Effect Of Zinc Deficiency And Supplementation On Insulin Signaling In Chickens
Advances in Environmental Biology, 7(1): 104-108, 2013
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed ORIGINALARTICLE
Corresponding Author
Ali Alkaladi, Department of Biological Sciences, Faculty of Science, King Abdulaziz University,
North Campus, PO Box 11508, Jeddah, 21463, Saudi Arabia.
: alkaladi@kau.edu.sa; Phone: +966 540424039; +966 26435219
Effect Of Zinc Deficiency And Supplementation On Insulin Signaling In Chickens
Ali Alkaladi
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, North Campus, PO Box
11508, Jeddah, 21463, Saudi Arabia.
Ali Alkaladi: Effect Of Zinc Deficiency And Supplementation On Insulin Signaling In Chickens
ABSTRACT
The aim of this study to investigate the effect of either zinc (Zn) deficiency or supplementation on insulin
synthesis and muscular insulin signals in chickens. A total of 90 one-day-old Hubbard male broiler were divided
in to three groups ; control group (GI), Zn deficiency group (GII) and Zn supplemented group (GIII). After 21
days, blood , pancreas , liver and thigh muscle samples were taken to investigate blood glucose, liver glycogen,
serum insulin, pancreatic cytosolic Zn, insulin receptor (IR), insulin receptor phosphorilation (IRP), insulin
receptor substrate-1 (IRS-1), serine/thereonine kinase (AKT), phosphoinositide-3-kinase (PI3K) and glucose
transporter protein 4( GLUT4) concentrations, IR and IRS-1 gene expressions. The results indicated that, Zn
deficiency leads to decrease of hepatic glycogen, serum insulin , pancreatic cytosolic Zn, IRP, AKT, PI3K and
GLUT4 concentrations and increase of blood glucose, while Zn supplementation reveres the result. So it can be
concluded that Zn deficiency adversely affect insulin synthesis and muscular insulin signals, while Zn
supplementation enforce both insulin synthesis and insulin signals in chickens.
Key wrods:
Introduction
Zinc is an essential trace element crucial for the
function of more than 300 enzymes and it is
important for cellular processes like cell division and
apoptosis. Hence, the disturbances of zinc
homeostasis have been associated with several
diseases including diabetes mellitus, a disease
characterized by high blood glucose concentrations
as a consequence of decreased secretion or action of
insulin. Zinc supplementation of animals and humans
has been shown to ameliorate glycemic control in
type 1 and 2 diabetes, the two major forms of
diabetes mellitus, but the underlying molecular
mechanisms have only slowly been elucidated. Zinc
seems to exert insulin-like effects by supporting the
signal transduction of insulin and by reducing the
production of cytokines, which lead to beta-cell
death during the inflammatory process in the
pancreas in the course of the disease. Furthermore,
zinc might play a role in the development of
diabetes, since genetic polymorphisms in the gene of
zinc transporter 8 and in metallothionein (MT)-
encoding genes could be demonstrated to be
associated with type 2 diabetes mellitus [11].
The total Zn2+ content of the mammalian
pancreas is high, and chiefly localized to the islet β-
cell. It plays an important role in both insulin
synthesis and storage. Indeed it’s concentrations
reach millimolar levels in the interior of the densecore
granule, where two Zn2+ ions coordinate six
insulin monomers to form the hexameric structure on
which insulin crystals are based [3].
Zinc plays a crucial role in many cell functions;
as a result, both zinc deficiency and excess of free
zinc are toxic to mammalian cells. The abundance of
zinc per cell is tissue dependent and the zinc content
of pancreatic beta cells is among the highest in the
body. In beta cells, zinc was proposed to be required
for multiple steps in insulin synthesis and release, but
conclusive evidence is lacking. After synthesis in the
ER, pro-insulin is transported into the Golgi
apparatus where immature, pale secretary
‘‘progranules’’ are formed . These granules contain
pro-insulin-zinc hexamers which are further
processed into mature insulin and C-peptide by the
prohormone convertases PC1/3 and PC2 . After
maturation, the zinc-insulin hexamers form waterinsoluble
crystals. It has been suggested that crystal
formation increases the degree of conversion of
soluble pro-insulin to insoluble insulin, but nearly
normal pro-insulin processing occurs in patients with
mutated histidine-B10 insulin, which cannot
crystallize [6]. There are many studies on the role of
zinc in insulin synthesis, storage and glucose
homeostasis in mammals but this role in chicken is
unknown , so this study was designed to monitor the
effect of zinc deficiency and supplementation on
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Adv. Environ. Biol., 7(1): 104-108, 2013
insulin concentration, synthesis and mechanism of
action on a molecular and cellular levels in chickens.
