Avian Neuropeptide Y: Beyond Feed Intake Regulation

Abstract

Neuropeptide Y (NPY) is one of the most abundant and ubiquitously expressed neuropeptides in both the central and peripheral nervous systems, and its regulatory effects on feed intake and appetite- have been extensively studied in a wide variety of animals, including mammalian and non-mammalian species. Indeed, NPY has been shown to be involved in the regulation of feed intake and energy homeostasis by exerting stimulatory effects on appetite and feeding behavior in several species including chickens, rabbits, rats and mouse. More recent studies have shown that this neuropeptide and its receptors are expressed in various peripheral tissues, including the thyroid, heart, spleen, adrenal glands, white adipose tissue, muscle and bone. Although well researched centrally, studies investigating the distribution and function of peripherally expressed NPY in avian (non-mammalian vertebrates) species are very limited. Thus, peripherally expressed NPY merits more consideration and further in-depth exploration to fully elucidate its functions, especially in non-mammalian species. The aim of the current review is to provide an integrated synopsis of both centrally and peripherally expressed NPY, with a special focus on the distribution and function of the latter.

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Citation: Greene, E.S.; Abdelli, N.;
Dridi, J.S.; Dridi, S. Avian
Neuropeptide Y: Beyond Feed Intake
Regulation. Vet. Sci. 2022,9, 171.
https://doi.org/10.3390/
vetsci9040171
Academic Editors: Lucianna
Maruccio and Carla Lucini
Received: 2 March 2022
Accepted: 29 March 2022
Published: 1 April 2022
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veterinary
sciences
Review
Avian Neuropeptide Y: Beyond Feed Intake Regulation
Elizabeth S. Greene 1, Nedra Abdelli 1,2, Jalila S. Dridi 3and Sami Dridi 1, *
1Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA;
esgreene@uark.edu (E.S.G.); nedra.abdelli@uab.cat (N.A.)
2Animal Nutrition and Welfare Service, Department of Animal and Food Sciences,
Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
3École Universitaire de Kinésithérapie, Universitéd’Orléans, Rue de Chartres, 45100 Orleans, France;
jaliladr2@gmail.com
*Correspondence: dridi@uark.edu; Tel.: +1-(479)-575-2853
Abstract:
Neuropeptide Y (NPY) is one of the most abundant and ubiquitously expressed neuropep-
tides in both the central and peripheral nervous systems, and its regulatory effects on feed intake
and appetite- have been extensively studied in a wide variety of animals, including mammalian
and non-mammalian species. Indeed, NPY has been shown to be involved in the regulation of feed
intake and energy homeostasis by exerting stimulatory effects on appetite and feeding behavior in
several species including chickens, rabbits, rats and mouse. More recent studies have shown that
this neuropeptide and its receptors are expressed in various peripheral tissues, including the thyroid,
heart, spleen, adrenal glands, white adipose tissue, muscle and bone. Although well researched
centrally, studies investigating the distribution and function of peripherally expressed NPY in avian
(non-mammalian vertebrates) species are very limited. Thus, peripherally expressed NPY merits
more consideration and further in-depth exploration to fully elucidate its functions, especially in
non-mammalian species. The aim of the current review is to provide an integrated synopsis of both
centrally and peripherally expressed NPY, with a special focus on the distribution and function of
the latter.
Keywords:
neuropeptide Y; feed intake regulation; adipose tissue; liver; immune system; gut; muscle;
bone; chickens
1. Introduction
Neuropeptide Y (NPY) is a 36-amino acid peptide, which along with peptide YY (PYY)
and pancreatic polypeptide (PP), belongs to the pancreatic polypeptide family. First isolated
from pig brain in 1982 [
1
], NPY is considered to be the most conserved peptide among
vertebrate species. It is one of the most abundant and ubiquitously expressed neuropeptides
in both the central and peripheral nervous systems [
2
], with the arcuate nucleus (also
known as the infundibular nucleus in birds) as its main source [
3
,
4
]. Considered as a
potent orexigenic neuropeptide, NPY is one of the major regulators of feed intake, energy
homeostasis and appetite in several species. Indeed, central injection of NPY has been
shown to increase food intake in rats [
5
,
6
], sheep [
7
], pigs [
8
], and chickens [
9
,
10
]. Moreover,
feed deprivation in rodents has been shown to up-regulate the hypothalamic expression of
NPY to incite feed intake and maintain homeostasis [
11
], while rats subjected to repeated
administration of NPY show hyperphagia and increased body weight gain leading to
obesity [
12
]. Aside from its role in the stimulation of food intake, NPY has been also shown
to be expressed in peripheral tissues but its functions are still not fully elucidated. NPY has
been shown to be highly expressed in the adrenal glands [
13
], white adipose tissue [
14
],
and bone [
15
,
16
] among others, indicating the potential involvement of this neuropeptide
in a wide range of physiological responses including adipogenesis, regulation of bone
mass, and energy metabolism [
17
], locomotion [
18
], anxiety [
19
] learning and memory [
20
],
Vet. Sci. 2022,9, 171. https://doi.org/10.3390/vetsci9040171 https://www.mdpi.com/journal/vetsci

Vet. Sci. 2022,9, 171 2 of 18
epilepsy [
21
], circadian rhythm [
22
], and cardiovascular function [
23
]. To exert its effects,
NPY binds to specific NPY receptors, which are members of the class A G-protein coupled
receptors [24].
Chicken is considered as an important scientific and commercial species and the focus
on studying the growth and metabolism of chicken, both of which are influenced by NPY,
led to the identification of avian NPY in the early 1990s [
25
]. Recently, six receptor subtypes
for NPY have been identified and cloned in diverse avian tissues (Y1, Y2, Y4, Y5, Y6, and
Y7) [26–28] and their characteristics have been investigated.
