Báo cáo khoa học: Role of peroxidase inhibition by insulin in the bovine thyroid cell proliferation mechanism pdf

Role of peroxidase inhibition by insulin in the bovine thyroid cell
proliferation mechanism
Leo
´
n Krawiec
1
, Ramo
´
n A. Pizarro
2
, Paula Aphalo, Elena M. V. de Cavanagh
3
, Mario A. Pisarev
1,2,4
,
Guillermo J. Juvenal
1,2
, Lucı
´
a Policastro
2
and Laura V. Bocanera
2
1
Argentine National Research Council (CONICET);
2
Department of Radiobiology, Argentine Atomic Energy Commission (CNEA);
3
Department of Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires and
4
Department of Human

Biochemistry, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina
Monolayer primary cultures of thyroid cells produce, in the
presence of insulin, a cytosolic inhibitor of thyroid peroxi-
dase (TPO), lacto peroxidase (LPO), horseradish peroxidase
(HRPO) and glutathione peroxidase (GPX). The inhibitor,
localized in the cytosol, is thermostable and hydrophylic.
Its molecular mass is less than 2 kDa. The inhibitory activity,
resistant to proteolytic and nucleolytic enzymes, disappears
with sodium metaperiodate treatment, as an oxidant of
carbohydrates, supporting its oligosaccharide structure. The
presence of inositol, mannose, glucose, the specific inhibition
of cyclic AMP-dependent protein kinase and the disap-
pearance of peroxidase inhibition by alkaline phosphatase
and a-mannosidase in purified samples confirms its chemical
structure as inositol phosphoglycan-like. Purification by
anionic interchange shows that the peroxidase inhibitor
elutes like the two subtypes of inositol phosphoglycans
(IPG)P and A, characterized as signal transducers of insulin
action. Insulin significantly increases the concentration of
the peroxidase inhibitor in a thyroid cell culture at 48 h. The
addition of both isolated substances to a primary thyroid
culture produces, after 30 min, a significant increase in
hydrogen peroxide (H
2
O
2
) concentration in the medium,
concomitantly with the disappearance of the GPX activity in
the same conditions. The presence of insulin or anyone of
both products, during 48 h, induces cell proliferation of the

thyroid cell culture. In conclusion, insulin stimulates thyroid
cell division through the effect of a peroxidase inhibitor, as its
second messenger. The inhibition of GPX by its action
positively modulates the H
2
O
2
level, which would produce,
as was demonstrated by other authors, the signal for cell
proliferation.
Keywords: insulin; IPG; inhibitor; peroxidase; proliferation.
Thyroid peroxidase (TPO) organifies iodide in the presence
of H
2
O
2
and is responsible for the synthesis of the thyroid
hormones. Bocanera et al. demonstrated that monolayer
primary cultures of thyroid cells, in the presence of insulin,
lose their iodide organification capacity several days before
the disappearance of TPO activity, due to the presence of
a cytosolic inhibitor, not detectable in fresh tissue [1].
However, the inhibitor is active on TPO isolated from fresh
tissue and inhibits the iodide organification when it is added
to free follicles. This inhibitor is thermostable, dialyzable,
has a molecular mass of less than 2 kDa and it has no
species specificity, as was shown in our previous work [1].
Several authors have demonstrated the remarkable role
of insulin in many aspects of cell metabolism. The observed
effects were ascribed to the modulation exerted by the

hormone on key enzymes of different metabolic pathways.
During the past decades it has been demonstrated that
inositol phosphoglycan (IPG) acts as a mediator of insulin
and insulin growth factor (IGF-I), mimicking their effects
[2–8]. Water-soluble IPG, results from the hydrolysis of
glycosyl phosphatidylinositol (GPI) [8,9]. Two related
substances, able to act as insulin mediators, were isolated
from hepatic membranes [10]. Both compounds have a
molecular mass of 1000–2000 Da. The isolation and partial
characterization, in bovine [11] and human liver [12], of two
subtypes of IPG (P and A) which act as signal transducers of
the insulin action, confirmed these findings. The aims of the
present study were to establish: (a) if insulin regulates the
peroxidase activity through the action of the cytosol
inhibitor as a second messenger; (b) if the inhibitor and
IPG are alike and (c) the implications of this regulatory
pathway in the mechanisms of cell proliferation.
Materials and methods
Cell cultures
Monolayer primary and free follicle cultures of bovine
thyroid cells were obtained according to Bocanera et al.[1].
Briefly, bovine thyroids were obtained from a local slaugh-
terhouse immediately after death and transported to the
laboratory in ice-cold saline containing 100 UÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin. The glands were
Correspondence to L. Krawiec, Divisio
´

