. Gen. 2013
17 64,555,868 64,680,839 46 0.2313 0.012 BMP7 Ovarian function3 21,596,232 22,794,763 70 0.1765 0.006 PAQP8 Puberty, swine Nonneman et al., Anim. Gen. 2013
15 65,197,162 65,381,481 80 0.1593 0.021 HS6ST1 IHH Tornberg et al, PNAS 20112 25,692,286 25,797,751 98 0.1280 0.053 PIK3R1 Menarche Demerath et al., 20131 183,533,664 183,678,408 176 0.0849 0.058 IQCH Menarche Elks et al., Nat. Gen. 20101 151,203,878 153,593,790 185 0.0828 0.044 IGF1R Puberty, cattle Fortes et al., Anim. Gen. 20132 58,719,232 59,243,471 193 0.0806 0.030 CRTC1 Menarche
Puberty, pigsElks et al., Nat Gen 2010Tart et al., Anim. Gen. 2013
10 9,289,183 9,488,500 197 0.0785 0.025 ESRRG Puberty, cattle Fortes et al., Anim. Gen. 20138 71,847,930 72,567,179 203 0.0769 0.029 NPFFR2 GnIH receptor5 78,429,610 78,537,181 208 0.0759 0.035 NELL2 Glutamatergic neurons Ha et al. Neuroendo 2008
224 5-SNP windows accounting for 59.3% of genetic variance 15 previously identified with puberty failure in gilts (Nonneman et al., 2013)7 in common with Tart et al., 2013 for age at puberty in gilts6 in common with GWAS for age at menarche in humans.
Output from GENSEL (http://bigs.ansci.iastate.edu)
Schneider et al., USMARC, unpulished
U.S. Meat Animal Research CenterReproduction Research Unit
•Neuroanatomical and in vitro data support a role for RFRP-3 in suppressing activity of the gonadotropic axis of mammals.
•Both centrally and at the pituitary gland
•Effects of RFRP-3 on LH secretion in vivo have been inconsistent (rodents, sheep, cattle, pigs, horses).
•Suppressive effect•No effect•Stimulatory effect
•The LH stimulatory action of NPFFR2 antagonist and the presence of NPFFR2 was candidate gene in GWAS are suggestive of a role of RFRP peptides in onset of puberty in the gilt.
U.S. Meat Animal Research CenterReproduction Research Unit
Nesfatin-1 is a product of the NUCB2 gene
Expressed (gene & protein) in adipose tissue and hypothalamus
negative energy balance
Produced and secreted by adipose tissue and 3T3L1 cells
PPARγ stimulated genecrosses blood-brain-barrierneg. correlated to BMI
Suppresses food intake and BW in the rat
ICV injection of antisense-morpholino oligonucleotidessuppressed LH secretion and delayed puberty in female rats
Nesfatin-1: Novel adipokine linking fat and reproduction
U.S. Meat Animal Research CenterReproduction Research Unit
Genomic association of NUCB2 with production traits
No association of NUCB2 gene with adiposity traits of pigs (barrows and gilts; n = 1,145) at 22 wk of age.
BW and ADG of pigs at 22 wk of age were modestly associated with SNP in the porcine NUCB2 gene.
Positive relationship of BW and body fatness with age a puberty in gilts.
Lents et al., 2013 Dom. Anim. Endocrinol.
U.S. Meat Animal Research CenterReproduction Research Unit
1 M K W R T I L L Q Y W F F L V T Y V F T 1 ATGAAGTGGAGGACCATCCTTCTACAATATTGGTTTTTCTTGGTTACATATGTATTTACT 21 A L E A V P I D I D K T K V K N T Q P V 61 GCTCTTGAAGCTGTGCCTATAGACATAGACAAAACAAAAGTAAAAAATACTCAGCCTGTG 41 D S A K I E P P D T G L Y Y D E Y L K Q 121 GACAGTGCGAAGATAGAACCACCAGATACTGGACTTTATTATGATGAATACCTCAAGCAA 61 V I D V L E T D N H F R E K L Q K A D I 181 GTGATTGATGTGCTGGAAACAGATAATCATTTCAGAGAAAAACTACAGAAAGCAGACATA 81 E E I K S G R L S R E L D L V S H H V R 241 GAGGAAATAAAGAGTGGAAGATTAAGCAGAGAGCTTGATTTAGTAAGTCACCATGTGAGG 101 T K L D E L K R Q E V A R L R 301 ACAAAACTTGATGAACTGAAAAGGCAAGAAGTGGCAAGGTTACGAA
cDNA and predicted peptide sequence of porcine nesfatin-1 and identity to the human and rat orthologs.
