The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration
Aim. To research the impact of agmatine on the redistribution of actin fractions, which are repre-sented by cytoskeletal actin filaments, short actin filaments of the plasma membrane skeleton and actin monomers (G-actin) in rat leukocytes under experimental diabetes mellitus (EDM). Methods. Leukocyt...
Збережено в:
| Дата: | 2017 |
|---|---|
| Автори: | , , |
| Формат: | Стаття |
| Мова: | English |
| Опубліковано: |
Інститут молекулярної біології і генетики НАН України
2017
|
| Назва видання: | Вiopolymers and Cell |
| Теми: | |
| Онлайн доступ: | http://dspace.nbuv.gov.ua/handle/123456789/153095 |
| Теги: |
Додати тег
Немає тегів, Будьте першим, хто поставить тег для цього запису!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration / I.V. Brodyak, I.I. Bila, N.O. Sybirna // Вiopolymers and Cell. — 2017. — Т. 33, № 6. — С. 403-414. — Бібліогр.: 36 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
irk-123456789-153095 |
|---|---|
| record_format |
dspace |
| spelling |
irk-123456789-1530952019-06-14T01:29:14Z The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration Brodyak, I.V. Bila, I.I. Sybirna, N.O. Structure and Function of Biopolymers Aim. To research the impact of agmatine on the redistribution of actin fractions, which are repre-sented by cytoskeletal actin filaments, short actin filaments of the plasma membrane skeleton and actin monomers (G-actin) in rat leukocytes under experimental diabetes mellitus (EDM). Methods. Leukocytes were lyzed in Triton X-100 and subjected to centrifugation to obtain cytoskeletal actin filaments, short actin filaments and actin monomers, which were separated in SDS-PAAG, followed by the immunoblot analysis of anti-actin antibodies. Results. Under EDM, an intensifi-cation of the process of short actin filament depolymerization and an increase in the G-actin con-tent were observed in leukocytes activated by sialospecific wheat germ lectin (WGA). Against a background of agmatine administration, the response dynamics to the WGA-stimulating effect was characterized by an increase in actin polymerization in the fraction of cytoskeletal filaments already after 0.5 min exposure to lectin, an exceptionally rapid depolymerization process after 1 min of the lectin treatment, and return to the initial indices after 3 min exposure to lectin. Conclusions. In leukocytes of animals with EDM against a background of agmatine administration, the transduction of WGA-induced signal through sialoglycoconjugates causes the actin redistribution. It indicates that this polyamine helps to restore and maintain a functional response of leukocytes to the activation signals. Мета. Дослідити вплив агматину на перерозподіл фракцій актину, які представлені актиновими філаментами цито-скелету, короткими актиновими філаментами скелету плазматичної мембрани і мономерами актину (G-актин), у лейкоцитах щурів з експериментальним цукровим діабетом (ЕЦД) Методи. Лейкоцити лізували в Тритоні Х-100 і піддавали центрифугуванню, у результаті якого було отримано актинові філаменти цитоскелету, короткі актинові філаменти і мономери актину, які розділяли в SDS-ПААГ, після чого проводили імуноблот аналіз із використан-ням анти-актинових антитіл. Результати. За умов ЕЦД у лейкоцитах, активованих сіалоспецифічним лектином зародків пшениці (WGA), спостерігається інтенсифікування процесу деполімеризації коротких актинових філаме-нтів і збільшення вмісту G-актину, а на фоні введення агматину динаміка формування відповіді на WGA-стимулювальний вплив характеризувалася посиленням полімеризації актину у фракції філаментів цитоскелету вже на 0,5 хв впливу лектину, дуже стрімким процесом деполімеризації на 1 хв після дії лектину та поверненням пока-зників до рівня у вихідному стані у разі дії лектину впродовж 3 хв. Висновки. У лейкоцитах тварин з ЕЦД на фоні введення агматину трансдукція WGA-індукованого сигналу через сіалоглікокон’югати зумовлює перерозподіл вмісту актину, вказуючи на те, що даний поліамін сприяє відновленню і підтриманню функціональної відповіді лейкоцитів на активаційні сигнали. Цель. Исследовать влияние агматина на перераспределение фракций актина, которые представлены актиновыми филаментами цитоскелета, короткими актиновыми филаментами скелета плазматической мембраны и мономера-ми актина (G-актин), в лейкоцитах крыс с экспериментальным сахарным диабетом (ЭСД). Методы. Лейкоциты лизировали в Тритоне Х-100 и подвергали центрифугированию, в результате которого было получено актиновые филаменты цитоскелета, короткие актиновые филаменты и мономеры актина, которые разделяли в SDS-ПААГ, после чего проводили иммуноблот анализ с использованием антиактиновых антител. Результаты. В условиях ЭСД в лейкоцитах, активированных сиалоспецыфическим лектином зародышей пшеницы (WGA), наблюдается интен-сификация процесса деполимеризации коротких актиновых филаментов и увеличение содержания G-актина, а на фоне введения агматина динамика формирования ответа на WGA-стимулирующее влияние характеризировалась усилением полимеризации актина во фракции филаментов цитоскелета уже на 0,5 мин влияния лектина, очень стремительным процессом деполимеризации на 1 мин после действия лектина и возвращением показателей к уро-вню в исходном состоянии в случае действия лектина в течение 3 мин. Выводы. В лейкоцитах животных с ЭСД на фоне введения агматина трансдукция WGA-индуцированого сигнала через сиалогликоконьюгаты приводит к пе-рераспределению содержания актина, указывая на то, что данный полиамин способствует восстановлению и по-ддержанию функционального ответа лейкоцитов на активационные сигналы. 2017 Article The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration / I.V. Brodyak, I.I. Bila, N.O. Sybirna // Вiopolymers and Cell. — 2017. — Т. 33, № 6. — С. 403-414. — Бібліогр.: 36 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000964 http://dspace.nbuv.gov.ua/handle/123456789/153095 576.321.36 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Brodyak, I.V. Bila, I.I. Sybirna, N.O. The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration Вiopolymers and Cell |
| description |
