Hypophagic effects of insulin is mediated via NPY1/NPY2

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Hypophagic effects of insulin is mediated via NPY1/NPY2 receptors in broiler cockerels

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2018-0470

Manuscript Type: Article

Date Submitted by the Author: 14-Aug-2018

Complete List of Authors: Yousefvand, Shiba; Ferdowsi University of Mashhad, Faculty of veterinary medicine, Department of basic sciencesHamidi, Farshid; Ferdowsi University of Mashhad, faculty of veterinary medicine, Department of basic sciencesZendehdel, Morteza; University of Tehran Faculty of Veterinary MedicineParham, Abbas; Ferdowsi University of Mashhad, faculty of veterinary medicine, Department of basic sciences

Is the invited manuscript for consideration in a Special

Issue:Not applicable (regular submission)

Keyword: Hypophagic effects, Insulin, Broiler cockerels, NPY receptors, ICV injection

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Title: Hypophagic effects of insulin is mediated via NPY1/NPY2 receptors in broiler

cockerels

Shiba Yousefvand1, Farshid Hamidi1*, Morteza Zendehdel2, Abbas Parham1

1Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of

Mashhad, Mashhad, Iran

2 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran,

Tehran, Iran

*Corresponding author: Farshid Hamidi1

+985138805599 - E-mail: [email protected]

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mailto:[email protected]

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Abstract

Neuropeptide Y (NPY) plays a mediatory role on cerebral insulin function in maintaining energy

balance. The current study was designed to determine the role of insulin in food intake and its

interaction with NPY receptors in 8 experiments of broiler cockerels, (each with 4 treatment

groups except 8). Chickens received control solution, 2.5, 5, and 10 ng of insulin in experiment 1

and control solution, 1.25, 2.5 and 5 μg of B5063, SF22 and SML0891 in experiments 2, 3 and 4

through intracerebroventricular (ICV) injection, respectively. In experiments 5, 6 and 7 chickens

received ICV injection of B5063, SF22, SML0891, co-injection of them +insulin, control

solution and insulin. In experiment 8, blood glucose was measured. Insulin, B5063 and

SML0891 decreased food intake, while SF22 led to increase in food intake. Also, hypophagic

effect of insulin was reinforced with injection of B560, but ICV injection of SF22 destroyed

hypophagic effect of insulin and increased food intake (p<0.05). However, SML0891 had no

effect on decreased food intake induced by insulin (p>0.05). 30 minutes after injection, blood

sugar in control group was higher than insulin group (p<0.05). Therefore, the NPY1 and NPY2

receptors act as mediators in hypophagic effect of insulin in broiler cockerels.

Key words: Hypophagic effects, Insulin, Broiler cockerels, NPY receptors, ICV injection.

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1. Introduction

The central regulation of food intake was modulated in different parts of the brain such as

striatum, nucleus tractus solitaries (NTS), amygdala, hypothalamus and arcuate nucleus (Hamidi

and Yousefvand 2017). The main hypothalamic area that regulates food intake is the arcuate

nucleus in the vicinity of the third ventricle of the brain (Zendehdel and Hassanpour 2014b).

Insulin in the central nervous system regulates processes such as regulation of glucose

metabolism (Duarte et al. 2012), energy homeostasis, nerve cell reproduction and survival (Plum

et al. 2005). The primary target for insulin is arcuate nucleus in the hypothalamus, a key area of

the brain that regulates energy homeostasis (Blevins et al. 2002). In fact, arcuate nucleus has

high density of insulin-binding sites (Shiraishi et al. 2008a). The arcuate nucleus has two large

neuronal populations including: 1) neuropeptide Y (NPY)/agouti related protein (AgRP), 2)

proopiomelanocortin (POMC) and cocaine/amphetamine regulated transcript (CART)

(Zendehdel and Hassanpour 2014b; Blevins et al. 2002). The central melanocortin system in

chicks seems to be important in regulating feeding behavior. It was shown that melanocortin

neurons are involved in reducing appetite caused by insulin. Central injection of insulin reduces

food intake in layer chicks. Insulin has no role in regulating food intake in broiler chickens

(Shiraishi et al. 2008a, 2011b). Obviously, many factors such as bird strain affects physiological

and nutritional state in response to neurotransmitters (Denbow et al. 1983).