Human 1 VPIDIDKTKVQNIHPVESAKIEPPDTGLYYDEYLKQVIDVLETDKHFREKLQKADIEEIKSGRLSKELDLVSHHVRTKLDEL 82Porcine 1 VPIDIDKTKVKNTQPVDSAKIEPPDTGLYYDEYLKQVIDVLETDNHFREKLQKADIEEIKSGRLSRELDLVSHHVRTKLDEL 82 rat 1 VPIDVDKTKVHNVEPVESARIEPPDTGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRSGRLSQELDLVSHKVRTRLDEL 82 .... ..... . .. .. .................. ..... .............. ..... ....... .. ....
Identity: Human = 92% Rat = 85%
Lents et al., Dom. Anim. Endocrinol. 2013
U.S. Meat Animal Research CenterReproduction Research Unit
Corresponds to 8, 24, 48, 72 h after ICV treatment.
*Saline different from all other treatments, P < 0.001
0
2
4
6
8
10
12
0 4 20 44 68
Cum
ulat
ive
feed
, kg
Hours after presentation of feed
Saline (n = 4)Leptin (100 µg; n = 4)Nesfatin-1 (100 µg; n = 4)
**
*
*
ICV injection of Nesfatin-1 suppresses feed intake in prepubertal gilts (treatment x time, P < 0.001).
Lents et al., 2013 Dom. Anim. Endocrinol.
U.S. Meat Animal Research CenterReproduction Research Unit
LH in serum of gilts after ICV injection of Nesfatin-1 (treatment x time, P < 0.01).
Lents et al., 2013 Dom. Anim. Endocrinol.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Saline Nesfatin‐1
pre‐icv
post‐icv
0.00.20.40.60.81.01.21.41.6
-180
-150
-120 -90
-60
-30 0 30 60 90 120
150
180
210
240
Saline
Nesfatin-1
Time from i.c.v. treatment (min)LH
(ng/
mL)
Mea
n LH
(ng/
mL)
2 h
bef
ore
and
afte
r i.c
.v. t
reat
men
t
a
bP < 0.001
U.S. Meat Animal Research CenterReproduction Research Unit
Nesfatin-1 identity highly conserved in the pig.
Expression of Nesfatin in pigs:Adipose tissue, depot dependent (Lents et al., 2013).
Hypothalamus and brain stem (Gaige et al., 2013).
ICV injection of nesfatin-1 in prepubertal gilts:Suppressed feed intake Increased LH secretion
Functional data supports the genomic association of NUCB2 with BW traits.
Summary
U.S. Meat Animal Research CenterReproduction Research Unit
Adiposity signalsLeptin, Insulin, Nesfatin-1
Feed IntakeGrowth
Oxidizable fuelsFFA, Glucose
Brain Stem
Livermetabolism
NTS
Vagus
2nd orderneurons
LH and FSHReproduction
Hypothalamus
+/-
ARC, PVNMSH – KiSSNPY – GnRH
+/-
Lents et al., 2013 Gonadotropin
U.S. Meat Animal Research CenterReproduction Research Unit
CollaboratorsC. Rick Barb, ARS Gary Hausman, ARS J. Joe Ford, ARSDan Nonneman, ARS Stan Hileman, WVU Kazuyoshi Tsutsui, Waseda Univ., JPJenny Thorson, ARS Bob Cushman, ARS Lea Remple, ARSKati Porter, WVU William Oliver, ARS Lique Coolen, Ole MissJeremy Miles, ARS Chrys Fergani, Ole Miss Elane Wright, ARSKrzysztof Czaja, WSU Lanny Meadows, WVU Mike Azain, UGAGary Rohrer, ARS Jeff Vallet, ARS Neely Heidorn, UGARomdhane Rekaya, UGA Jack Dekkers, ISU Chris Tuggle, ISU
Technical SupportSuzy Hassler Sue HauverShanda Watts Carl RogersMichelle McManus L. Lee-RutherfordTroy Gramke John GambleSherie Hulsey T.C. TsaiRobert Strickland Bruce LarsenScott Whitcomb Swine Operations
Grant no. 2005-35203-1685 and 2011-67015-30059 (Lents) 2013-00896 (Hileman)
Acknowledgments
U.S. Meat Animal Research CenterReproduction Research Unit
Neuropeptide Feeding Effect Reg. by Leptin* NPYOrexinMCHGalaninAGRP*α-MSH/POMCCRH/UrocortinCARTNeurotensin 1
Regulation of appetite by neuropeptids
Barb et al., ICAFE 2008