Aim. To research the impact of agmatine on the redistribution of actin fractions, which are repre-sented by cytoskeletal actin filaments, short actin filaments of the plasma membrane skeleton and actin monomers (G-actin) in rat leukocytes under experimental diabetes mellitus (EDM). Methods. Leukocytes were lyzed in Triton X-100 and subjected to centrifugation to obtain cytoskeletal actin filaments, short actin filaments and actin monomers, which were separated in SDS-PAAG, followed by the immunoblot analysis of anti-actin antibodies. Results. Under EDM, an intensifi-cation of the process of short actin filament depolymerization and an increase in the G-actin con-tent were observed in leukocytes activated by sialospecific wheat germ lectin (WGA). Against a background of agmatine administration, the response dynamics to the WGA-stimulating effect was characterized by an increase in actin polymerization in the fraction of cytoskeletal filaments already after 0.5 min exposure to lectin, an exceptionally rapid depolymerization process after 1 min of the lectin treatment, and return to the initial indices after 3 min exposure to lectin. Conclusions. In leukocytes of animals with EDM against a background of agmatine administration, the transduction of WGA-induced signal through sialoglycoconjugates causes the actin redistribution. It indicates that this polyamine helps to restore and maintain a functional response of leukocytes to the activation signals. |
| format |
Article |
| author |
Brodyak, I.V. Bila, I.I. Sybirna, N.O. |
| author_facet |
Brodyak, I.V. Bila, I.I. Sybirna, N.O. |
| author_sort |
Brodyak, I.V. |
| title |
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration |
| title_short |
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration |
| title_full |
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration |
| title_fullStr |
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration |
| title_full_unstemmed |
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration |
| title_sort |
dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2017 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/153095 |
| citation_txt |
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus against the background of agmatine administration / I.V. Brodyak, I.I. Bila, N.O. Sybirna // Вiopolymers and Cell. — 2017. — Т. 33, № 6. — С. 403-414. — Бібліогр.: 36 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
AT brodyakiv thedynamicsofactinfilamentpolymerizationinactivatedleukocytesunderexperimentaldiabetesmellitusagainstthebackgroundofagmatineadministration AT bilaii thedynamicsofactinfilamentpolymerizationinactivatedleukocytesunderexperimentaldiabetesmellitusagainstthebackgroundofagmatineadministration AT sybirnano thedynamicsofactinfilamentpolymerizationinactivatedleukocytesunderexperimentaldiabetesmellitusagainstthebackgroundofagmatineadministration AT brodyakiv dynamicsofactinfilamentpolymerizationinactivatedleukocytesunderexperimentaldiabetesmellitusagainstthebackgroundofagmatineadministration AT bilaii dynamicsofactinfilamentpolymerizationinactivatedleukocytesunderexperimentaldiabetesmellitusagainstthebackgroundofagmatineadministration AT sybirnano dynamicsofactinfilamentpolymerizationinactivatedleukocytesunderexperimentaldiabetesmellitusagainstthebackgroundofagmatineadministration |
| first_indexed |
2025-07-14T04:44:57Z |
| last_indexed |
2025-07-14T04:44:57Z |
| _version_ |
1837596198416941056 |
| fulltext |
403
I. V. Brodyak, I. I. Bila, N. O. Sybirna
© 2017 I. V. Brodyak et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited
UDC: 576.321.36
The dynamics of actin filament polymerization in activated leukocytes
under experimental diabetes mellitus against the background
of agmatine administration
I. V. Brodyak, I. I. Bila, N. O. Sybirna
Ivan Franko National University of Lviv
4, Hrushevskoho Str., Lviv, Ukraine, 79005
iryna_brodyak@yahoo.com
Aim. To research the impact of agmatine on the redistribution of actin fractions, which are
represented by cytoskeletal actin filaments, short actin filaments of the plasma membrane
skeleton and actin monomers (G-actin) in rat leukocytes under experimental diabetes mellitus
(EDM). Methods. Leukocytes were lyzed in Triton X-100 and subjected to centrifugation to
obtain cytoskeletal actin filaments, short actin filaments and actin monomers, which were
separated in SDS-PAАG, followed by the immunoblot analysis of anti-actin antibodies. Results.
Under EDM, an intensification of the process of short actin filament depolymerization and an
increase in the G-actin content were observed in leukocytes activated by sialospecific wheat
germ lectin (WGA). Against a background of agmatine administration, the response dynamics
to the WGA-stimulating effect was characterized by an increase in actin polymerization in the
fraction of cytoskeletal filaments already after 0.5 min exposure to lectin, an exceptionally
rapid depolymerization process after 1 min of the lectin treatment, and return to the initial
indices after 3 min exposure to lectin. Conclusions. In leukocytes of animals with EDM against
a background of agmatine administration, the transduction of WGA-induced signal through
sialoglycoconjugates causes the actin redistribution. It indicates that this polyamine helps to
restore and maintain a functional response of leukocytes to the activation signals.
K e y w o r d s: actin, leukocytes, agmatine, experimental diabetes mellitus.
Introduction
The experimental studies on leukocytes in the
case of diabetes mellitus demonstrate signifi-
cant violations of the morphofunctional state
of the blood cells. The reduction of chemo-
taxis capacity, phagocytic activity of leuko-
cytes, the dysfunction of adhesion, aggregation
and migration abilities of these cells correlate
with the level of hyperglycemia of peripheral
blood. In turn, the morphofunctional state of
leukocytes is addicted to the complex interac-
Structure and Function
of Biopolymers
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2017. Vol. 33. N 6. P 403–414
doi: http://dx.doi.org/10.7124/bc.000964
404
I. V. Brodyak, I. I. Bila, N. O. Sybirna
tion among proteins of the membrane, the
cytoskeleton and the network of intracellular
signaling [1–3].