NPY is one of the most abundant signalling peptides in the central nervous system of

vertebrates (Brome´e et al. 2006) and one of the strongest endogenous stimulants of food intake

in mammals and birds as well (Bi et al. 2012). Among NPY receptors, Y1, Y2 and Y5 are

involved in the central regulation of food intake (Levens et al. 2004).

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Glucose has different functions in the birds, its main function is to produce energy in the

body. Chickens are not sensitive to insulin and have a high plasma glucose level, so require high

doses of exogenous insulin to produce metabolic responses to insulin therapy (Ashwell et al.

2003).

So far, no studies have been reported on the effects of insulin via NPY receptors on

broiler cockerels’ feeding behaviours. Hence, the present study aims to evaluate the effect of

insulin via NPY (Y1, Y2 and Y5) receptors on the food intake of 3-h food-deprived (FD3)

cockerels. Due to the high levels of blood sugar in chicks and need a high dose of insulin

exogenous to produce hypoglycaemia, effect of ICV injection of central insulin in blood sugar

broiler cockerels were investigated.

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2. Materials and methods

2.1 Animals

The present study was carried out on 324 male broiler chickens (Ross 308) with an approximate

weight of 750 g from Mahan Company (Mahan Co, Tehran, Iran). The chicks were kept in flocks

until two weeks of age, then divided randomly and kept in individual cages. During the study,

birds have free access to fresh water and a mash diet containing 23 % crude protein and 2850

kcal/kg of metabolizeable energy (Chineh Co, Tehran, Iran) provided for them. Chicken were

maintained under standard conditions (at 22±1 °C, 50% relative humidity and continuous

lighting condition) (Olanrewaju et al. 2006). Three hours before ICV injections, the chickens

were deprived of food (FD3), but they had free access to water. Animal handling and

experimental procedures were performed according to the Guide for the Care and Use of

Laboratory Animals by the National Institutes of Health (USA) as well as the current laws of the

Iranian government for animal care.

2.2 Experimental drugs

Drugs used were: I5523 (Insulin from porcine pancreas), B5063 (selective NPY1 receptor

antagonist), SF22 (selective NPY2 receptor antagonist), SML0891 (selective non-peptide NPY5

receptor antagonist) and Evans blue which were obtained from Sigma Co (Sigma, St. Louis,

USA). Xylazine and ketamine were prepared by Alfasan CO (Alfasan, Woerden, Netherlands)

and lincospectin by Razak Co (Razak, Karaj, Iran). NPY receptor’s antagonists were dissolved in

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absolute dimethyl sulfoxide (DMSO) and insulin was dissolved in HCL 0.01 M. Then, all drugs

were diluted in 0.85% saline containing 0.1% Evans blue solution (Mahzouni et al. 2016).

2.3 Surgical preparation

When chickens were approximately 750 g of weight (at 20 days of age) , Stereotactic surgery

was performed. six hours before surgery (stereotaxic) the chicks were deprived of food (Hamidi

and Zendehdel 2015), but the water was freely available to them. Chickens were anesthetized

with xylazine (2 mg/Kg im) and ketamine (10 mg/Kg im) (Maitia et al. 2006) and a 23-gauge

thin-walled stainless steel guide cannula was stereotaxically implanted into the right lateral

ventricle. The stereotaxic coordinates were AP = 6.7, L = 0.7, H = 3.5–4 mm below the

duramater with the head oriented as described (Zendehdel et al. 2012c). The cannula was secured

with three stainless steel screws placed in the calvaria surrounding each guide cannula, and then

acrylic dental cement (Acro pars CO, Tehran, Iran) was applied to the screws and guide cannula.