n Bioquı
´
mica Nuclear, Unidad
de Actividad Radiobiologı
´
a, Comisio
´
n Nacional de Energı
´
aAto
´
mica,
Avenida del Libertador 8250, 1429-Buenos Aires, Argentina.
Fax: + 54 11 67727121; Tel.: + 54 11 6772 7185;
E-mail:
Abbreviations: GPX, glutathione peroxidase; HRPO, horseradish
peroxidase; LPO, lacto peroxidase; TPO, thyroid peroxidase.
(Received 5 April 2004, accepted 28 April 2004)
Eur. J. Biochem. 271, 2607–2614 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04189.x
dissected carefully, cut into small pieces under sterile condi-
tionsanddigestedwith1 mgÆmL
)1
collagenase type 1 A and
DNAse 20 lgÆmL
)1
in 199 medium (Sigma Chemical Co.)
with 2.2 mgÆmL
)1
NaHCO
3

,5 lgÆmL
)1
transferrin, 0.85 l
M
bovine insulin, 2.5 lgÆmL amphotericin B, 100 UÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin, in a relation of
10 mLÆg
)1
tissue, at 39 °C, during 90 min. The digested
material was filtered through sterile gauze and centrifuged at
700 g during 30 s, washed twice with the medium and
resuspended in the complete M-199 medium containing 5%
fetal bovine serum. The follicles were seeded in standard
plates for monolayer cultures or in dishes supplied with a
hydrophobic layer, to keep the follicles in suspension. After
48 h, the medium was replaced by another containing 0.1%
fetal bovine serum. Previous to each treatment, insulin was
suppressed for 48 h. The cultures were stopped at different
days and assays were performed as indicated below. All cells
were grown at 37 °C, under an atmosphere of 95% air and
5% CO
2
, in a humidified incubator.
Bovine TPO isolation
Thyroid peroxidase was obtained according to Bocanera
et al. [1]. Briefly, fresh glands were dissected, cut into small
pieces and homogenized in 0.1

M
phosphate buffer, 0.1 m
M
KI, pH 8.0 [1 : 5 (w/v)]. The homogenate was filtered
through gauze and centrifuged at 900 g during 10 min
at 4 °C. The resulting supernatant was centrifuged at
105 000 g during 60 min. The pellet was resuspended [1/1,
(w/v)] in the same buffer containing 0.6% Triton X-100 and
kept on ice, with frequent agitation, during 180 min. After
centrifugation at 105 000 g, during 60 min, the resulting
supernatant was utilized as the source of solubilized TPO.
Isolation of cytosolic inhibitor
The 105 000 g supernatant obtained from monolayer cells
cultures, homogenized in milli Q water, was analyzed for its
inhibitory activity on TPO extracted from fresh glands.
Thyroid peroxidase (TPO), lacto peroxidase (LPO),
horseradish peroxidase (HRPO), glutathione peroxidase
(GPX), catalase and cyclic AMP-dependent protein kinase
activity assays
TPO, LPO and HRPO activities were determined following
the tyrosine iodination assay, described by DeGroot and
Davis [13]. GPX was assayed according to Flohe
´
and
Gunzler [14]. Catalase was determined by the method of
Aebi [15] and cyclic AMP-dependent protein kinase
according to Villalba et al. [16].
Hydrogen peroxide assay
Hydrogen peroxide was determined according to Ravindra-
nath [17].

Effect of the peroxidase inhibitor on glucose transport
in free follicles
The free follicles, cultured as described above, were washed
three times with Krebs–Ringer 20 m
M
Hepes (KRH),
pH 7.4, without glucose. The experiments were performed
in 24-well plates, with 0.1 mL of the follicles suspension in
KRH (5 mg of proteinÆmL
)1
) and aliquots corresponding
to 0; 10; 20; 50 lg of cytosolic protein, obtained from
confluent monolayer thyroid cell culture, in each well.
After preincubation for 60 min at 37 °C,theuptakeof
2-deoxyglucose (nonmetabolic substrate) was assessed
according to Krawiec et al.[18].
Solubility of the peroxidase inhibitor in organic solvents
The cell supernatant (2 mL corresponding to 1 mg of
protein) was mixed with 2 mL of chloroform, by stirring the
mixture vigorously, at room temperature. Both phases,
discarding the interphase, were separated by centrifugation
and evaporated to dryness. The dry residues were resus-
pended in the original volume of water and the TPO activity
assayed as described above.
Action of proteolytic enzymes on the peroxidase
inhibitor
Trypsin and chimotrypsin, resuspended in 200 m
M
ammo-
nium bicarbonate, pH 8.0, were incubated with the cyto-