The actin cytoskeleton is a dynamic system
involved in the adhesion and migration of
leukocytes [4]. The process of leukocyte
transfer from the peripheral blood into the
subendotelium is triggered by the molecules
of adhesion [5]. The formation of stable adhe-
sion structures as a result of actin polymeriza-
tion leads to the formation of actin сomet
tails, which are associated with integrins at
the front end of leukocytes and induce β1-
integrin clustering, causing the formation an
actin rafts on the cell surface in the area of
lamelopodia and filopodia [6]. Lamelopodia
contain a highly dense and extensive network
of actin filaments, which are orientated from
the sharp ends (positively charged) to the
edge of the cell. The polymerization of actin
generates the force that pushes the membrane
forward, creating preconditions for new adhe-
sion contacts of leukocytes with the endothe-
lium involving filopodia [7]. Filopodia are
made of densely staffed parallel filaments of
actin (10 or more), the sharp ends of which
are directed towards the membrane. The
grouping of actin filaments is mediated by
fascin, a binding protein. The polarized nature
of actin filaments allows motor proteins to
actively transport monomers of actin to the
ends of filopodia, thus increasing the local
actin polymerization [8]. The mechanism for
increasing the level of polymerized actin
(F-actin) involves an increase in the pool of
actin monomers of globular actin (G-actin),
which is required for the polymerization of
new actin filaments, or may be caused by
increased affinity of already existing filaments
of F-actin to G-actin. Free negatively charged
ends of actin filaments (where the lengthening
of actin filaments occurs) are developed in
several ways: by removing cap proteins,
which in the attached position at growing
ends of actin block the process of polymeriza-
tion; by depolymerizing actin fragments ow-
ing to the disconnection of non-covalent
bonds between actin monomers within actin
filaments, which leads to the formation of
shorter filaments with negatively charged free
ends; by producing actin oligomers de
novo [9].
The reorganization of the cytoskeleton oc-
curs when Ca2+-dependent signaling cascades,
involving Src family kinases and low-molec-
ular GTFases, are activated [10]. GTFases of
Rap1 family perform the activation of PI-3'-
kinase, which is required for the Rap1-
dependent activation of Rac (a member of
small GTFase of the Rho family), through
Vav2, and it is Rac that directly affects the
organization of actin cytoskeleton [11]. PI-3'-
kinase enzyme plays a key role not only in the
activation of adhesion molecules, but also in
chemokine-induced cell adhesion and cell po-
larization [12]. In contrast, the relationship
between L-selectins, CD44, CD43, ICAM-
1–3 and the cytoskeleton is performed through
ezrin / radixin / moesin (ERM) protein family.
Proteins of ERM family have both membrane
and actin-binding domains, which enable their
function as linkers between membrane proteins
and the actin cytoskeleton [13]. Leukocytic
adhesion molecules (selectin, LFA-1, VLA-4,
ICAM-1) also interact with cytoskeletal pro-
teins using cytoplasmic actin-binding proteins
(ABPS: actinin, paxillin, vinculin and tallinn),
which activate Arp2/3 complex triggering the
405
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus
actin polymerization [9]. Arp2/3 complex is a
matrix where new actin filaments are formed,
in addition, the complex joins in the middle of
actin filaments and promotes their bran-
ching [14]. Actin filaments in this case are
formed at an angle of 70° to previously formed
actin filaments. Those filaments push the mem-
brane forward from the side of their growth
(from leader (growing) ends). As a result of
ATP hydrolysis, the macroergic phosphate
necessary for the polymerization of new actin
filaments and actin filaments at the lagging
end, involving ADP/kofilin complex in the
lamel region, experiences depolymeriza-
tion [15]. Thus the transfer of leukocytes is
dynamic and multistage cascade process re-
quiring the activation of various adhesion mol-
ecules and signalling pathways, as well as the
restructuring of actin cytoskeleton [9].
Therefore, the study on mechanisms of the
actin polymerization-depolymerization is es-
sential for understanding the migratory abili-
ties and functional activity of leukocytes under
diabetes.
It has been previously stated [16] that in
animal leukocytes under experimental diabetes
mellitus (EDM) the total content of actin is
reduced, whereas actin polymerization is in-
tensified in the membrane skeleton filaments.
Agmatine administration to animals with EDM
increases both the total content of actin and
the level of polymerized actin in the fraction
of cytoskeletal filaments, which are caused by
the depolymerization of short actin filaments
of the plasma membrane skeleton. This poly-
amine either directly or indirectly affects the
functional state of leukocytes. As an endoge-
nous ligand for the imidazole receptors (I1 /
I2), and with less affinity with α2-adrenergic
receptors, N-methyl-D-aspartate and serotonin
receptors, agmatine lowers the glucose levels
in plasma under DM [17–19]. The hypoglyce-
mic mechanism of agmatine is provided
throught direct insulin-like effects on peri phe-
ral organs, interaction with β-cells of the pan-
creatic islets leading to an increased release of
insulin, and increased secretions of endorphins
by adrenals owing to the activation of imi da-
zo le receptors, which cause the increased glu-
cose uptake by the cells of the body [17, 20].
Therefore, agmatine can be used as a thera-
peutic agent for the treatment of DM and re-
lated metabolic disorders [21].
The aim of our study was to investigate the
effects of agmatine on the redistribution of
actin fractions represented by cytoskeletal ac-
tin filaments, short actin filaments of the plas-
ma membrane skeleton and actin monomers
(G-actin) in leukocytes of the control group of
animals and rats with EDM. Since there are
adhesion molecules and receptors on the sur-
face of leukocytes, which by their biochemical
nature are glycoconjugates with a high level
of sialization and interact with cytoskeletal
proteins through the membrane structures and
signalling proteins, the dynamics of actin re-
organization was evaluated after 0.5 min,
1 min and 3 min of leukocyte preincubation
with wheat germ lectin (WGA) under both
normal and diabetic conditions. WGA lectin
interacts with sialic acid residues that are pres-
ent in terminal positions of N-linked glycopro-
teins, as well as with the residues of sialic
acids and N-acetyl-β,D-glucosamine as part of
O-glycans of glycoproteins and glyco li-
pids [22]. Incubating leukocytes with WGA
lectin, we have modelled in vitro the activation
state of leukocytes.