An orthodontic No. 014 wire (American Orthodontics) trimmed to the exact length of the guide

cannula was inserted into the guide cannula while the chicks were not being used for

experiments. Lincospectin was applied to the incision to prevent infection. The birds were

allowed a minimum of 5 days of recovery prior to injection.

2.4 Experimental procedures

7 experiment in this study was carried out in two stages and each stage of the experiment in four

experimental groups (a control group and three treatment groups) and Experiment 8 (Blood

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glucose measurement) in two experimental groups (control group and insulin group). The first

stage consisted of four experiments to determine the effective and sub-effective doses of the

compounds used and the second stage involved three experiments using compound interactions.

In 7 experiments Forty-four birds were used in each experiment (n=11 for each group but finally,

6 chicks in each group were used for statistical analysis) and in experiment 8, 16 chicks (n=8 for

each group but finally, 6 chicks in each group were used for statistical analysis) were used.

Injections were done with a 29-gauge, thin-walled stainless steel injecting cannula which extends

1.0 mm beyond the guide cannula. This injecting cannula was connected through a 60-cm-long

PE-20 tubing to 10-μl Hamilton syringe. Injections were performed manually and at slow and

uniform speeds in 60 seconds. In the first stage the volume of each injection was 10 μl. A

subsequent 60-s period was allowed to permit the solution to diffuse from the tip of the cannula

into the ventricle. Tubing and syringes were kept in 70% ethanol, and the glassware was

autoclaved to render materials pyrogen-free. In these experiments, normal saline was used as a

control solution. Placement of the guide cannula into the right ventricle was verified by the

presence of cerebrospinal fluid and ICV injection of methylene blue followed by slicing the

frozen brain tissue at the end of the experiments. Only the data from the correct injections

(Correct planting of cannula) were used in statistical analysis.

At each stage of the study, only one experiment was performed on each bird. After

injections in experiments 1-4 (or after the second injections for experiment 5-7) , the chicks were

immediately returned to the cages and fresh food and water were provided to the chickens, so

cumulative food intake at 30, 60 and 120 minutes after injection were recorded. All injections

were performed at 11 am to 13:15 pm.

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The first stage involved experiments1-4. The first experiment was designed to evaluate

the effect of insulin on the central regulation of the food intake and obtain the effective dose of

insulin on the food intake in FD3 chickens. Chickens in each group received 2.5, 5 and 10 (ng/10

µl) of I5523 (insulin) via ICV injection, and the control group was intracerebroventriculary

injected with the control solution. The experiment 2 evaluated the effect of ICV injection of

B5063 (NPY1 receptor antagonist) on the central regulation of the feeding behaviour. The sub-

effective dose of B5063 on the food intake was obtained, too. Via ICV injection, FD3 chickens

received the control solution and 1.25, 2.5 and 5µg doses of B5063 respectively. Experiments 3

and 4 were performed parallel to the experiment 2 but fasted chickens received control solution

and 1.25, 2.5 and 5µg doses of SF22 (NPY2 receptor antagonist) and SML 0891 (NPY5 receptor

antagonist) respectively to determine these sub-effective doses in fasted chickens.

The second stage involved experiments 5-7. In experiment 5, FD3 chickens received ICV

injection of the compounds according to the Table 1. Experiments 6 and 7 were done parallel to

the experiment 5 but fasted chickens received in the same way the doses of 1.25 ng of SF22 and

SML0891 respectively in 6 and 7 experiments. These doses of drugs were calculated based on

previous studies (Shiraishi et al. 2008a; Stengel et al. 2010a).

Experiment 8 was conducted in two experimental groups and blood samples were

collected from each group. Pre-injection blood samples were collected from all chickens in two

groups. After first blood samples, control group 10 μl of control solution and insulin group 10 ng

of insulin received via ICV injection. 30 minutes after injection, blood samples were collected

again from each chicken in both groups. In both steps, blood samples were taken from jugular

vein (Stevens and Ridgway 1966).