Although central administration of NPY seems to have a similar effect on feed intake
regulation [
29
] in mammalian and avian species [
30
], much effort is still needed to under-
stand the physiological functions of peripheral NPY in chickens. Therefore, the purpose
of the current paper is to review the physiological functions of avian NPY at both the
central and peripheral level, for subsequent identification of research gaps that need to be
addressed in the future.
2. Structure of NPY
The NPY system is an ancient signaling pathway, as it is found in both vertebrates
and invertebrates, highlighting a potential evolutionarily conserved function [
31
]. Struc-
turally similar to PYY and PP, the amino acid sequence of NPY is one of the most highly
conserved neuropeptides. As shown in Table 1, there is over 90% identity in the amino
acid sequence among mammalian species, and greater than 80% identity between chicken
and other species (Figure 1A) [
32
]. Additionally, phylogenic analysis indicates that the
non-mammalian species share a common ancestor that diverged from mammals in their
NPY sequence [
33
] (Figure 1B). The molecular structure contains numerous hydrophobic in-
teractions, as well as and N-terminal polyproline-II-like helix and a C-terminal
α
-helix [
34
].
The N-terminal portion is responsible for interactions with various receptors, as studies
have shown this segment interacts with Y1 but not Y2 [
35
,
36
]. Additionally, NPY contains
two translation initiation sequences, allowing for the production of both full-length and a
truncated NPY, containing only peptides 17–36 [
37
], which can further differentially bind
to receptors.
Table 1. Amino acid sequence homology of NPY among several species.
Zebrafish Mouse Rat Human Bovine Sheep Xenopus Chicken
Zebrafish 100.00 67.71 67.71 66.67 65.62 64.58 62.50 65.62
Mouse 67.71 100.00 98.97 92.78 90.72 89.69 74.23 81.44
Rat 67.71 98.97 100.00 93.81 91.75 90.72 75.26 82.47
Human 66.67 92.78 93.81 100.00 94.85 93.81 78.35 84.54
Bovine 65.62 90.72 91.75 94.85 100.00 98.97 76.29 84.54
Sheep 64.58 89.69 90.72 93.81 98.97 100.00 75.26 83.51
Xenopus 62.50 74.23 75.26 78.35 76.29 75.26 100.00 84.54
Chicken 65.62 81.44 82.47 84.54 84.54 83.51 84.54 100.00
Numbers indicate percent identity between species, as determined by Clustal Omega 1.2.4 [26].

Vet. Sci. 2022,9, 171 3 of 18
Vet. Sci. 2022, 9, x 3 of 18
Figure 1. NPY amino acid sequence alignments (A) and phylogeny (B). Amino acid sequences were
aligned using Clustal Omega 1.2.4 [32]. * positions with a single, fully conserved residue. “:” (colon)
conservation between groups of strongly similar properties. “.” (period) conservation between
groups of weakly similar properties. Phylogenetic tree generated with MEGA7: Molecular Evolu-
tionary Genetics Analysis version 7.0 for bigger datasets [33].
3. NPY Receptors
The physiological effects of NPY are exerted through binding to specific Y receptors,
which are part of the G-protein-coupled (GPCR) family [24]. To date, 7–8 different recep-
tors have been identified, though their presence and functionality differs among species.
In mammals, Y1, Y2, Y4, Y5, and Y6 have been identified [38], whereas in fish, chicken
and other avian species, Y7 is additionally present [27], and in frogs [39] and telost fish
[40], Y8a and Y8b may also be present. The Y receptors have a long evolutionary history
and are grouped into three subfamilies based on the homology and similarity of their
amino acid sequences. The Y1 subfamily consists of Y1, Y4, Y6, and Y8, with sequence
homology ranging from 40 to 60% [41]. Y1 only binds to intact NPY and PYY peptides
[42,43], whereas Y4 preferentially binds PP over NPY or PYY [44]. Additionally, Y4 shows
low sequence homology among different species, making it one of the most rapidly evolv-
ing GPCR known [41]. Interestingly, Y6 is the most variable in expression and functional-
ity across species, with a complete absence in rat [45], it is truncated in many other mam-
mals or in specific tissues [46], and present and functional in chicken [27,28,47]. The Y2
family consists of Y2 and Y7, and likely arose from a gene duplication of Y1 in an inverte-
brate ancestor, creating Y2 [31]. Unlike Y1, Y2 can bind truncated forms of NPY in mam-
mals and chicken [48,49], though with less affinity in fish [50]. The Y5 subfamily consists
of a single member, Y5, that similarly came from a duplication of Y1, after the creation of
Y2 [31], but only has approximately 20% sequence homology with Y1 or Y2 [51]. Along
with Y1, Y5 is the receptor responsible for the canonical orexigenic effects of NPY [52–55].
4. NPY Downstream Signaling Cascades
Mammalian NPY receptors couple primarily through Giα to inactivate adenylate
cyclase (AC) and decrease cAMP synthesis, which in turn leads to a reduction in protein
Figure 1.
NPY amino acid sequence alignments (
A
) and phylogeny (
B
). Amino acid sequences were
aligned using Clustal Omega 1.2.4 [
32
]. * positions with a single, fully conserved residue. “:” (colon)
conservation between groups of strongly similar properties. “.” (period) conservation between groups
of weakly similar properties. Phylogenetic tree generated with MEGA7: Molecular Evolutionary
Genetics Analysis version 7.0 for bigger datasets [33].