406
I. V. Brodyak, I. I. Bila, N. O. Sybirna
Materials and Methods
Animal preparation
The experiments were based on white outbred
male rats weighing 150–180 g. The animals
had free access to food and water during their
stay under standard vivarium conditions. The
experiments were conducted in compliance
with the General Ethical Principles for
Conducting Experiments on Animals adopted
at the First National Congress on Bioethics
(Kyiv, 2001), which agree with the provisions
of the European Convention for the Protection
of Vertebrate Animals used for Experimental
and Other Scientific Purposes (Strasbourg,
1985). The animals were divided into four
groups: (1) control, (2) control + agmatine, (3)
experimental diabetes mellitus (EDM), and (4)
EDM + agmatine. EDM was induced by the
intraperitoneal administration of 6 mg strep-
tozotocin (Sigma, United States) per 100 g of
body mass, dissolved in 10 mM citrate buffer
(pH 5.5). The development of diabetes was
controlled by the blood glucose level deter-
mined 72 h after administering streptozotocin.
Animals with a glucose level above 14 mM
were considered eligible for experiments.
Starting from the third day from the moment
of inducing diabetes, the animals from the
second and fourth groups were intramuscu-
larly administered agmatine (Sigma, United
States) at a concentration of 20 g/kg body
weight for 14 days, while the animals from the
first and third groups were intramuscularly
administered saline solution for 14 days.
Blood collection
Blood was collected after light anesthesia with
diethylether. Heparin was added beforehand
to prevent coagulation (the end point heparin:
whole blood dilution = 1 : 100).
Isolation of blood leukocytes
Leukocytes were isolated from blood by cen-
trifugation in gradient of ficolltriombrast den-
sity (r = 1.076–1.078). Afterwards, the cells
were washed twice in phosphate buffered sa-
line (PBS: (137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4 × 7H2O,1.8 mM KH2PO4,
pH 7.4)). Cell viability was controlled by try-
panblue (0.1 % w/v solution) exclusion test.
Cell activation
Cells (1.5 ∙ 106) were preincubated for 0.5, 1
or 3 min at 37 °C with WGA lectin (Lectinotest
Laboratory, Lviv, Ukraine) in the final concen-
tration of 32 mg/ml. Then cells were washed
with PBS+ (137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4 × 7H2O, 1.8 mM KH2PO4,
1 mM CaCl2, 0.5 mM MgCl2 × 6H2O, рН 7.5).
Fractionation of actin cytoskeleton
Cells (1.5 ∙ 106) were lysed in 250 ml of the
buffer composed of 0.5% Triton X-100,
100 mM KCl, 5 mM MgCl2, 2 mM EGTA,
25 mM Tris, pH 7.5 and a protease inhibitor
cocktail (“Sigma”, USA) to separate cytoske-
le tal actin filaments from solubilized actin
[23]. After 10 min (4 °C), cell lysates were
centrifuged for 10 min at 10 000 g (Fig. 1).
The yielded pallet contained long cytoskeletal
actin filaments, whereas the supernatant con-
tained short actin filaments and actin mono-
mers. After withdrawing 10 ml samples of
supernatant for SDS–PAGE analysis, the su-
pernatants were subjected to high-speed cen-
trifugation (1 h, 100 000 g, Fig. 1) to separate
actin monomers (supernatant) and plasma
407
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus
membrane skeleton (pellet). The pellet was
dissolved in 30 ml of 8 M urea and analyzed
by SDS–PAGE. To quantify actin in isolated
fractions, equal volume of each fraction was
applied onto a gel [23]. After immunoblotting
with an anti-actin antibody, actin bands were
Fig. 1. The immunoblot analysis of dif fe-
rent actin fractions in leukocytes of heal thy
rats, rats with experimental diabetes melli-
tus (EDM) and after agmatine admi ni stra-
tion. The scheme for frac tio nating lysates
obtained after cell lysis through 0.5 % Tri-
ton X-100 covers the process of separation
of those fractions that contain cytoskeletal
actin filaments, short actin filaments of the
plasma membrane skeleton and actin mo-
no mers. To determine the level of actin, we
carried out the immunoblot analysis of frac-
tions containing cytoskeletal actin fila-
ments, short actin filaments and actin mo-
no mers: А – fraction from leukocytes acti-
vated for 0.5 min. with wheat germ lectin
(WGA) and immunoblotted for actin ; B –
fraction from leukocytes activated for
1 min. with WGA and immunoblotted for
actin; С – fraction from leukocytes activa-
ted for 3 min. with WGA and immunoblot-
ted for actin. Equal volume of each fraction
was applied onto the gel. Proteins from
1.5 × 106 cells were loaded per lane.
408
I. V. Brodyak, I. I. Bila, N. O. Sybirna
analyzed densitometrically using Gelpro32.
The actin content of each fraction at different
time-points of WGA activation was expressed
as a percentage of the total actin amount.
Immunoblotting
The lysates as well as cellular fractions were
subjected to SDS–PAGE, transferred onto ni-
trocellulose sheets and immunoblotted essen-
tially as described [24]. The membranes were
probed with mouse IgG anti-actin antibody
(MP Biomedicals, OH) followed by anti-mouse
IgG-peroxidase (Sheep anti-Mouse Ig
Antibody, (H+L) HRP conjugate, Millipore,
USA). The immunoreactive bands visualized
by chemiluminescence (Pierce, IL) were ana-
lyzed densitometrically using Gelpro32.