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2.5 Statistical analysis

Cumulative food intake in each experiment was calculated by repeated measurement of two-way

analysis of variance (ANOVA). Data were presented as the mean ± SEM. Whenever there was a

significant difference between the treatments, the Tukey test (as post hoc test) was used to

determine any significant difference between the treatments. Independent Sample T Test was

used to compare the blood glucose in control and insulin groups before and after injection. Paired

Sample t test was used to compare the blood glucose of each group before injection and with the

corresponding group after injection. The significance level was considered as p<0.05.

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3. Results

The effects of insulin on cumulative food intake via NPY receptors and ICV injection of 10 ng

dose of insulin on blood glucose levels in FD3 broiler cockerels were investigated and the results

were shown in figures1-8.

In experiment 1, ICV injection of insulin (doses of 5 and 10 ng) resulted in a dose-

dependent decrease in cumulative food intake compared with the control group (p<0.05).

However, 2.5 ng of insulin did not affect food intake in comparison with the control group

(p>0.05) (Fig. 1).

In experiment 2, ICV injection of 1.25 μg of B5063 did not influence food intake in FD3

chicks compared with the control group (p>0.05), whereas the doses of 2.5 and 5 μg of B5063

reduced food intake in a dose-dependent manner compared with the control group (p<0.05) (Fig.

2).

In experiment 3, ICV injection of 2.5 and 5 μg of SF22 significantly increased food

intake in a dose-dependent manner in fasted broiler cockerels in comparison with the control

group (p<0.05), but 1.25 μg of SF22 did not affect food intake compared with the control group

(p>0.05) (Fig. 3).

In experiment 4, the effect of ICV injection of SML0891 on food intake in broiler

cockerels was evaluated. Doses of 2.5 and 5 μg of SML0891 dose-dependently decreased

cumulative food intake compared with the control group (p<0.05); while, 1.25 μg of SML0891

had no effect on food intake compared with the control group (p>0.05) (Fig. 4).

On the basis of experiments 1-4, 10 ng of insulin and 1.25 μg of NPY receptors

antagonists were selected for experiments 5-8.

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In experiment 5, ICV injection of insulin (10 ng) significantly decreased the food intake

compared with the control group. Hypophagic effect of insulin was significantly reinforced due

to the injection of 1.25 μg of B5063 (p<0.05). However, 1.25 μg of B5063 had no effect on food

intake compared with the control group (p>0.05) (Fig. 5).

In experiment 6, ICV injection of insulin (10 ng) significantly decreased cumulative food

intake compared with the control group (p<0.05). Injection of SF22 (1.25 μg) + insulin (10 ng)

significantly increased food intake compared with the insulin group in FD3 broiler chickens at

30, 60 and 120 minutes post injection (p<0.05). 1.25 μg of SF22 had no effect on the food intake

compared with the control group (p>0.05) (Fig. 6).

In experiment 7, 1.25 μg of SML0891 did not influence cumulative food intake in fasted

chicks compared with the control group (p>0.05). However, ICV injection of insulin (10 ng)

significantly decreased food intake compared with the control group (p<0.05). Also, 1.25 μg of

SML0891 did not affect hypophagic effect of insulin (10 ng) in broiler cockerels (p>0.05) (Fig.

7).

In none of the experiments conducted in this study, the doses have caused sleepiness or

behavioural changes in chicken.

In experiment 8, there was no significant difference in blood sugar between the control

and insulin groups before injection (p>0.05), but in 30 minutes after injection, the blood sugar in

the control group was significantly higher than the insulin group. Blood sugar in both groups of

insulin and control at 30 minutes post injection were significantly higher than those of two

groups before injection (p<0.05).

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4. Discussion

Previous studies have investigated the effects of insulin on controlling the feeding behaviour

through different neural pathways and signalling pathway in liver in mammals and birds. It has

been shown that insulin causes a reduction in the central food intake via melanocortin neurons in

rats (Benoit et al. 2002). ICV injection of insulin in the layer chicks stimulates expression of

POMC and inhibits expression of NPY (Shiraishi et al. 2008a). In fasted (5 hours of hunger) and

full-fed chicks, administration of insulin anti-serum causes turn off insulin signals in liver such

AKT phosphorylation (Dupont et al. 2008). Inhibition of the PI3K/AKT pathway almost inhibits

all insulin functions, so this signalling pathway plays an important role in metabolic functions of

insulin (Dupont et al. 2009). The present study provides the first evidence that central insulin and

NPY interact via NPY1 receptor to regulate food intake in broiler cockerels.