3. NPY Receptors
The physiological effects of NPY are exerted through binding to specific Y receptors,
which are part of the G-protein-coupled (GPCR) family [
24
]. To date, 7–8 different receptors
have been identified, though their presence and functionality differs among species. In
mammals, Y1, Y2, Y4, Y5, and Y6 have been identified [
38
], whereas in fish, chicken and
other avian species, Y7 is additionally present [
27
], and in frogs [
39
] and telost fish [
40
],
Y8a and Y8b may also be present. The Y receptors have a long evolutionary history and
are grouped into three subfamilies based on the homology and similarity of their amino
acid sequences. The Y1 subfamily consists of Y1, Y4, Y6, and Y8, with sequence homology
ranging from 40 to 60% [
41
]. Y1 only binds to intact NPY and PYY peptides [
42
,
43
], whereas
Y4 preferentially binds PP over NPY or PYY [
44
]. Additionally, Y4 shows low sequence
homology among different species, making it one of the most rapidly evolving GPCR
known [
41
]. Interestingly, Y6 is the most variable in expression and functionality across
species, with a complete absence in rat [
45
], it is truncated in many other mammals or
in specific tissues [
46
], and present and functional in chicken [
27
,
28
,
47
]. The Y2 family
consists of Y2 and Y7, and likely arose from a gene duplication of Y1 in an invertebrate
ancestor, creating Y2 [
31
]. Unlike Y1, Y2 can bind truncated forms of NPY in mammals and
chicken [
48
,
49
], though with less affinity in fish [
50
]. The Y5 subfamily consists of a single
member, Y5, that similarly came from a duplication of Y1, after the creation of Y2 [
31
], but
only has approximately 20% sequence homology with Y1 or Y2 [
51
]. Along with Y1, Y5 is
the receptor responsible for the canonical orexigenic effects of NPY [52–55].
4. NPY Downstream Signaling Cascades
Mammalian NPY receptors couple primarily through Gi
α
to inactivate adenylate cy-
clase (AC) and decrease cAMP synthesis, which in turn leads to a reduction in protein kinase

Vet. Sci. 2022,9, 171 4 of 18
A (PKA) activity [
56
]. NPY receptors can also, through Gq or Gi
β
/
γ
, activate phospholipase
C (PLC) and protein kinase C (PKC), which in turn induces mitogen-activated protein ki-
nase (MAPK) activation including the phosphorylation of the extracellular signal-regulated
kinase (ERK1/2) [
57
]. A phosphatidylinositol-3-kinase (PI-3-K) pathway upstream of
ERK1/2 activation has also been identified [
58
] (Figure 2). Although such downstream
signaling is not well confirmed in chickens, phosphorylated levels of protein kinase b (Akt),
forkhead box protein O1 (FOXO1), and ribosomal protein S6 kinase (S6K) were increased in
the hypothalamus of fasted and re-fed chicks, which correlated with increases in the plasma
concentration of insulin [
59
]. Similarly, central administration of insulin by ICV injection
increased the phosphorylation of Akt, FOXO1, and S6K in the hypothalamus of chicken.
Central inhibition of PI3K (by LY294002) or mTOR (by rapamycin) was able to increase the
feed intake, further highlighting possible NPY involvement [
59
]. However, further studies
are warranted to fully determine whether or not these downstream mediators are activated
by NPY.
Vet. Sci. 2022, 9, x 4 of 18
kinase A (PKA) activity [56]. NPY receptors can also, through Gq or Giβ/γ, activate phos-
pholipase C (PLC) and protein kinase C (PKC), which in turn induces mitogen-activated
protein kinase (MAPK) activation including the phosphorylation of the extracellular sig-
nal-regulated kinase (ERK1/2) [57]. A phosphatidylinositol-3-kinase (PI-3-K) pathway up-
stream of ERK1/2 activation has also been identified [58] (Figure 2). Although such down-
stream signaling is not well confirmed in chickens, phosphorylated levels of protein ki-
nase b (Akt), forkhead box protein O1 (FOXO1), and ribosomal protein S6 kinase (S6K)
were increased in the hypothalamus of fasted and re-fed chicks, which correlated with
increases in the plasma concentration of insulin [59]. Similarly, central administration of
insulin by ICV injection increased the phosphorylation of Akt, FOXO1, and S6K in the
hypothalamus of chicken. Central inhibition of PI3K (by LY294002) or mTOR (by rapamy-
cin) was able to increase the feed intake, further highlighting possible NPY involvement
[59]. However, further studies are warranted to fully determine whether or not these
downstream mediators are activated by NPY.
Figure 2. NPY downstream signaling pathways. The representation shows the potential main path-
ways through which NPY signals. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate;
CREB, cAMP response element binding protein; ERK, extracellular signal-regulated kinase; FoxO,
Forkhead Box O; PI3K, phosphatidylinositol-3-kinase; PKA, protein kinase A; PKC, protein kinase
C; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding
protein. The figure was made using Biorender.com.
5. Tissue Distribution of NPY System
NPY was first discovered in the porcine brain [1], and has since been well character-
ized in the brain and central nervous system of numerous species [60–64], with the great-
est concentration seen in the arcuate nucleus of the hypothalamus [65]. In the chicken,
Figure 2.
NPY downstream signaling pathways. The representation shows the potential main path-
ways through which NPY signals. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate;
CREB, cAMP response element binding protein; ERK, extracellular signal-regulated kinase; FoxO,
Forkhead Box O; PI3K, phosphatidylinositol-3-kinase; PKA, protein kinase A; PKC, protein kinase C;
PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein.
The figure was made using Biorender.com.
5. Tissue Distribution of NPY System
NPY was first discovered in the porcine brain [
1
], and has since been well characterized
in the brain and central nervous system of numerous species [
60
–
64
], with the greatest
concentration seen in the arcuate nucleus of the hypothalamus [
65
]. In the chicken, NPY
is widely distributed across the brain [
66
], with in situ hybridization studies identifying
high abundances of NPY mRNA in specific neurons, including the hippocampus, nucleus

Vet. Sci. 2022,9, 171 5 of 18
commissurae pallii, infundibular hypothalamic nucleus, nucleus pretectalis pars ventralis
and neurons around the nucleus rotundus [67].