Data analysis
The significance of differences between groups
was calculated using Student’s t-test. P < 0.05
was considered to be statistically significant.
Results and Discussion
The comparative analysis of actin fraction
redistribution (represented by cytoskeletal
filaments, short actin filaments of the plasma
membrane skeleton and actin monomers
(Fig. 1A–C)) was performed on the basis of
immunoblot densitometric analysis using anti-
actin antibodies. In the cells of the control
group, those three actin forms amounted to
72 ± 7, 9 ± 2, and 20 ± 3 % respectively
(Fig. 2).
After 0.5 min of stimulation by WGA lectin,
the percentage of polymerized actin repre-
sented by cytoskeletal filaments decreased by
18%, whereas the content of the short actin
filament increased by 20 % in leukocytes of
the control group (Fig. 2). After 1 min of WGA
lectin exposure, we observed exactly the op-
posite effect: the fraction of cytoskeletal fila-
ments increased up to 70 %, whereas short
actin filaments accounted for only 9 % of po-
lymerized actin. The level of actin monomers
in leukocytes after 0.5 and 1 min exposure to
lectin did not undergo any significant changes.
In 3 min after WGA lectin exposure, F-actin
fraction of short actin filaments of leukocytes
increased from 9 % to 15 %, which correlated
with a decrease in the content of G-actin
against a background of unchanged levels of
cytoskeletal actin filaments. The acquired data
indicate that in the control group of animals
the process of actin polymerization under leu-
kocyte activation by preincubation with WGA
lectin for 0.5 min is intensified in the fraction
of short actin filaments, and in 1 min – in the
cytoskeletal filaments (Fig. 2). Such dynamics
of actin polymerization in leukocytes of the
control group apparently corresponds to the
activation of those blood cells under stimula-
tory factors [25, 26].
Analyzing the redistribution of polymerized
actin fractions, i.e. cytoskeletal actin filaments
and short actin filaments in the initial state
(Fig. 2, 0 min without WGA exposure), it was
revealed that there is a decrease in the number
of cytoskeletal actin filaments and an increase
in short actin filaments of plasma membrane
skeleton in leukocytes of diabetic animals
compared with the control group in similar
conditions (Fig. 2).
In 0.5 min of the WGA lectin stimulation,
the leukocytes of diabetic animals contained
66 ± 5 % of actin in the cytoskeletal filament
fraction, 25 ± 2 % in short actin filaments, and
9 ± 1 % accounted for actin monomers (Fig. 2).
409
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus
The obtained results indicate that under EDM
the stimulation with WGA lectin leads to the
intensification of actin polymerization process
in the fraction of leukocyte cytoskeletal fila-
ments due to the depolymerization of short
actin filaments. The content of actin monomers
Legends: cytoskeletal actin filaments, shot actin filaments, actin monomers
Fig. 2. Redistribution dynamics of actin fractions in lysates of leukocytes containing cytoskeletal actin filaments, short
actin filaments and actin monomers at rest (0 min) and in an activation state (after preincubation with WGA lectin for
thirty seconds, one minute and three minutes): A – control; B – experimental diabetes mellitus (EDM); C – control
against a background of agmatine administration; D – experimental diabetes mellitus against a background of agma-
tine administration. The data were obtained by immunoblot densitometry (Fig. 1A–C). The actin content of each
fraction at different time-points of WGA stimulation was expressed as a percentage of the total actin amount. The data
are presented as the mean ± standard mean error of five experiments: * Significantly different from unstimulated cells
(0 min) at P < 0.05.
410
I. V. Brodyak, I. I. Bila, N. O. Sybirna
under such conditions did not change (Fig. 2).
Although the level of F-actin fraction of cyto-
skeletal filaments did not undergo significant
changes in leukocytes under EDM after 1 and
3 min of WGA lectin exposure, the process of
actin depolymerization (represented by actin
of short filaments) accelerated, thus increasing
the content of actin monomers up to 30 %
after 3 min of WGA lectin exposure (Fig. 2).
After cell activation, there is a rapid and
short-term increase in F-actin, which correlates
with a decrease in the content of G-actin [27].
However, under EDM the opposite processes
of intensified depolymerization of short actin
filaments and increased contents of G-actin
take place in leukocytes activated by WGA
lectin. During the activation of leukocytes,
short actin filaments are involved in changing
the morphology and functional capacity of the
cells, whereas the reduction of their content in
leukocytes under diabetes mellitus clearly
causes the impairment of all those functions.
Such changes may be a result of the distur-
bances of transmembrane and intracellular
signaling through sialoglycoconjugates of leu-
kocytes, the number and structural organiza-
tion of oligosaccharide chains of which are
changed under EDM [28].
The results are consistent with our earlier
research [29], which revealed that under type
1 DM the PI-3'-kinase signaling pathway,
which in fact causes the delay of WGA-
stimulating signal transduction, in blood leu-
kocytes is misaligned and slowed. PI-3'-kinase
signaling pathway is involved in cellular re-
sponse to external stimuli by regulating the
activity of proteins of the cytoskeleton [14]
and forming stress fibrils, lamellipodia and
filopodia of leukocytes [30, 31]. Changes in
the level of actin polymerization-depolymer-
ization in leukocytes under EDM may indicate
a breach in systems of activation, adhesion and
phagocytosis, as well as manifestation of cy-
totoxicity, because the actin cytoskeleton of
leukocytes is continually reorganized, being a
dynamic system that determines the morpho-
logical changes in the cell [32, 33].
After agmatine administration, actin frac-
tions in leukocytes were distributed as follows
in the control group of rats: 72 ± 6 % in the
fraction containing membrane filaments close-
ly associated with the cytoskeleton, 18 ± 3 %
represented short actin filaments, and actin
monomers accounted for 10 ± 2 % (Fig. 2).
Thus, the administration of agmatine to the
control group of animals leads to the reduction
of actin monomers due to the intensification
of actin filament polymerization in the plasma
membrane skeleton in comparison with un-
stimulated leukocytes from the control group
of animals (Fig. 2).