According to the results of the first experiment, ICV injection of insulin (at doses of 5

and 10 ng) decreased the food intake (Fig. 1). In line with the results of our study, ICV injection

of insulin in rats and leghorn chicks had an anoroxygenic effect (Carvalheira et al. 2003; Honda

et al. 2007b; Shiraishi et al. 2008a, 2011b). Therefore, it can be said that insulin in birds like

mammals is a hypophagic neuropeptide. But ICV injection of insulin does not affect broiler’s

food intake (Shiraishi et al. 2011b). That results contradict with our study. This contradiction

may be due to the difference between chicken strain investigated in Shiraishi's study (Chunky)

and the one studied in our paper (ROS 308). Obviously, many factors such as bird strain affect

physiological and nutritional state in response to neurotransmitters (Denbow et al. 1983), such

as, ICV injection of insulin reduces food intake in high fat-fed rats in comparison with low-fat

fed rats (Kelly et al. 2008). Continuous administration (for 4 weeks) of detemir (insulin

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analogue) in rats with high fat-fed reduced food intake and fat mass in the body (Rojas et al.

2011).

ICV injection of B5063 and SML0891 in experiments 2 and 4 respectively (at doses of

2.5 and 5 μg) led to a dose-dependent decrease in food intake in fasted cockerels (Figures 2 and

4). In the third experiment, 2.5 and 5 μg doses of SF22 significantly increased food intake (Fig.

3). In many research consistent with our study, the effects of excitation and inhibition of NPY1

and NPY5 receptors on food intake have been investigated. The activation of NPY1 receptor

stimulates food intake in hamster (Keen-Rhinehart and Bartness 2007). The NPY5 and NPY1

receptor antagonist inhibits food intake in rodents (Duhault et al. 2000; Kanatani et al. 2000).

They are post-synaptic receptors in NPY neurons (Furuse 2002). In experiment 3, NPY2 receptor

antagonist significantly increased food intake compared with the control group, because NPY2

receptor is an autoreceptor (Brome´e et al. 2006). Some studies have been conducted on the

effect of NPY2 receptor on food intake and their results are consistent with our study (Levens et

al. 2004; Ortiz et al. 2007).

In the fifth experiment, ICV injection of B5063 significantly reinforced Hypophagic

effect of insulin (Fig. 5). These results suggest that there is an interaction between insulin and

NPY1 receptor. Various studies confirm our results. Insulin reduces food intake by inhibiting the

expression of NPY (Shiraishi et al. 2008a). Insulin interacts with NPY neurons, especially in the

arcuate nucleus (Levin et al. 1998) and Insulin receptors are located in NPY neurons in the

arcuate nucleus (Shiraishi et al. 2011a). The NPY expressing neurons act as a vital mediator of

central insulin role in regulating appetite and energy homeostasis (Yulyaningsih et al. 2014;

Steculorum et al. 2016; Loh et al. 2017).

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In experiment 6, ICV injection of SF22 destroyed hypophagic effect of insulin and

increased the food intake (fig. 6). In fact, the results of this experiment confirm our results and

support the relationship between insulin and NPY1 receptor. On the other hands, this receptor

directly inhibits the release of NPY in paraventricular nucleus (PVN) and reduces food intake

(Levens et al. 2004).

In experiments 7, ICV injection of SML0891 did not influence hypophagic effects of

insulin (Fig. 7). The results of this study indicate that there is no relationship between insulin

and NPY5 receptor in central regulation of the food intake. In line with our result, it was

indicated that NPY- induced feeding in mice was prominently performed via NPY1 receptor, not

through NPY5 receptor (Kanatani et al. 2000).