solic fraction (1 lg of enzyme per 15 lgofprotein)at37 °C,
during 12 h.
Proteinase K (1 lg of enzyme per 10 lgofprotein)was
incubated with the cytosolic fraction in a medium contain-
ing (final concentration): 10 m
M
Tris/HCl; 0.1
M
NaCl;
1m
M
EDTA, pH 7.5, at 56 °C, during 12 h. In all cases, the
proteolytic enzymes were inactivated at 95 °C, during
10 min and the TPO control assay was performed in the
presence of the inactivated enzymes.
Effects of DNAse and RNAse on the peroxidase inhibitor
The incubations were performed in both cases in NaCl/P
i
,
pH 7.4, at 37 °C during 12 h, with an enzymatic concen-
tration 1 : 10 of the protein present in the assay. The
reactions were continued as indicated for the proteolytic
assays.
Purification of the peroxidase inhibitor by liquid–liquid
partition
Purification was performed according to the method of
Folch et al. [19] modified, handled with a rotary evaporator
(e.g. Heidolph WB 2000), under reduced pressure and low
N
2

stream, at 65 °C in a water bath. The cell supernatant
was extracted first, in a separating funnel, with 20 volumes
of chloroform/methanol [2 : 1 (v/v)] and the partition was
made with water (1 : 6 of the total volume). After centrif-
ugation, the hydrophilic upper phase was removed. The
hydrophobic lower phase was re-extracted with theoretical
upper phase from the mixture chloroform/methanol/water
[8 : 4 : 3 (v/v)]. The second upper phase was combined with
the first for further processing. The evaporated upper and
lower phases were resuspended in water (50% of the original
volume) and the inhibitory effects were compared. The
combined upper phases were submitted to a liquid–liquid
partition in a column of Sephadex G-25 coarse (dry bead
diameter 100–300 lm), according to Siakotos and Rouser
2608 L. Krawiec et al. (Eur. J. Biochem. 271) Ó FEBS 2004
[20]. The beads were soaked overnight in methanol/water
[1 : 1 (v/v)] and then rinsed four or five times with the same
solvent. The Sephadex was packed into a 1.5 · 20 cm
column and successively washed twice in the same order and
with the same volumes of the four mixtures of solvents then
used for the elution. After seeding the sample, the elution
was made in the following order of increasing polarity:
(a) Fifty mililiters chloroform/methanol 19:1 (v/v), saturated
with 0.5 percentage water for the elution of non-ganglioside
lipids. (b) One hundred mililiters chloroform/methanol 19:1
(v/v) five volumes, acetic acid one volume, saturated
with 2.5 percentage water, for the elution of gangliosides.
(c) Fifty mililiters chloroform/methanol 9:1 (v/v) five
volumes, acetic acid one volume, saturated with 4.2
percentage water, for the elution of traces of gangliosides.

(d) One hundred mililiters methanol/water 1:1 (v/v) for the
elution of non-lipid polar compounds soluble in water.
After evaporation the fractions were resuspended in water
(50% of the volume submitted to the partition).
Effect of sodium metaperiodate (NaO
4
I) on the activity
of the peroxidase inhibitor
The inhibitor, partially purified by liquid–liquid partition,
was evaporated to dryness, resuspended in NaCl/P
i
with
2m
M
NaO
4
Iin0.1
M
phosphate buffer 0.1 m
M
KI, pH 7.0,
and maintained at room temperature in the darkness during
12 h. The remnant inhibitory action was assayed on the
TPO enzyme as described above. The control activity of
TPO was determined in the presence of NaO
4
I.
Action of alkaline phosphatase on the inhibitor
The inhibitor purified as described above, was evaporated to
dryness and resuspended in 50 mm Tris/HCl (pH 9.3 at

25 °C), 1 m
M
MgCl
2
,0.1 m
M
ZnCl
2
, 1 mm spermidine, and
maintained at 37 °C during 12 h. After inactivating the
enzyme at 95 °C, during 10 min, in the presence of 5 mm
EDTA, the remnant inhibitory action was assayed on the
TPO enzyme as described above. The control activity of
TPO was determined in the presence of inactivated alkaline
phosphatase.
Action of a-mannosidase on the inhibitor
The inhibitor purified as described above, was evaporated to
dryness and resuspended in 100 m
M
acetate buffer (pH 5.0),
and incubated in the presence of 100 mU of a-mannosidase
at 37 °C during 12 h. After inactivating the enzyme at
95 °C, during 10 min, the mixture was neutralized with
KOH. The remnant inhibitory action was assayed on the
TPO enzyme as described above. The control activity of
TPO was determined in the presence of inactivated
a-mannosidase.
Thin layer chromatography (TLC) of the peroxidase
inhibitor and its components
Samples, obtained by liquid–liquid partition, were applied