In leukocytes of the control group of ani-
mals against the background of agmatine ad-
ministration after 0.5 min of WGA lectin
stimulation, the percentage of polymerized
actin represented by cytoskeletal filaments and
short actin filaments, decreased by 50 % and
11 % respectively. Such an intensive process
of depolymerization led to a sharp increase in
G-actin – from 10 % in the state of inactivation
to 72 % in the state of leukocyte activation by
WGA lectin for thirty seconds. After 1 min
WGA lectin exposure, we observed just the
opposite effect – an intensive process of actin
polymerization caused an increase in F-actin
filaments of the cytoskeletal fraction up to
45 %, whereas the fraction of short actin fila-
ments increased up to 20 %. The level of actin
411
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus
monomers in leukocytes after 1 min WGA
lectin exposure decreased twofold. In 3 min
after WGA lectin exposure, the level of F-actin
filaments of the leukocyte cytoskeleton
dropped to 7 %, which correlated with an in-
crease in G-actin content up to about 80 %.
The obtained data indicate that in the control
group of animals the agmatine administration
causes the formation of leukocyte response to
activation signals after 1 min exposure to
WGA lectin, and it terminates the process of
actin filament cell reorganization if the cells
are preincubated for 3 min (Fig. 2). Conse qu-
en tly, the dynamics of actin redistribution in
this group of animals shows that major chan-
ges in activated leukocytes occur in the cyto-
skeletal filaments, correlating with the chan ges
in actin monomers. Since the level of F-actin
fraction of short actin filaments after 1 and
3 min. of WGA lectin stimulating effects does
not undergo significant changes as compared
to the initial state of leukocytes (0 min. – with-
out WGA exposure), it can be assumed that in
the control group of animals agmatine causes
the cytoskeletal actin filaments polymerization
de novo at the exposure of actin monomers at
1 min of WGA lectin action, involving the
activation of Arp2/3 complex [34].
If agmatine is administered to animals with
EDM, the process of polymerization in leuko-
cytes intensifies in the fraction of cytoskeletal
filaments and depolymerization of short actin
filaments occurs compared to animals under
EDM without agmatine administration (Fig. 2,
see 0 min.). Under EDM, against a background
of agmatine administration, the stimulating
effect of WGA lectin exposure for 0.5 min.
leads to the intensification of actin polymeriza-
tion process in the cytoskeletal filament frac-
tion of leukocytes due to depolymerization of
short actin filaments and usage of actin mono-
mers (Fig. 2). After 1 min WGA lectin expo-
sure, the level of F-actin filaments of the leu-
kocyte cytoskeleton reduced by 40 %, which
correlated with an increase in G-actin content
up to 30 % and short actin filaments up to 20 %
(Fig. 2). After 3 min of WGA lectin stimulation
in leukocytes of animals with EDM against the
background of agmatine administration, actin
was redistributed among the three fractions in
the following ratio – 67 ± 8, 23 ± 3 and 10 ± 1,
reaching the level of performance in an inac-
tive state (Fig. 2, see 0 and 3 min.).
Therefore, in leukocytes of animals with
EDM, against the background of agmatine
administration, the dynamics of response to
stimulating effects of WGA lectin were cha-
rac terized by the intensification of polymeriza-
tion of cytoskeletal actin filaments already
after 0.5 min exposure to lectin, by exceptio-
nal ly rapid depolymerization processes after
1 min exposure to lectin, and by the return to
the performance level in the initial state after
3 min exposure to lectin (Fig. 2). Under those
conditions, the WGA lectin binds to comple-
mentary glycoconjugates on the surface of
leukocytes and lectin-induced signal transduc-
tion, which causes the reorganization of the
actin cytoskeleton, reach actin content in each
fraction similarly to the activated leukocytes
of the control group of animals. The findings
indicate that agmatine positively affects the
functional state of the animal leukocytes under
EDM. Perhaps the normalization of glucose
level in animals with EDM in response to
agmatine [35, 36] is one of the mechanisms
for mediating those functional changes in leu-
kocytes under EDM.
412
I. V. Brodyak, I. I. Bila, N. O. Sybirna
Conclusions
Based on our previous results [22, 28] and
obtained data, we can conclude that under
EDM the changes in the number and struc-
tural organization of sialoglycoconjugates on
the surface of leukocytes lead to the disruption
of WGA-induced transmembrane and intracel-
lular signaling, resulting in the enhanced de-
polymerization processes of short actin fila-
ments and an increase in the content of G-actin.
In leukocytes of the animals with EDM,
against the background of agmatine adminis-
tration, the transduction of lectin-induced sig-
nal through sialoglycoconjugates causes a
quantitative redistribution of actin fractions. It
indicates that polyamine helps to restore and
maintain the functional response of leukocytes
to the activation signals.
REFERENCES
1. Alba-Loureiro TC, Munhoz CD, Martins JO, Cer-
chiaro GA, Scavone C, Curi R, Sannomiya P. Neu-
trophil function and metabolism in individuals with
diabetes mellitus. Braz J Med Biol Res. 2007;
40(8):1037–44.
2. McManus LM, Bloodworth RC, Prihoda TJ, Blod-
gett JL, Pinckard RN. Agonist-dependent failure
of neutrophil function in diabetes correlates with
extent of hyperglycemia. J Leukoc Biol. 2001;70(3):
395–404.
3. Tipu HN, Ahmed TA, Bashir MM. Human leukocyte
antigen class II susceptibility conferring alleles
among non-insulin dependent diabetes mellitus pa-
tients. J Coll Physicians Surg Pak. 2011;21(1):26–9.
4. Vicente-Manzanares M, Sánchez-Madrid F. Role of
the cytoskeleton during leukocyte responses. Nat
Rev Immunol. 2004;4(2):110–22.