Material and Methods
Birds, Diets, and Treatments:
A total of 90 one-day-old male chicks were used
in the 21-d experiment. Birds were randomly divided
into three group; Control group, kept on the basal
diet supplemented with 20 mg /kg added Zn from
ZnSO4.7H2O to be contain (48.37 mg/Kg) Zn (NRC,
1994). Zn deficient group, kept on basal diet that
contain 28,37 mg/kg Zn and Zn supplemented group,
kept on basal diet supplemented with 60 mg/kg
added Zn from ZnSO4.7H2O to be contain (88.37
mg/Kg) Zn. ( Table I). The basal cornsoybean meal
diet was formulated to meet or exceed the
requirements for starter broilers (NRC, 1994) except
for Zn and contained 28.37 mg of Zn/kg of diet on an
as-fed basis, by analysis [7]. Chicks were maintained
on a 24-h constant light schedule and allowed
adlibitum access to experimental diets and tap water,
which contained no detectable Zn.
Table 1: Composition of the basal diet for 1- to 21-day-old broilers(A)
Ingredient Percentage Calculated composition
Corn 55.97 ME ( Kcal/Kg) 2993
Soybean meal 36.00 CP(e) (%) 21.56
Soybean oil 3.60 Lys (%) 1.19
CaHPO4 H2O ( b) 1.95 Met (%) 0.54
CaCO3
( b) 1.16 Met + Cys (%) 0.91
NaCL( b) 0.30 Ca(e) (%) 1.10
Met 0.20 Nonphytatephosphate 0.46
Micronutrient (c) 0.32 Zn (e) 28.37
Cornstarch + Zinc (d) 0.50
(A) ingredient and nutrient composition reported
on an as-fed basis
(b) reagent-grade
(c) provided per kilogram of diet: vitamin A (as
all-trans retinol acetate), 15,000 IU; cholecalciferol,
3 900 IU; vitamin E (as all-rac-α-tocopherol acetate),
30 IU; vitamin K (as menadione sodium bisulfate),
3.0 mg; thiamin (as thiamin mononitrate), 2.4 mg;
riboflavin, 9.0 mg; vitamin B6, 4.5 mg; vitamin B12,
0.021 mg; calcium pantothenate, 30 mg; niacin,
45 mg; folic acid, 1.2 mg; biotin, 0.18 mg;
choline (as choline chloride), 700 mg; Cu, 8 mg; Mn,
100 mg; Fe, 80 mg; I, 0.35 mg; Se, 0.15 mg
(d) zinc supplement added in place of equivalent
weight of cornstarch
(e) determined by analysis; each value based on
triplicate determinations
Sample Collections and Analysis:
Blood samples were taken from each bird via
cardiac puncture and then centrifuged to harvest
serum for determination of insulin and glucose
concentrations. Chicks were immediay killed by
cervical dislocation. Pancreas and thigh muscle
sample was frozen in liquid nitrogen until be used for
laboratory investigation.
Assays:
Plasma glucose was quantitated by glucose
oxidase-peroxidase method using the kit supplied by
SPINREACT, Spain (Ref: 1001190). Serum insulin
was determined using Ultra Sensitive Chickens
Insulin ELISA Kit (Cat.No. E-EL-ch 1528,
Elabscience, Beijin) following manufacturer
instructions, liver Glycogen content was determined
according to Caruso et al, [1] Zinc concentrations in
pancreatic cell cytoplasm was determined by
inductively coupled argon plasma spectroscopy
(model 9000, Thermo Jarrell Ash, Waltham, MA) as
described by Li et al. [7]. Muscular Insulin receptor,
Insulin receptors phosphorylation, insulin receptor
substrate-1, serine/thereonine kinase.
phosphoinositide-3-kinase and glucose transporter
protein 4 were determined using ultra sensitive
chickens ELISA kits ( Cat. No E-EL-ch 1110,
Elabscience, Beijin; KHR9121, Invitrogen, USA;
KT-56519, Kamiga biomedical, USA; JM-K453-
40,MBL, USA; E-EL-ch0531, Elabscience, Beijin
and AMSE12G0201, AMSbio, UK.) respectively
following the manufacturer instructions.