Outside of the central nervous system, NPY and its receptors have more recently been
identified in the periphery, though its role in these tissues is still being elucidated. Bone [
15
],
adipose tissue [
14
], platelets [
68
] and intestine are all known mammalian sources of NPY.
In chicken, NPY has also been identified in numerous tissues, including ovary, testes, heart,
kidney, lung, skeletal muscle, fat, pancreas, liver, and intestine [
28
,
47
,
69
], though it has not
been detected in spleen. The Y1 receptor is expressed in all chicken tissues studied to date,
though at differing relative abundance, with greater expression in the ovary, heart, kidney,
liver, and muscle [
28
,
47
]. The Y2 receptor has similar tissue expression in chicken as in
rainbow trout [
50
] and frog [
39
], with the heart, duodenum, liver, lung, muscle, ovary, testes,
pituitary, spleen, and pancreas [
26
] all expressing this receptor. Interestingly, this differs
from humans, where Y2 is often lowly detected or undetectable [
70
]. These differences
likely relate to some of the different effects seen in mammalian and non-mammalian species,
such as NPY-mediated lipid accumulation in chickens [
71
]. Likely due to their similarity
in structure [
27
], Y7 shows a comparable pattern of wide tissue distribution, and likely
serves an overlapping functional role as well. There have been few studies examining Y5
in the periphery. However, gene transcripts have been amplified in pancreas, testes, ovary,
duodenum [26], and muscle [47] of chicken.
6. Physiological Functions of NPY
6.1. Central Functions of NPY
The balance between feed intake and energy homeostasis is a complex system of regu-
latory mechanisms and pathways in multiple organ systems. These mechanisms include
signaling molecules such as nutrients, metabolites, hormones, neuropeptides, and receptors
that originate from the central and peripheral nervous system, as well as other tissues
such as the gut, muscle, liver, and adipose. Together, these molecules interact via feedback
mechanisms to convey signals and information about the whole-body nutrient status of an
organism [
72
,
73
]. In the arcuate nucleus (the equivalent of the infundibular nucleus in chick-
ens) of the hypothalamus, feeding signals are integrated by two groups of neurons with
opposing functions [
74
]. Stimulation of the NPY/agouti-related protein (AgRP) neurons
results in an orexigenic response and increased energy intake and storage, whereas stimu-
lation of the proopiomelanocortin (POMC) and cocaine/amphetamine-regulated transcript
(CART) neurons induces a decrease in energy intake and storage [
72
] (Figure 3). Indeed,
intraperitoneal injection of recombinant NPY increased feed intake in broiler (meat-type)
chickens [
47
], being in concordance with previous studies using central administration of
NPY in broilers [
30
,
75
–
77
] and layer (egg-type) chickens [
78
,
79
]. The effects of NPY on
feeding behavior of chickens are mediated by Y1 and Y5 [
80
]. On the contrary, the encoded
precursor protein of POMC in chickens was shown to produce bioactive alpha-melanocyte
stimulating hormone (
α
MSH). The injection of this hormone has been shown to inhibit
feed intake in chickens [
77
], an effect antagonized by AgRP through binding and signaling
to specific melanocortin receptor subtypes (MC3R and MC4R) [
72
]. On the other hand,
previous studies have shown NPY mRNA levels to be up-regulated in the hypothalamus
by fasting [81], feed restriction [82], and down-regulated by insulin [83] and leptin [84].

Vet. Sci. 2022,9, 171 6 of 18
Vet. Sci. 2022, 9, x 6 of 18
Figure 3. A proposed model describing central feed intake regulation in chickens through hypotha-
lamic (an)orexigenic neuropeptides. AgRP, agouti-related peptide; NPY, neuropeptide Y; POMC,
proopiomelanocortin. (-) inhibition; (+) stimulation. The figure was made using Biorender.com.
6.2. Peripheral Functions of NPY
6.2.1. NPY in Adipose Tissue
The factors that regulate energy balance are complex and are not limited to the central
nervous system. Energy storage, particularly in adipose tissue, the primary energy reser-
voir for the body, is an important component of the overall energy status of an organism.
White adipose tissue contains a heterogeneous mixture of mature adipocytes, preadipo-
cytes, mesenchymal stem cells, immune cells, and a matrix of collagen fibers, and is the
main adipose depot for energy storage in the form of triacylglycerol. Brown adipose, on
the other hand, is responsible for heat production and non-shivering thermogenesis; how-
ever, this form of fat has not been found in chicken [85]. Concurrently, with the known
role of NPY in centrally regulating feed intake and energy balance, it is not surprising that
it also plays a role in cross-talk between the hypothalamus and adipose tissue. Several
studies have shown that the sympathetic nerve terminals in adipose tissue, as well as ad-
ipocytes themselves [86], can secrete NPY and promote adipogenesis and inhibit lipolysis
[87].
Much of our understanding of avian NPY in adipose comes from in vitro studies
using culture of pre-adipocytes isolated from the abdominal fat of chickens. In these cells,
treatment with NPY promotes differentiation and lipid accumulation, an effect mediated
by Y2 [71]. During differentiation, the addition of NPY is associated with increased glyc-
erol-3-phosphate dehydrogenase (GAPDH) activity, which leads to the production of
glycerol-3-phosphate, a key molecule in triacylglycerol synthesis. Additionally, gene ex-
pression of several important transcription factors involved in adipocyte differentiation
(peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding
protein alpha (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1)), are
all affected by NPY [71] (Figure 4). Similarly, NPY treatment promotes preadipocyte ac-
tivity during the early phase of chick development. This results in increased lipid accu-
mulation, enhanced expression of SREBP, C/EBPβ, and fatty acid binding protein 4
(FABP4), increased GAPDH activity, and decreased expression of Krüppel-like factor 7
Figure 3.