5. Ley K, Laudanna C, Cybulsky MI, Nourshargh S.
Getting to the site of inflammation: the leukocyte
adhesion cascade updated. Nat Rev Immunol.
2007;7(9):678–89.
6. Galbraith CG, Yamada KM, Galbraith JA. Polyme-
rizing actin fibers position integrins primed to probe
for adhesion sites. Science. 2007;315(5814):992–5.
7. Narita A, Mueller J, Urban E, Vinzenz M, Small JV,
Maéda Y. Direct determination of actin polarity in
the cell. J Mol Biol. 2012;419(5):359–68.
8. Arjonen A, Kaukonen R, Ivaska J. Filopodia and
adhesion in cancer cell motility. Cell Adh Migr.
2011;5(5):421–30.
9. Schnoor M. Endothelial actin-binding proteins and
actin dynamics in leukocyte transendothelial migra-
tion. J Immunol. 2015;194(8):3535–41.
10. Rullo J, Becker H, Hyduk SJ, Wong JC, Digby G,
Arora PD, Cano AP, Hartwig J, McCulloch CA,
Cybulsky MI. Actin polymerization stabilizes α4β1
integrin anchors that mediate monocyte adhesion.
J Cell Biol. 2012;197(1):115–29.
11. Fukuyama T, Ogita H, Kawakatsu T, Inagaki M,
Takai Y. Activation of Rac by cadherin through the
c-Src-Rap1-phosphatidylinositol 3-kinase-Vav2
pathway. Oncogene. 2006;25(1):8–19.
12. Heit B, Liu L, Colarusso P, Puri KD, Kubes P. PI3K
accelerates, but is not required for, neutrophil che-
motaxis to fMLP. J Cell Sci. 2008;121(Pt 2):205–14.
13. Snapp KR, Heitzig CE, Kansas GS. Attachment of
the PSGL-1 cytoplasmic domain to the actin cyto-
skeleton is essential for leukocyte rolling on P-se-
lectin. Blood. 2002;99(12):4494–502.
14. Welch MD. The world according to Arp: regulation
of actin nucleation by the Arp2/3 complex. Trends
Cell Biol. 1999;9(11):423–7.
15. Pollard TD, Blanchoin L, Mullins RD. Molecular
mechanisms controlling actin filament dynamics in
nonmuscle cells. Annu Rev Biophys Biomol Struct.
2000;29:545–76.
16. Brodyak IV, Bila II, Overchuk M, Sybirna NO. Effect
of agmatine on actin polymerization in leukocytes
of strep-tozotocin-induced diabetic rats. Studia Bio-
logica. 2014; 8(3–4):17–30.
17. Chang CH, Wu HT, Cheng KC, Lin HJ, Cheng JT. In-
crease of beta-endorphin secretion by agmatine is in-
duced by activation of imidazoline I(2A) receptors in
adrenal gland of rats. Neurosci Lett. 2010;468(3):297–9.
18. Hwang SL, Liu IM, Tzeng TF, Cheng JT. Activation
of imidazoline receptors in adrenal gland to lower
413
The dynamics of actin filament polymerization in activated leukocytes under experimental diabetes mellitus
plasma glucose in streptozotocin-induced diabetic
rats. Diabetologia. 2005;48(4):767–75.
19. Jou SB, Liu IM, Cheng JT. Activation of imidazoline
receptor by agmatine to lower plasma glucose in
streptozotocin-induced diabetic rats. Neurosci Lett.
2004;358(2):111–4.
20. Lee JP, Chen W, Wu HT, Lin KC, Cheng JT. Metfor-
min can activate imidazoline I-2 receptors to lower
plasma glucose in type 1-like diabetic rats. Horm
Metab Res. 2011;43(1):26–30.
21. Piletz JE, Aricioglu F, Cheng JT, Fairbanks CA,
Gilad VH, Haenisch B, Halaris A, Hong S, Lee JE,
Li J, Liu P, Molderings GJ, Rodrigues AL, Satria-
no J, Seong GJ, Wilcox G, Wu N, Gilad GM. Agma-
tine: clinical applications after 100 years in transla-
tion. Drug Discov Today. 2013;18(17–18):880–93.
22. Sybirna NО, Shevtsova AI, Ushakova GО, Bro-
dyak IV, Pismenetzka IY. Fundamentals of glycobi-
ology. Monograph; Lviv: Ivan Franko National
University of Lviv, 2015. 492 p.
23. Kleveta G, Borzęcka K, Zdioruk M, Czerkies M,
Kuberczyk H, Sybirna N, Sobota A, Kwiatkowska K.
LPS induces phosphorylation of actin-regulatory
proteins leading to actin reassembly and macrophage
motility. J Cell Biochem. 2012;113(1):80–92.
24. Kwiatkowska K, Frey J, Sobota A. Phosphorylation
of FcgammaRIIA is required for the receptor-in-
duced actin rearrangement and capping: the role of
membrane rafts. J Cell Sci. 2003;116(Pt 3):537–50.
25. Fenteany G, Zhu S. Small-molecule inhibitors of
actin dynamics and cell motility. Curr Top Med
Chem. 2003;3(6):593–616.
26. Lin BH, Tsai MH, Lii CK, Wang TS. IP3 and cal-
cium signaling involved in the reorganization of the
actin cytoskeleton and cell rounding induced by
cigarette smoke extract in human endothelial cells.
Environ Toxicol. 2016;31(11):1293–1306.
27. Watts RG, Crispens MA, Howard TH. A quantitative
study of the role of F-actin in producing neutrophil
shape. Cell Motil Cytoskeleton. 1991;19(3):159–68.
28. Ferents I, Brodyak I, Lyuta M, Klymyshyn N, Bur-
da V, Sybirna N. Sialylation status of leukocyte
cell-surface glycoconjugates in streptozotocin-in-
duced diabetic rats and after treatment with agma-
tine. Curr Iss Pharm Med Sci. 2013; 26(4):390–2.