RNA isolation, reverse transcription, and
polymerase chain reaction:
Total RNA was prepared from the frozen
muscular powder using the E.Z.N.A ™.spin column
RNA extraction kit (Omega Bio-Tech, Cat NO
R6834-01, Canada) following the manufacturer
instructions. Concentrations of RNA were measured
by spectrophotometry (OD 260 nm), and RNA
integrity was electrophoretically verified using
ethidium bromide. After DNAse treatment (Ambion,
Clinisciences, Montrouge, France), RNA was reverse
transcribed using Super Script II RNase H Reverse
Transcriptase (Invitrogen, Carlsbad, CA, USA) in the
presence of Random Primers (Promega,
Charbonnièresles- Bains, France). Polymerase chain
reaction (PCR) was performed using a 2720
thermocycler (Applied Biosystems, USA). Using
PCR master mix (Qiagen USA) following the
manufacturer instructions and using the specific
primer (Table 2). PCR products were analyzed on a
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Adv. Environ. Biol., 7(1): 104-108, 2013
2% agarose gel in 90 mM Trisborate, 2 mM EDTA
buffer (TBE), pH 8, and visualized by staining with
ethidium bromide and UV transillumination, For
quantitative evaluation, absolute optical densities
(OD) of RT-PCR signals were obtained by
densitometric scanning using an image analysis
system (1-D Manager; TDI Ltd.). The values for the
specific targets were normalized according to those
of β actin to express arbitrary units of relative
abundance of the specific messages (i.e., relative
expression).
Statistical analysis:
The data were statistically analyzed by SPSS
version 20. statistical packages (IBM 1 New Orchard
Road Armonk, New York 10504-1722 United
States). Data were presented as a mean ± SD, n = 10.
Statistical differences between groups were
performed using student's t-test. Differences
considered significant when p < 0.05 [14].
Table 2: primers used for polymerase chain reaction:
Gene
Primer sequence
Product
size bp
Annea
ling
(°C)
Accession No Reference
IR F 5\ TTTGGGATGGTTTATGAGGG 3\
383 58 XM_00123339
8.1
R 3 [2] \GCCAGGTCTCTGTGAACAAA 5\
IRS1 F 5\ GCCCGGCCCACGAGGCTG 3\
490 58 NM_00103157
R 3\ GTACGCTTGTCCGTAACG 5\ 0.1
Βactin F 5\ AGCCATGTACGTAGCCATCC3\
230 55 NM_ 205518.1 Afifi and
5\ CTCTCAGCTGTGGTGGTGAA3\ Alkaladi 2011
Results:
Table 3: Effect of Zn deficiency and supplementation on serum glucose, serum insulin, muscular glycogen and pancreatic Zn.
Group Blood glucose (mg/dl) Serum insulin (ng/ml) Glycogen (mg/kg) Pancreatic Zn(μg/ml cytosol)
I 275 ± 13.2 0.76 ±0.07 53.7 ± 4 15.7 ± 2.5
II 486.6 ± 7.6a 0.25 ± 0.05b 27.7 ± 2.5b 10.3 ± 2b
III 225 ± 5fg 0.58 ± 0.8fk 47 ± 3.6fh 26.3 ± 1.5fh
a,b,c represent the statistical difference of group II relative group I at ( 0.001, 0.01 and 0.05) respectively. d,e,f represent the statistical
difference of group III relative group I at ( 0.001, 0.01 and 0.05) respectively. g,h,k, represent the statistical difference of group III
relative group II at ( 0.001, 0.01 and 0.05) respectively.