A proposed model describing central feed intake regulation in chickens through hypotha-
lamic (an)orexigenic neuropeptides. AgRP, agouti-related peptide; NPY, neuropeptide Y; POMC,
proopiomelanocortin. (-) inhibition; (+) stimulation. The figure was made using Biorender.com.
6.2. Peripheral Functions of NPY
6.2.1. NPY in Adipose Tissue
The factors that regulate energy balance are complex and are not limited to the central
nervous system. Energy storage, particularly in adipose tissue, the primary energy reservoir
for the body, is an important component of the overall energy status of an organism. White
adipose tissue contains a heterogeneous mixture of mature adipocytes, preadipocytes,
mesenchymal stem cells, immune cells, and a matrix of collagen fibers, and is the main
adipose depot for energy storage in the form of triacylglycerol. Brown adipose, on the
other hand, is responsible for heat production and non-shivering thermogenesis; however,
this form of fat has not been found in chicken [
85
]. Concurrently, with the known role of
NPY in centrally regulating feed intake and energy balance, it is not surprising that it also
plays a role in cross-talk between the hypothalamus and adipose tissue. Several studies
have shown that the sympathetic nerve terminals in adipose tissue, as well as adipocytes
themselves [86], can secrete NPY and promote adipogenesis and inhibit lipolysis [87].
Much of our understanding of avian NPY in adipose comes from
in vitro
studies
using culture of pre-adipocytes isolated from the abdominal fat of chickens. In these cells,
treatment with NPY promotes differentiation and lipid accumulation, an effect mediated by
Y2 [
71
]. During differentiation, the addition of NPY is associated with increased glycerol-3-
phosphate dehydrogenase (GAPDH) activity, which leads to the production of glycerol-3-
phosphate, a key molecule in triacylglycerol synthesis. Additionally, gene expression of
several important transcription factors involved in adipocyte differentiation (peroxisome
proliferator-activated receptor gamma (PPAR
γ
), CCAAT/enhancer binding protein alpha
(C/EBP
α
), and sterol regulatory element-binding protein 1 (SREBP1)), are all affected by
NPY [
71
] (Figure 4). Similarly, NPY treatment promotes preadipocyte activity during the
early phase of chick development. This results in increased lipid accumulation, enhanced
expression of SREBP, C/EBP
β
, and fatty acid binding protein 4 (FABP4), increased GAPDH
activity, and decreased expression of Krüppel-like factor 7 (KLF7) and DNA topoisomerase
II alpha (TOP2A), all indicative of greater adipogenic activity [88].

Vet. Sci. 2022,9, 171 7 of 18
Vet. Sci. 2022, 9, x 7 of 18
(KLF7) and DNA topoisomerase II alpha (TOP2A), all indicative of greater adipogenic
activity [88].
Figure 4. Peripheral physiological functions of NPY. VLDL, very low-density lipoprotein. The
figure was made using Biorender.com.
These results have more recently been verified in vivo, as NPY, Y1, and lipolytic fac-
tors adipose triglyceride lipase (ATGL) and FABP4 were decreased in the subcutaneous
fat depot from day 0 to 4 post-hatch, indicating the involvement of the NPY system in the
mobilization of fat from this early-life energy reservoir [89].
NPY has also been shown to inhibit lipolysis via Y1. This was evidenced through the
reduction in plasma non-esterified fatty acids (NEFAs) at 1 and 12 h post injection of NPY
in chickens [90]. On the other hand, peripheral NPY has been shown to differentially affect
adipogenesis and lipolysis in chicks from lines selected for low (LWS) or high body weight
(HWS) [90]. The results of this study indicated higher rates of lipolysis in LWS and adipo-
genesis in HWS.
6.2.2. NPY in the Liver
The regulation of hepatic lipid homeostasis is at least partially controlled by interac-
tion with the central nervous system, and defects within this interaction have been asso-
ciated with dyslipidemia, such as obesity, diabetes, and metabolic syndrome [91]. As such,
nerve fibers within the liver have been shown to express NPY in mammalian (mouse, rat,
guinea pig, dog, monkey, and human) and non-mammalian (carp, bullfrog, turtle, and
chicken) species [92]. In mammalian studies, centrally produced NPY has been shown to
be involved in lipid [91,93,94] and glucose [94,95] metabolism regulation, both biological
processes regulated by the liver. Central administration of NPY increased very low-den-
sity lipoprotein (VLDL) secretion in rats, independently of food intake [93]. This effect is
mediated by Y1 as the Y1 agonist, [F7, P34]-NPY, increased VLDL secretion via activation
of stearoyl-CoA desaturase 1 (SCD1), ADP-ribosylation factor 1 (ARF1), and lipin1, all
necessary factors for VLDL maturation and secretion [91]. More recently, it has been
shown that hepatic stellate cells can secrete NPY and that NPY is important in the fibro-
genic response that can be seen in diseased liver states [96].
In chicken, NPY and its receptors are expressed in the liver [69,97], except for Y7 [27],
though their role has yet to be fully explored. NPY and Y1, Y2, and Y5 all increase in
Figure 4.
Peripheral physiological functions of NPY. VLDL, very low-density lipoprotein. The figure
was made using Biorender.com.
These results have more recently been verified
in vivo
, as NPY, Y1, and lipolytic factors
adipose triglyceride lipase (ATGL) and FABP4 were decreased in the subcutaneous fat
depot from day 0 to 4 post-hatch, indicating the involvement of the NPY system in the
mobilization of fat from this early-life energy reservoir [89].