29. Sybirna NO, Zdioruk MI, Brodiak IV, Bars’ka ML,
Vovk OI. [Activation of the phosphatidylinositol-3’-
kinase pathway with lectin-induced signal through
sialo-containing glycoproteins of leukocyte mem-
branes under type 1 diabetes mellitus]. Ukr Biokhim
Zh. (1999). 2011;83(5):22–31.
30. Aiba Y, Kameyama M, Yamazaki T, Tedder TF, Kuro-
sa ki T. Regulation of B-cell development by BCAP
and CD19 through their binding to phosphoinositide
3-kinase. Blood. 2008;111(3):1497–503.
31. Fais S, Malorni W. Leukocyte uropod formation and
membrane/cytoskeleton linkage in immune interac-
tions. J Leukoc Biol. 2003;73(5):556–63.
32. Pollard TD, Borisy GG. Cellular motility driven by
assembly and disassembly of actin filaments. Cell.
2003;112(4):453–65. Review. Erratum in: Cell.
2003;113(4):549.
33. Zarbock A, Kempf T, Wollert KC, Vestweber D.
Leukocyte integrin activation and deactivation:
novel mechanisms of balancing inflammation. J Mol
Med (Berl). 2012;90(4):353–9.
34. Samstag Y, Eibert SM, Klemke M, Wabnitz GH. Actin
cytoskeletal dynamics in T lymphocyte activation and
migration. J Leukoc Biol. 2003;73(1):30–48.
35. Ferents IV, Brodyak IV, Lyuta MYa, Sybirna NО.
Effect of agmatine on the blood system parameters
of rats un-der the condition of experimental diabetes
mellitus. Studia Biologica. 2012; 6(3):65–72.
36. Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc
Rhoads J, Carey Satterfield M, Smith SB, Spen-
cer TE, Yin Y. Arginine metabolism and nutrition in
growth, health and disease. Amino Acids. 2009;
37(1):153–68.
Динаміка полімеризації актинових філаментів
в активованих лейкоцитах за умов
експериментального цукрового діабету на фоні
введення агматину
І. В. Бродяк, І. І. Біла, Н. О. Сибірна
Мета. Дослідити вплив агматину на перерозподіл
фракцій актину, які представлені актиновими філамен-
тами цитоскелету, короткими актиновими філамента-
ми скелету плазматичної мембрани і мономерами ак-
тину (G-актин), у лейкоцитах щурів з експерименталь-
414
I. V. Brodyak, I. I. Bila, N. O. Sybirna
ним цукровим діабетом (ЕЦД) Методи. Лейкоцити
лізували в Тритоні Х-100 і піддавали центрифугуван-
ню, у результаті якого було отримано актинові філа-
менти цитоскелету, короткі актинові філаменти і моно-
мери актину, які розділяли в SDS-ПААГ, після чого
проводили імуноблот аналіз із використанням анти-
актинових антитіл. Результати. За умов ЕЦД у лейко-
цитах, активованих сіалоспецифічним лектином за-
родків пшениці (WGA), спостерігається інтенсифіку-
вання процесу деполімеризації коротких актинових
філаментів і збільшення вмісту G-актину, а на фоні
введення агматину динаміка формування відповіді на
WGA-стимулювальний вплив характеризувалася по-
силенням полімеризації актину у фракції філаментів
цитоскелету вже на 0,5 хв впливу лектину, дуже стрім-
ким процесом деполімеризації на 1 хв після дії лекти-
ну та поверненням показників до рівня у вихідному
стані у разі дії лектину впродовж 3 хв. Висновки.
У лейкоцитах тварин з ЕЦД на фоні введення агмати-
ну трансдукція WGA-індукованого сигналу через
сіалоглікокон’югати зумовлює перерозподіл вмісту
актину, вказуючи на те, що даний поліамін сприяє від-
новленню і підтриманню функціональної відповіді
лейкоцитів на активаційні сигнали.
К л юч ов і с л ов а: актин, лейкоцити, агматин, екс-
периментальний цукровий діабет.
Динамика полимеризации актиновых
филаментов в активированных лейкоцитах
в условиях экспериментального сахарного
диабета на фоне введения агматина
И. В. Бродяк, И. И. Била, Н. А. Сибирна
Цель. Исследовать влияние агматина на перераспре-
деление фракций актина, которые представлены
актиновыми филаментами цитоскелета, короткими
актиновыми филаментами скелета плазматической
мембраны и мономерами актина (G-актин), в лейко-
цитах крыс с экспериментальным сахарным диабетом
(ЭСД). Методы. Лейкоциты лизировали в Тритоне
Х-100 и подвергали центрифугированию, в результате
которого было получено актиновые филаменты цитос-
келета, короткие актиновые филаменты и мономеры
актина, которые разделяли в SDS-ПААГ, после чего
проводили иммуноблот анализ с использованием
антиактиновых антител. Результаты. В условиях ЭСД
в лейкоцитах, активированных сиалоспецыфическим
лектином зародышей пшеницы (WGA), наблюдается
интенсификация процесса деполимеризации коротких
актиновых филаментов и увеличение содержания
G-актина, а на фоне введения агматина динамика
формирования ответа на WGA-стимулирующее влия-
ние характеризировалась усилением полимеризации
актина во фракции филаментов цитоскелета уже на 0,5
мин влияния лектина, очень стремительным процессом
деполимеризации на 1 мин после действия лектина и
возвращением показателей к уровню в исходном со-
стоянии в случае действия лектина в течение 3 мин.
Выводы. В лейкоцитах животных с ЭСД на фоне
введения агматина трансдукция WGA-индуцированого
сигнала через сиалогликоконьюгаты приводит к пере-
распределению содержания актина, указывая на то,
что данный полиамин способствует восстановлению
и поддержанию функционального ответа лейкоцитов
на активационные сигналы.
К л юч е в ы е с л ов а: актин, лейкоциты, агматин,
экспериментальный сахарный диабет.
Received 20.09.2017
|