Chickens are not sensitive to insulin and have a high plasma glucose level, so require

insulin high doses to produce metabolic responses to insulin therapy (Ashwell et al. 2003). It has

been shown that injection of 40 μg/Kg of insulin in 8 days old chicken (Tokushima et al. 2005)

and 80 μg/Kg of insulin in pigeon (Sweazea et al. 2006) reduced blood glucose levels. In current

study, 30 minutes after injection, the control group and insulin group (10 ng) had a significant

increase in blood sugar compared to pre-injection, which could be due to re-feeding the chickens

immediately after injection (Denbow and Cline 2015). In the 30 minutes after injection, blood

sugar decreased significantly in the insulin group compared to the control group. ICV injection

of insulin causes an increase in plasma insulin levels (Denbow and Cline 2015) and Insulin in the

blood inhibits glucose production (Obici et al. 2002). Therefore in the present study, maybe ICV

injection of insulin Causes insulin to enter the bloodstream and decreased plasma sugar levels.

Of course, further studies in this field can clarify the pathway involved.

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In conclusion, the results of the present study indicate that the hypophagic effect of

insulin may be modulated by NPY1 and NPY2 receptors (not NPY5 receptor) and central insulin

reduces blood sugar in broiler cockerels.

further investigation is required to elucidate the underlying cellular and molecular signaling

pathways (such as Expression of NPY in brain and phosphorylation AKT in liver) in the

interconnection between insulin and NPY on feeding behavior in broiler cockerels. The authors

plan to investigate the expression of insulin-induced NPY in the brain and AKT phosphorylation

in liver, in their later studies. Also, with regard to the change in the type of response to insulin in

different diets in rats, authors suggest that this study be conducted in high-fat and low-fat diets

broiler cockerels.

Acknowledgements

The authors thank the central laboratory (Dr. Rastegar Lab.) of the Faculty of Veterinary

Medicine, University of Tehran for cooperation. This research is conducted as a part of the PhD

thesis of the first author. This study was approved by license number 3/40151 at Ferdowsi

University of Mashhad.

Conflicts of interest

The authors declare that they have no conflict of interest.

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Fig. 1 Effects of intracerebroventricular injection of control solution and different levels of the

insulin (2.5, 5 and 10 ng) on cumulative feed intake (g) in broiler cockerels. Data are

expressed as mean ± SEM. Different letters (a-c) indicate significant differences between

treatments at each time (p<0.05).


B5063 (NPY1 receptor antagonist; 1.25, 2.5 and 5 µg) on cumulative feed intake (g) in

broiler cockerels. Data are expressed as mean ± SEM. Different letters (a-c) indicate

significant differences between treatments at each time (p<0.05).


SF22 (NPY2 receptor antagonist; 1.25, 2.5 and 5 µg) on cumulative feed intake (g) in

broiler cockerels. Data are expressed as mean ± SEM. Different letters (a-c) indicate



SML 0891 (NPY5 receptor antagonist; 1.25, 2.5 and 5 µg) on cumulative feed intake (g)

in broiler cockerels. Data are expressed as mean ± SEM. Different letters (a-c) indicate


Fig. 5 Effects of intracerebroventricular injection of control solution, B5063 (NPY1 receptor

antagonist; 1.25 µg), insulin (10 ng) and co-injection of the B5063 + insulin on

cumulative feed intake (g) in broiler cockerels. Data are expressed as mean ± SEM.

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Different letters (a, b and c) indicate significant differences between treatments at each

time (p<0.05).

Fig. 6 Effects of intracerebroventricular injection of control solution, SF22 (NPY2 receptor

antagonist; 1.25 µg), insulin (10 ng) and co-injection of the SF22 + insulin on cumulative

feed intake (g) in broiler cockerels. Data are expressed as mean ± SEM. Different letters

(a, b and c) indicate significant differences between treatments at each time (p<0.05).