Table 4: Effect of Zn deficiency and supplementation on muscles insulin signals
G IR
(ng/ml)
IRP
(ng/ml)
IRS
(ng/ml)
AKT
(ng/ml)
PI3K
(ng/ml)
GLUT4
(ng/ml)
IR gene expression
(arbitrary unit)
IRS1gene expression
(arbitrary unit)
I 23 ± 2.6 4.3 ± 1.2 33.3±1.5 2.2 ± 0.3 16.3 ± 1.5 2.5 ± 0.2 3.1 ± 0.62 11.3 ± 1.32
II 25 ± 6.1 2.5 ± 0.5c 32±2 1.5 ± 0.2c 6.3 ± 1.5c 1.3 ± 0.3c 2.9 ± 0.71 10.6 ± 1.22
III 21 ± 2.1 5.3 ±
0.8k
34.7±1.5 3.2 ±
0.3fh
25.3 ±
2.5fh
4 ± 1k 3.2 ± 0.42 12.3 ± 2.45
G; group. IR; insulin receptor. IRP; insulin receptor phosphorilation. IRS; insulin receptor substrate-1. AKT; serine/thereonine kinase. PI3K;
phosphoinositide-3-kinase. GLUT4; glucose transporter protein 4. a,b,c represent the statistical difference of group II relative group I at
( 0.001, 0.01 and 0.05) respectively. d,e,f represent the statistical difference of group III relative group I at ( 0.001, 0.01 and 0.05)
respectively. g,h,k, represent the statistical difference of group III relative group II at ( 0.001, 0.01 and 0.05) respectively.
Effect of either Zn deficiency or supplementation on
serum glucose, muscular glycogen, serum insulin
and pancreatic cytosolic Zn concentrations:
Zinc deficiency in chickens accompanied with a
significant increase of blood glucose ( 0.001),
decrease of muscular glycogen, serum insulin and
pancreatic cytosolic zinc concentrations ( 0.01).
In contrast Zn supplementation to chicken
significantly decrease blood glucose and increase
Muscular glycogen, serum insulin and pancreatic
cytosolic Zn concentrations, when either compared to
control or Zn deficient chicks ( table 3).
Effect of either Zn deficiency or supplementation on
muscular insulin signal molecules:
Either Zn deficiency or supplementation not
significantly affect on either concentrations or gene
expression of both IR and IRS-2. While Zn
deficiency significantly decreases the concentrations
of muscular IRP, AKT, PI3K and GLUT4, Zn
supplementation significantly increase the above
mentioned parameters ..
Discussion:
Chicken rearing nowadays becomes a high
established manufacture due to the growing high
demands of a ship protein, that can be get from the
high growth rat chicken. The main column of this
manufacture is the diet, that mainly a carbohydrate
dependant "the carbohydrates metabolism mainly
controlled by insulin hormone". In mammals , insulin
synthesis, storage, secretion and signaling modulated
by Zn status but that not established in chickens. This
work is a trial to know the modulatory effect of Zn
status on insulin synthesis and insulin signals in
chickens.
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Fig. I: The expression level of mRNA for IR, IRS-1 and Betactin, M; DNA marker, 1; control group, 2 Zn
deficient group, 3; Zn supplemented group.
The current results indicated that, In contrast to
Zn supplemented chickens, Zn deficient chickens
showed a decrees in pancreatic Zn, Serum insulin,
Liver glycogen and increase in blood glucos. Indeed
the results are correlated and explain each other. The
decrease in pancreatic cytosol Zn concentration
related to Zn deficiency in diet, where pancreas
contains large amount of Zn and is the first organ
affected by Zn deficiency. The decrease of serum
insulin in Zn deficient group and it's increase in Zn
supplemented one indicates the importance of Zn in
regulation of serum insulin level , this may be
through regulating insulin gene expression, or insulin
modification, or storage, or excretion or may be all
this processes. Insulin is important for entrance of
glucose to hepatic cells and glycogen synthesis this
explain the increase of blood glucose and the
decrease of hepatic glycogen concentration. The
above explanations are enforced by the results
obtained in Zn supplemented group where, the Zn
supplementation disappear all Zn deficiency effects,
this indicated that, Zn is the cause of this effects (
table 3).
