The Potential of Protein Ghrelin as Material for Energy Balance

Setting for Feed Efficency in Broiler Chicken

Nove Hidajati

, Anwar Ma’ruf

and Ratna Damayanti

Department Basic of Veterinary Medicine, Faculty Veterinary Medicine, Universitas Airlangga, Surabaya,

Postgraduate School, Universitas Airlangga, Surabaya

Keywords: Ghrelin, energy, feed efficiency, broiler.

Abstract: The purpose of this study was to determine the molecular weight of the protein ghrelin as a basis to

determine amino acid composition of protein ghrelin and subsequently to make synthetic ghrelin protein

whose function is to control energy balance in broilers. Samples were isolated from the digestive tract and

brain tissue of the broilers and then examined by SDS Page and Western blot. Based on the results, it can

be concluded that the protein ghrelin had the molecular weight of 44 kDa and neuropeptide Y of 11 kDa.

1 INTRODUCTION

Ghrelin and leptin are complementary but work

antagonistically. Their signals reflect acute or

chronic energy balance changes and their effects are

mediated by hypothalamic neuropeptides such as

neuropeptide Y (NPY) and augouti related peptide

(AgRP) (Inui et al., 2004).

Gastric distension and gastric

hyposensitisation are insufficient to stimulate ghrelin

response. This possibility is a postgastric process

involving insulin secretion, either directly or

indirectly, through the incretin stimulation of the

hormone glucagon such as peptide 1 and gastric

inhibitory peptide. Most studies suggest that insulin

will lower ghrelin concentrations that are

independent of glucose. The insulin mechanism

inhibiting the effect of ghrelin concentration is not

fully known. These insulin effects may be mediated

by the direct effects of ghrelin secreting cells or the

effects of humoral mechanisms or central

mechanisms (Bloom, 2005).

Association between ghrelin, stomach,

hypothalamus and the implications of ghrelin on

gastrointestinal function control, energy balance, and

current growth has not been entirely clear.

Therefore, a study is needed to find ghrelin amino

acid from broiler chickens so that we can create

synthetic

ghrelin protein that can be used to regulate the

energy balance and growth of the livestock.

2 MATERIALS AND METHODS

This study used samples of male Lohman (MB 202

P) broiler chickens which were maintained from the

age of 1 day up to 21 days in letter cages, as many as

25 chicken.

Day old chicken were placed in a letter

cage for 21 days with food and drink ad libitum.

After reaching the age of 21 days, the chickens were

sacrificed to be sampled in the form of

gastrointestinal and brain tissue for the following

tests (1) Isolation of ghrelin and neuropeptide Y

(NPY) proteins from the gastrointestinal tract and

brains, (2) Identification of ghrelin and neuropeptide

proteins (NPY) of the gastrointestinal tract and

brains of broilers using SDS-PAGE (sodium dudecyl

sulphate polyacrylamide gel electrophoreses)

method, (3) Analysis of molecular weight of ghrelin

protein and Neuropeptide Y by blotting method i.e.

Western Blot technique using proteins described

electrophoresis of polyacrylamide gel, (4)

Examination of amino acid structure of ghrelin and

neuropeptide Y by MALDI-TOP method.

528

Hidayati, N., Ma’ruf, A. and Damayanti, R.

The Potential of Protein Ghrelin as Material for Eenergy Balance Setting for Feed Efﬁcency in Broiler Chicken.

DOI: 10.5220/0007546305280531

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 528-531

ISBN: 978-989-758-348-3

3 RESULTS AND DISCUSION

3.1 SDS Page for Ghrelin and

Neuropeptide Y Proteins

Results SDS-PAGE ghrelin and neuropeptide Y

(NPY) proteins in the broiler's gastrointestinal tract

and brain showed the presence of ghrelin and

neuropeptide Y proteins, as shown in Figure 1.

Figure 1 : SDS page of ghrelin and NPY proteins from the

digestive tract and brain.

The results of SDS-PAGE on gastrointestinal

tract and brains of broilers revealed ghrelin protein

and neuropeptide Y. SDS-PAGE results showed that

there were several visible bands. One protein band

was found each in the markers between 260 and 140

kDa, 140 and 100 kDa, 100 kDa and 50 kDa, 50 kDa

and 40 kDa, and between 25 kDa and 10 kDa.

Protein bands formed between 50 kDa and 40

kDa markers and between 25 kDa and 10 kDa

markers were suspected as ghrelin and neuropeptide

Y proteins. The protein bands formed on the

gastrointestinal tract and the broiler's brain were

very clear, indicating that the tissue appears to

induce the strongest antibody antigen reaction.

SDS-PAGE protein of gastrointestinal tract and

brain of broiler chicken showed protein band

between 50 kDa and 40 kDa markers, which were

protein with molecular weights of 44 kDa and 11

kDa, but it has not been certain whether it was

ghrelin protein and neuropeptide Y as several other

protein bands were also formed between these

markers. To prove that the formation of protein

bands with molecular weight of 44 kDa and 11 kDa

was ghrelin and neuropeptide Y protein, it was

necessary to perform further examination.

3.2

Western Blot for Protein Ghrelin

from Broiler’s Digestive Tract

The Western blot of ghrelin protein in

gastrointestinal tissue showed the presence of a 44

kDa molecular weight of ghrelin protein, as diplayed

in Fig. 2.