NPY has also been shown to inhibit lipolysis via Y1. This was evidenced through
the reduction in plasma non-esterified fatty acids (NEFAs) at 1 and 12 h post injection of
NPY in chickens [
90
]. On the other hand, peripheral NPY has been shown to differentially
affect adipogenesis and lipolysis in chicks from lines selected for low (LWS) or high body
weight (HWS) [
90
]. The results of this study indicated higher rates of lipolysis in LWS and
adipogenesis in HWS.
6.2.2. NPY in the Liver
The regulation of hepatic lipid homeostasis is at least partially controlled by interaction
with the central nervous system, and defects within this interaction have been associated
with dyslipidemia, such as obesity, diabetes, and metabolic syndrome [
91
]. As such,
nerve fibers within the liver have been shown to express NPY in mammalian (mouse, rat,
guinea pig, dog, monkey, and human) and non-mammalian (carp, bullfrog, turtle, and
chicken) species [
92
]. In mammalian studies, centrally produced NPY has been shown to
be involved in lipid [
91
,
93
,
94
] and glucose [
94
,
95
] metabolism regulation, both biological
processes regulated by the liver. Central administration of NPY increased very low-density
lipoprotein (VLDL) secretion in rats, independently of food intake [
93
]. This effect is
mediated by Y1 as the Y1 agonist, [F7, P34]-NPY, increased VLDL secretion via activation
of stearoyl-CoA desaturase 1 (SCD1), ADP-ribosylation factor 1 (ARF1), and lipin1, all
necessary factors for VLDL maturation and secretion [
91
]. More recently, it has been shown
that hepatic stellate cells can secrete NPY and that NPY is important in the fibrogenic
response that can be seen in diseased liver states [96].
In chicken, NPY and its receptors are expressed in the liver [
69
,
97
], except for Y7 [
27
],
though their role has yet to be fully explored. NPY and Y1, Y2, and Y5 all increase in
hepatic expression from day 4 to 14 post-hatch [
98
], a time during which lipogenesis is
increasing. Unlike most mammals, where the liver and adipose share the role of de novo
lipogenesis, in chicken over 95% of de novo fatty acids are synthesized by the liver [
99
],
making this a key organ in overall energy homeostasis. Therefore, further research into the

Vet. Sci. 2022,9, 171 8 of 18
role of hepatic NPY in chicken may impact not only the poultry industry, but has potential
effects as a clinically relevant human model for lipid dysmetabolism.
6.2.3. NPY in the Muscle
The modern chicken has been selected for fast growth rate and feed efficiency [
100
],
with the majority of this increase seen in the commercially important breast muscle [
101
].
As NPY has a significant role in energy balance regulation, it is conceivable that it could be
an important mediator of muscle growth. Indeed, several recent studies have characterized
the expression of the NPY system in avian muscle and myoblast cells. Similar to the
effect seen centrally, fasting increased the gene expression of NPY and its receptors in
both breast and leg muscle of 9-day-old broilers [
47
]. In the same study, intraperitoneal
administration of NPY up-regulated gene expression of the NPY system in leg and breast
muscle, particularly Y1, Y2, Y4, Y6, and Y7, though this effect was dose dependent in leg
muscle, with increases seen at the low dose and decreases at the higher doses [
47
]. This
suggests that NPY regulates its own system and that NPY might have paracrine/autocrine
functions. Of particular interest, NPY was also able to regulate mitochondrial function in
this metabolically active, fast-growing tissue, indicating that NPY plays a crucial role in
muscle energy metabolism.
Satellite cells are multi-potent cells that are important in muscle fiber growth and
regeneration. These cells fuse with existing muscle fibers and donate their nuclei, thereby
increasing protein production and hypertrophy, and allowing for muscle growth [
102
].
Studies using isolated chicken [
103
] and turkey [
103
,
104
] satellite cells have shown that
NPY is expressed by these cells and can be regulated by environmental factors. The effects
of thermal manipulation on the expression of the NPY system seem to be dependent on the
bird lines from which the cells were isolated, as well as the state of the cells (proliferative vs.
differentiating). As modern commercial broilers and turkeys have a relatively narrow range
of thermotolerance and are thereby susceptible to heat stress [
105
], these changes in NPY
and NPY receptors suggests that, in muscle, this neuropeptide may act as a hypothermic
regulatory agent. In chicken-derived satellite cells, the response to incubation temperature
was dependent on the type of birds from which the cells were isolated. In proliferating
cells isolated from two lines of chicken, neither NPY nor its receptors were greatly affected
by temperature. However, NPY, Y2, and Y5 were increased by elevated temperature
in differentiating satellite cells from Ross 708 broilers [
103
]. Similarly, NPY expression
was increased in turkey-derived satellite cells when incubated at 41 [
104
] or 43
◦
C [
103
]
as compared to lower temperatures. Concurrently, expression of receptors Y2 and Y5
were increased in proliferating satellite cells, whereas only Y2 was affected by increased
temperature in differentiating satellite cells [103].
6.2.4. NPY in the Bone
More than just a structural support for the body, bone is a dynamic tissue that under-
goes remodeling throughout the life of an organism. Bone homeostasis balances resorption
and formation, which when in disequilibrium, can lead to changes in the microarchitecture
and integrity of bone tissue [
106
]. The bone is innervated [
107
], giving the potential for a
direct link with centrally-mediated processes. Indeed, the NPY system has more recently
been identified as one of the key regulators of this important process as it is secreted by
nerve fibers in the marrow and vascular canals, and the Y1 [
15
,
108
] and Y2 [
109
] receptors
have been implicated in bone homeostasis. The regulation of bone mass via NPY differs
at the hypothalamic and bone level. Osteoblast specifc-Y1 knockout studies in mice have
shown that this receptor is critical for the actions of NPY directly at the bone [
108
], whereas
Y2 is critical for the central regulation of bone mass [
110
]. In further support of these
distinct roles, the expression of Y1 has been reported in osteoblastic bone marrow-derived
mesenchymal stem/stromal cells (BMSCs) and osteocytes, whereas Y2 is yet undetected
in bone cells. Interestingly, the effects of NPY
in vivo
are different from that seen in iso-
lated BMSCs.