The total Zn2+ content of the mammalian
pancreas is high, and these ions are chiefly localized
to the islet β-cell . Correspondingly, Zn2+ plays an
important role in both insulin synthesis and storage.
Indeed, total Zn2+ concentrations reach millimolar
levels in the interior of the dense-core granule ,
where two Zn2+ ions coordinate six insulin
monomers to form the hexameric structure on which
insulin crystals are based [3]. It has been reported
that pancreas is the most sensitive soft tissue to
dietary Zn for chicks, and pancreas Zn concentration
was shown to be a useful indicator for Zn
requirement of broilers [5,13] reported that, in
contrast to Zn supplementation, db/db mice fed the
low-Zn diet had higher serum fasting glucose (17%)
and lower serum fasting insulin (63%) concentrations
than db/db mice fed the Zn-adequate diet . The
interactions among Zn, insulin, and glucose
homeostasis are complex, and Zn deficiency might
induce a state of insulin deficiency by interfering
with either insulin storage or activation [8].
Either Zn deficiency or supplementation not
affect on IR and IRS-1 gene expression and
concentrations, but IRP, PI3P, KAT and GLUT4
were inhibited by Zn deficiency and activated by Zn
supplementation ( taple 4 and fig 1). This indicts that,
Zn not affect on action of insulin on insulin receptors
but its action apeare postreceptor either through
activation of receptor tyrosine kinase
phosphorylation or Activation of PI3K/KAT pathway
leading to activation of GLUT4 that increase the
entrance of glucose to muscle cells. Several modes of
action have been described to explain the improved
action of insulin by Zn. It appears that
Zn can have direct insulin-like effects, which
may be due to stimulation of the postreceptor
proteins Akt and PI3-kinase [10] Several potential
mechanisms have been suggested for Zn affecting
insulin action, including a role for Zn to enhance
tyrosine kinase phosphorylation [13].
Some of the insulinomimetic effects of zinc can
be explained by the induction of translocation of
GLUT to the plasma membrane, through activation
of one zinc-dependent molecule, insulin-responsive
aminopeptidase (IRAP), which is expressed and
characterized in fat and muscle as insulin target
tissues, resulting in an increased uptake of glucose
into tissue cells, thereby lowering the blood glucose
level [11].
Like insulin, zinc enhances glucose uptake into
fibroblasts and adipocytes, which suggests an
involvement of zinc in this pathway. Examining the
effects of zinc on the insulin signal transduction, it
was observed that zinc leads to tyrosine
phosphorylation of the β subunit of the insulin
receptor, but to a lower extent compared to insulin,
and that IRS does not seem to play a role in
enhancing glucose uptake as a response to zinc
stimulus. According to this model, which proposes
an activation of PI3Kwithout involvement of IRS,
zinc may induce the production of H2O2 by
epididymal cells, which in turn causes the activation
of focal adhesion kinase (FAK) and FAK can finally
activate the PI3K-Akt pathway [11].
Support for the involvement of zinc in
phosphorylation of the insulin receptor was provided
by Haase and Maret [4] who identified PTP1B as a
sensitive target of zinc ions and an important
regulator of the phosphorylation state of the insulin
receptor. Inhibition of PTP1B by zinc ions, which
might be released from Metalothionine (MT), leads
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Adv. Environ. Biol., 7(1): 104-108, 2013
to an increased phosphorylation status of the insulin
receptor triggering the post-receptor events.
Considering that oxidative stress leads to a release of
zinc from MT and to cellular zinc depletion, this
condition as well as zinc deficiency due to decreased
absorption, increased excretion or increased
requirements could possibly lead to diabetes mellitus
Furthermore, zinc increased phosphorylation of
serine residues and therefore activation of Akt in
preadipocytes and adipocytes thereby enhancing
GLUT translocation. This effect could be blocked by
wortmannin, an inhibitor of PI3K, underlining the
importance of PI3K for the activation of Akt by zinc
[13].
Conclusion:
It can be concluded that, like mammals Zn
activate ß cells for production of insulin, and increase
insulin signals in muscle through activation of PI3KAKT
pathway and GLUT4. So it play important role
in glucose homeostasis in chickens.
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