Fig. 7 Effects of intracerebroventricular injection of control solution, SML 0891(NPY5 receptor

antagonist; 1.25 µg), insulin (10 ng) and co-injection of the SML 0891+ insulin on

cumulative feed intake (g) in broiler cockerels. Data are expressed as mean ± SEM.

Different letters (a and b) indicate significant differences between treatments at each time

(p<0.05).

Fig. 8 Effect of ICV injection of insulin on blood sugar levels in broiler cockerels. different

letters indicate a significant difference between the control and insulin (10 ng) groups before and

after the injection. Different indices indicate a significant difference between the groups in the

pre-injection and post-injection (p<0.05). Data are expressed as mean ± SEM.

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Table caption

Table 1. Treatment procedure in experiment 5.

Groups The first injection (5μl)The second injection

(after 15 minutes) (5μl)

Group 1 Control solution Control solution

Group 2 Control solution Insulin (10 ng)

Group 3NPY1 receptor antagonist

(1.25 μg)Control solution

Group 4NPY1 receptor antagonist

(1.25 μg)Insulin (10 ng)

Normal saline was used as a control solution.

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Fig. 1 Effects of intracerebroventricular injection of control solution and different levels of the insulin (2.5, 5 and 10 ng) on cumulative feed intake (g) in broiler cockerels. Data are expressed as mean ± SEM. Different

letters (a-c) indicate significant differences between treatments at each time (p<0.05).

258x122mm (300 x 300 DPI)

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Fig. 2 Effects of intracerebroventricular injection of control solution and different levels of the B5063 (NPY1 receptor antagonist; 1.25, 2.5 and 5 µg) on cumulative feed intake (g) in broiler cockerels. Data are

expressed as mean ± SEM. Different letters (a-c) indicate significant differences between treatments at each time (p<0.05).

259x119mm (300 x 300 DPI)

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Fig. 3 Effects of intracerebroventricular injection of control solution and different levels of the SF22 (NPY2 receptor antagonist; 1.25, 2.5 and 5 µg) on cumulative feed intake (g) in broiler cockerels. Data are


259x118mm (300 x 300 DPI)

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Fig. 4 Effects of intracerebroventricular injection of control solution and different levels of the SML 0891 (NPY5 receptor antagonist; 1.25, 2.5 and 5 µg) on cumulative feed intake (g) in broiler cockerels. Data are


256x117mm (300 x 300 DPI)

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Fig. 5 Effects of intracerebroventricular injection of control solution, B5063 (NPY1 receptor antagonist; 1.25 µg), insulin (10 ng) and co-injection of the B5063 + insulin on cumulative feed intake (g) in broiler

cockerels. Data are expressed as mean ± SEM. Different letters (a, b and c) indicate significant differences between treatments at each time (p<0.05).

259x119mm (300 x 300 DPI)

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Fig. 6 Effects of intracerebroventricular injection of control solution, SF22 (NPY2 receptor antagonist; 1.25 µg), insulin (10 ng) and co-injection of the SF22 + insulin on cumulative feed intake (g) in broiler cockerels.

Data are expressed as mean ± SEM. Different letters (a, b and c) indicate significant differences between treatments at each time (p<0.05).

255x118mm (300 x 300 DPI)

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Fig. 7 Effects of intracerebroventricular injection of control solution, SML 0891(NPY5 receptor antagonist; 1.25 µg), insulin (10 ng) and co-injection of the SML 0891+ insulin on cumulative feed intake (g) in broiler cockerels. Data are expressed as mean ± SEM. Different letters (a and b) indicate significant differences

between treatments at each time (p<0.05).

260x118mm (300 x 300 DPI)

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Fig. 8 Effect of ICV injection of insulin on blood sugar levels in broiler cockerels. different letters indicate a significant difference between the control and insulin (10 ng) groups before and after the injection. Different indices indicate a significant difference between the groups in the pre-injection and post-injection (p<0.05).

Data are expressed as mean ± SEM.

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310x219mm (300 x 300 DPI)

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Hypophagic effects of insulin is mediated via NPY1/NPY2