In vivo
, during fasting when hypothalamic NPY is high, bone formation is

Vet. Sci. 2022,9, 171 9 of 18
reduced [
16
]. However, when studies are conducted
in vitro
, particularly with BMSCs, the
results can be controversial [
111
]. For instance, some studies showed an inhibition of BMSC
proliferation and osteoblast differentiation by NPY [
112
], while others reported enhanced
BMSC proliferation and osteoblastic activity with NPY treatment [
113
–
115
]. Regardless,
the effects of NPY seem to be conferred through the Wnt signaling pathway [
111
]. This
pathway is activated by NPY in a dose-dependent manner, where downstream activation
of
β
-catenin and phospho- glycogen synthase kinase-3 beta (p-GSK-3
β
) occurs, as well
as an up-regulation of the osteoblastic genes alkaline phosphatase (ALP), collagen type I,
osteocalcin and Runx2 [
111
]. The role of NPY in chicken and other avian species has yet to
be explored. Given the importance of bone disorders, such as bacterial chondronecrosis
with osteomyelitis (BCO), as well as the importance of bone health and metabolism to egg
production [
116
] in the poultry industry, this provides an open avenue for future research.
6.2.5. NPY in Macrophage and Immune System
Innervation of immune organs constitutes one of the primary ways in which NPY reg-
ulates immune function [
117
]. Additionally, though basal levels are low, upon stimulation
or immune activation, immune cells can also directly produce NPY and up-regulate NPY
receptors, leading to autocrine and paracrine effects [
118
]. The Y1 receptor is present in al-
most every type of immune cell, including lymphocytes, natural killer cells, dendritic cells,
granulocytes, and monocytes/macrophages [
119
]. Early studies in mice showed that NPY
can modulate the immune response by acting as a chemical attractant, decreasing adhesion
and promoting migration and phagocytosis of peritoneal macrophages [
120
,
121
]. These
effects are mediated by the Y1 receptor; however, under different physiological or patho-
logical conditions, activation of the Y2 receptor can increase adhesion [
122
] and decrease
migration of monocytes and leukocytes [
123
]. The differences in downstream effects upon
receptor binding are related to dipeptidyl peptidase 4 activity, which specifically terminates
NPY-Y1 interactions and also changes with age [
122
,
124
]. These differences highlight the
complexity of the NPY response and the importance of NPY-receptor interactions. Based
on the known differences in sequence homology of these receptors among species, it is
quite likely that the effects of NPY in chicken immune cells differ, or exert effects through
different receptors, compared to mammalian species.
NPY also exerts inflammo-modulatory effects through cytokine production. These
effects can be either pro- or anti-inflammatory, depending on the cell type and mode of
activation. For instance, activated RAW246.7 macrophages showed increased expression
of tumor necrosis factor alpha (TNF
α
), C-reactive protein (CRP), and monocyte chemoat-
tractant protein 1 (MCP1), all of which were decreased by co-incubation with an Y1 an-
tagonist [
125
]. In addition, in isolated mouse macrophages and human whole blood from
healthy subjects, NPY increases interleukins (IL-1b, IL-6) and TNF
α
[
126
,
127
]. However,
NPY produced by adipose tissue macrophages inhibits the expression of IL-6 and TNF
α
through the autocrine and paracrine systems [128].
Finally, NPY has also been shown to indirectly regulate immune function through
pathways that affect obesity, diabetes, mood, and thermoregulation, all of which can then
modulate the immune response. Of particular importance in chicken is the interaction
between NPY and heat stress. Indeed, NPY has been shown to induce hypothermia in
birds [
129
,
130
], and is known to be modulated by heat stress in birds [
131
]. As this state
also induces inflammation, further study of the role of NPY and its interaction with the
immune system during this critical physiological state may provide future insights into
helping the poultry industry manage heat stress.
6.2.6. NPY in the Gut
With feed intake controlled by the hypothalamus and nutrients absorbed by the
gastrointestinal tract, the term “gut–brain axis” refers to the critical and complex communi-
cation that controls energy homeostasis. The system is bidirectional, in that signals from
the brain regulate motility, secretion, digestion, absorption in the gut, and the gut sends

Vet. Sci. 2022,9, 171 10 of 18
signals relating to nutrient and energy status back to the central nervous system. NPY is
present at all levels of this axis, and within the gut, it is primarily expressed by the enteric
neurons [
132
]. Because of this, NPY is able to regulate a wide range of functions of the
intestine, including motility and epithelial permeability, as well as the immune-related
functions of cytokine production and inflammation [
133
]. In mammals, centrally adminis-
tered NPY delays gastric emptying, likely via interaction with Y2 receptors, as determined
by receptor-inhibition studies [
134
,
135
]. Additionally, NPY has an anti-secretory and pro-
absorptive effect [
136
,
137
], particularly in the retention of chloride ions [
133
,
138
]. In Caco2
cells, it has been shown that NPY exerts this effect by increasing the association between the
Cl
¯
/HCO3
¯
(OH
¯
) transporter (SLC26A3) and membrane lipid rafts [
139
]. To date, most
of the gastric effects of NPY have been attributed to its interactions with Y1 or Y2; however,
multiple NPY receptor subtypes are present within the intestine, suggesting that the vari-
ety of functions of NPY may result from this diversity, though the exact interactions and
consequences are yet to be elucidated. Y4, in particular, is present in both the mammalian
and chicken [28,97] gut, though it may mediate the effects of PP over NPY [133].