Figure 2 : Western blot for ghrelin protein from broilers’

digestive tract

The result of ghrelin protein molecular weight

calculation showed that the molecular weight of

ghrelin protein was 44 kDa. The formation of

protein bands between 50 kDa and 40 kDa markers,

after being calculated, apparently showed a molecule

with molecular weight of 44 kDa. This suggested

that the protein produced by SDS-PAGE tested with

Western blot was a ghrelin protein of growing-phase

broiler with a molecular weight of 44 kDa. The

formation of the protein band with 44 kDa molecular

weight was definite because there was a binding

between protein ghrelin resulted from SDS-PAGE

and rabbit pAb ghrelin (data Sheet Rev. 102203F).

3.3

Western Blot for NPY Protein from

Broiler’s Brain

The result of Western blot protein of neuropeptide Y

on brain tissue showed the existence of Y

neopopeptide protein with 11 kDa molecular weight,

as shown in Figure 3.

The Potential of Protein Ghrelin as Material for Eenergy Balance Setting for Feed Efﬁcency in Broiler Chicken

529

Figure 3 : Western blot for neuropeptide Y protein from

broiler’s brain.

The results of the molecular weight calculation

of neuropeptide Y protein showed that it had a

molecular weight of 11 kDa. The formation of

protein bands between 25 kDa and 10 kDa markers,

after being calculated, was found to be 11 kDa. This

suggested that the SDS-PAGE protein tested with

Western blot was a neuropeptide Y protein of

growing phase broiler chicken with a molecular

weight of 11 kDa. The formation of a protein band

of 11 kDa molecular weight was definite because

there was a binding between the protein ghrelin

resulting from SDS-PAGE with neupeptide Y

antibody (data Sheet ab30914).

Ghrelin is a gastric peptide that plays an

important role in the regulation of food into the body

(food intake). Before eating the plasma, ghrelin

concentration rises gradually and immediately goes

down after eating. The addition of ghrelin

intravenously increases food intake and appetite,

which proves that ghrelin plays a role in hunger and

the beginning of a meal initiation. Ghrelin is also

involved in weight control because the body mass

index is negatively controlled by plasma ghrelin

concentrations at the time of fasting. Abnormalities

of the signal from the stomach signal is related to

energy balance disorders and growth, and this is

related to gastrointestinal and neuroendockrine

function.

Ghrelin and leptin are complementary but work

antagonistically, their signals reflect acute or chronic

energy balance changes and their effects are

mediated by hypothalamic neuropeptides such as

neuropeptide Y (NPY) and augouti related peptide

(AgRP).

4 CONCLUSIONS

The molecular weight of ghrelin protein was 44 kDa

with amino acid structure consisted of methionine,

phenylalanine, leucine, arginine, valine, isoleucine,

leucine and neuropeptide Y molecular weight was

11 kDa with threonine, methionine, arginine,

leucine, tryptophan, valine, serine, valine, leucine,

threonine, leucine, alanine, glutamate, alanine,

tyrosine, proline, and serine. By identifying the

molecular weight and the arrangement of amino

acids, we can create synthetic ghrelin protein to

regulate the energy balance of broiler chickens.

REFERENCES

Cummings DE, Purnell JQ. Frayo RS, Schmidova

K.Wisse BE and Weigle DS. Apreprandial

rise in plasma ghrelin level suggests a role in

meal initiation in humans. Diabetes. 2001.50.

1714-1719.

Haqq AM, Farooqi IS, O’Rahilly S, Stadler DD,

Rosenfeld RG, Pratt KL, LaFranchi SH,

Purnell JQ. 2003. Serum ghrelin levels are

inversely correlated with body mass index,

age, and insulin concentrations in normal

children and are markedly increased in

Prader-Willi syndrome. J Clin Endocrinol

Metab 88:174–178.

Hiejima H, Nishi Y, Hosoda H. 2009. Regional

distribution and the dynamics of n-decanoyl

ghrelin, another acyl-form of ghrelin, upon

fasting in rodents. Regulatory Peptides, vol.

156, no 1-3, pp 47-56.

Kaiya Hiroyuki, Saito Ei-Suke, Tachibana Tetsuya,

Furuse Mitsuhiro, Kangawa Kenji. 2007.

Changes in ghrelin levels of plasma and

proventriculus and ghrelin mRNA of

proventriculus in fasted and refed layer

chicks. Domestic Animal Endocrinology.

Elsevier. 32 (2007) 247-259.

Kojima M, Hosoda H & Kangawa K.2004. Clinical

endocrinology and metabolism. Ghrelin, a

novel growth-hormone-releasing and

appetite-stimulating peptide from stomach.

ICPS 2018 - 2nd International Conference Postgraduate School

530

Best Pract Res Clin Endocrinol Metab 18,

517–530.

Saito E, Kaiya H, Takagi T, Yamasaki I, Denbow

DM, Kanagawa K, and Furuse

M.2002.Chicken ghrelin and growth

hormone-releasing peptide-2 inhibit feed

intake of neonatal chicks. Eur. J. Pharmacol.

453:75-79.

Saito E, Kaiya H, Tachibana T, Tomonaga S, DM

Denbow DM, Kanagawa K and Furuse

M.2005. Inhibitory effect of ghrelin on food

intake is mediated by the corticotrophin

releasing factor system in neonatal chicks.

Reg. Pept. 125: 201-208.

Wang HJ, Geller F, Dempfle A, Schauble N, Friedel

S, Litchner P, fontenla-Horro F, Wudy S,

Hagemann S, Gortner L, Huse K,

Remschmidt H, Bettecken T, Meitinger T,

Schafer H, Hebebrand J and Hinney A,

Ghrelin receptor gene: identification of

several sequence variants in extremely obese

children and adolescents, healthy normal-

weight and underweight students, and

children with short normal stature. Journal of

Clinical Endocrinology and Metabolism

2004.89.157-162.

The Potential of Protein Ghrelin as Material for Eenergy Balance Setting for Feed Efﬁcency in Broiler Chicken

531