As the importance of the microbiota in whole-body health has become recognized in
recent years, it is not surprising that neuropeptides also play a role in the gut microbiome.
NPY has been shown to have anti-microbial properties, with the ability to inhibit the
growth of E. coli
in vitro
[
140
]; however, studies with other organisms such as S. aureus
and C. albicans have shown conflicting results [
140
–
142
]. The mechanism behind this
potential inhibition seems to come from both direct disruption and depolarization of the
cell membrane [
143
–
145
], and indirectly via modulation of intestinal inflammation [
146
].
This effect has yet to be studied in avian species, but does present an interesting and
potentially impactful area for future research.
7. Regulation of Avian NPY Expression
The regulation of avian NPY expression involves nutritional, hormonal, genetic, and
environmental factors. Indeed, early studies showed that negative energy conditions such
as food restriction and deprivation enhance hypothalamic NPY mRNA expression [
82
]
and neuron activity [
147
]. A study conducted by Zhou et al. [
148
] showed that chickens
subjected to fasting for up to 72 h exhibited increased NPY content in the hypothalamic
infundibular nucleus (IN) and paraventricular nucleus (PVN), but not in the lateral hypotha-
lamic area (LHA). In the PVN, NPY returned to pre-fasting levels after 24 h of re-feeding.
However, the level of NPY was unaffected in the IN, suggesting that fasting and re-feeding
of broiler chickens can differentially affect NPY in the brain. A more recent study showed
an increased NPY expression associated with lowered feed intake, particularly in 3-week-
old cockerels, confirming that NPY is associated with the nutritional state of chickens [
148
].
Additionally, gene expression of NPY and other orexigenic molecules were up-regulated
in low growth rate as compared to high growth rate cockerels, corroborating the findings
reported by previous studies [
149
,
150
]. Moreover, the increase in NPY was associated with
an overexpression of brain-specific homeobox protein (BSX). This confirms the requirement
of BSX for physiological expression of NPY/AgRP and stimuli of hyperphagic response in
avian species as demonstrated in mice [151].
In long-term divergently selected chickens, for the ratio of abdominal fatness to live
weight, Dridi’s group found that the hypothalamic expression of NPY was higher in fat
compared to lean bird lines under both fed and fasted conditions [
152
]. The same group
found that the hypothalamic expression of NPY was lower in high- compared to low-feed
efficient male quails, but it remained unchanged between female lines [
153
], indicating a
potential gender-dependent effects.
NPY expression is also regulated by hormonal factors such as insulin, leptin, and gluco-
corticoids (GCs). These peripheral hormonal signals are integrated in the hypothalamus at
the arcuate nucleus of mammals or infundibular nucleus of birds [
154
,
155
]. Intracerebroven-
tricular (ICV) injection of GCs increases feed intake in chicks [
156
] in a dose-dependent
manner, whereas infusion of recombinant leptin over a 6 h period significantly reduced

Vet. Sci. 2022,9, 171 11 of 18
feed intake in 3-week-old broiler chickens. This effect was mediated via selective down-
regulation of the hypothalamic expression of NPY [157].
Moreover, a study was conducted with the aim to evaluate the effect of dietary energy
level and feeding state on the GC-induced gene expression of hypothalamic feeding-related
neuropeptides, including NPY [
158
]. The results showed that dexamethasone treatment
significantly increased hypothalamic NPY expression under fasting conditions. This effect
was observed in chickens fed a high-fat diet but not in their counterparts receiving a low-fat
diet, suggesting that the effect of peripheral GCs injection on NPY expression is dependent
on dietary energy concentration. The same study showed a decrease in hypothalamic NPY
levels under re-feeding conditions.
ICV injection of insulin has been shown to inhibit feed intake in young chickens via
the central melanocortin system [
83
]. Similarly, ICV injection of insulin had an anorexgenic
effect on leghorn and broiler chicks [
159
,
160
], indicating that insulin in birds, like mammals,
is an anorexigenic neuropeptide. More recent studies have further explored the interaction
between NPY and insulin, and have indicated that the hypophagic effect of insulin is likely
mediated by the Y1 and Y2 receptors [161,162].
Environmental conditions may also alter NPY expression; however, the data are con-
troversial and a matter of debate. For instance, heat-stressed chickens showed an increased
NPY mRNA [
163
,
164
], decreased NPY mRNA [
125
], or no change when compared to
controls [
165
], though these effects may differ based on age and strain of the birds studied,
as well as the temperature and duration of the heat stress. Similarly, ICV injection of NPY
during heat exposure diminished the orexigenic response of broiler chicks to NPY [
131
],
while heat-stressed layer-type chicks, ICV-injected with NPY responded similarly to ther-
moneutral chicks [
129
]. Moreover, NPY treatment has been shown to exert a hypothermic
effect on layer-type chickens [
129
,
166
], however this has yet to be explored in broilers,
but does suggest that NPY additionally inhibits energy expenditure. The reduction in
NPY abundance and function observed in some studies is a plausible explanation for the
decrease in food intake during heat stress, whereas the increase reported by other authors
may be induced by the increase in plasma corticosterone under stressful conditions.
8. Conclusions and Perspectives
In summary, avian NPY plays a key role in feed intake regulation, consistent with the
results obtained in mammals. Several studies demonstrated that this neuropeptide is also
expressed in various peripheral tissues, suggesting a pleotropic physiological functions.
However, much effort is still required to determine the exact physiological functions and
their associated downstream mechanisms in such tissues.
Author Contributions:
Writing—original draft, N.A. and E.S.G.; constructing the figures, J.S.D.;
writing—review and editing, S.D. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All the data are included in the review.
Conflicts of Interest: The authors declare no conflict of interest.
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