in duplicate plates of silica gel 60 on polyester. The
developing solvent consisted in a mixture of chloroform/
methanol/0.2% calcium chloride [60 : 40 : 9 (v/v/v)]. After
drying the plates, one of them was (a) submerged in a
solution of sulfuric acid 5% in ethanol; (b) dried and (c)
heated at 120 °C in a stove, until the appearance of brown
spots, typical of carbohydrates. The silica of the second
plate, with no detection reagent, was scraped at the position
where the brown spots had appeared on the first plate. This
material was then resuspended in water and extracted by
shaking. After centrifugation, the supernatant was assayed
on the TPO activity.
The inhibitor components were determined in the sam-
ples, previously hydrolized in 6
M
HCl, during 8 h at 100 °C
and evaporated under nitrogen. The samples were applied in
a plate of silica gel 60 on polyester. The developing solvent
consisted of a mixture of propanol/ethanol/water (7 : 1 : 2).
The procedure continued as was described above.
D
-Mannose,
D
-galactose,
D
-trehalose, inositol and
D
-glucose
were run in parallel, as standards.
High performance liquid chromatography (HPLC)

for protein isolation
The protein content of the same fraction purified by TLC
was analyzed by reverse-phase chromatography, utilizing:
column C8, 30 · 2.1 mm (Brownlee). Solvent A, trifluoro-
acetic acid (TFA) 0.1%; Solvent B, acetonitrile 80% in TFA
0.08%; Gradient, 5 min at 10% of solvent B and 30 min
from 10% to 100% of solvent B. The isolated protein
fractions were assayed for the inhibitory effect on the TPO
activity.
Isolation of peroxidase inhibitor as inositol
phosphoglycan (IPG)-like compound
The samples, purified by liquid–liquid partition, were
processed following the protocols described previously by
Caro et al. [12].
Cell proliferation
Bovine thyroid cells were cultured as described previously.
After the different treatments (48 h in the presence of
insulin, IPG P or IPG A-like) the cultures were stopped.
Cell counting. Dishes of cells were detached from the
monolayer with trypsin and counted in a hemocytometer.
[
3
H]Thymidine incorporation. To measure thymidine
incorporation into TCA-precipitable material, [
3
H]thymi-
dine (5 lCiÆmL
)1
) was added together with insulin, IPG P
or IPG A-like during all the treatment. At the end of the

culture period the medium was removed and the cells were
washed with phosphate-buffered saline (NaCl/P
i
). TCA
(0.5 mL, 10%) was added to the wells for 1 h at 4 °C. After
the removal of TCA the treatment was repeated. The TCA-
precipitable material was dissolved using 0.3
M
NaOH,
overnight at 37 °C and the radioactivity was counted in a
liquid scintillation counter.
Protein was determined according to Lowry et al.[21].
All reagents were obtained from Sigma Chemical Co
(St. Louis, MO, USA). Na
125
Iand[
3
H]thymidine were
purchased from New England Nuclear. Each experiment
was repeated at least four times and each point was
Ó FEBS 2004 Peroxidase inhibition and proliferation (Eur. J. Biochem. 271) 2609
run in quadruplicate. Statistical significance of the
differences among groups was calculated according to
Dunnet [22].
Results
The 105 000 g supernatant (cytosolic fraction) obtained
from monolayer bovine thyroid cells cultured in the
presence of 0.85 l
M
insulin, usual concentration for thyroid

cultures, causes significant inhibitory effects on the semi-
purified TPO activity from fresh tissue. Values of TPO
activity in the monolayer thyroid cell cultures were below
the blanks.
In order to isolate the peroxidase inhibitor, the 105 000 g
supernatant was submitted to a purification procedure by a
liquid–liquid partition method. In the first stage, the
hydrophylic upper phase contained the total original
activity of the peroxidase inhibitor. In the second stage of
the purification, 70% of the inhibitory activity was recov-
ered in fraction 4 (F4) (methanol/water 1 : 1), which
contains nonlipid water-soluble compounds. Fractions 3
and 2 contributed with 25 and 5%, respectively, of total
inhibitory activity, while no activity was recovered in
fraction 1, which extracts lipids (results not shown).
The physicochemical nature of the peroxidase inhibitor
was assessed by different methods. The biological activity,
as a TPO inhibitor, was used as an end-point to monitor the
effect of different treatments.
Table 1 shows the effect of several enzymes, including
trypsin, chymotrypsin and proteinase K, on the 105000·g
supernatant from the thyroid monolayer cell culture. None
of these proteolytic enzymes affected the inhibitory power.
These results were confirmed by the HPLC analysis of the
semipurified fraction F4 demonstrating the absence of
inhibitory effect on TPO in the protein fractions (data not
shown).
The absence of lipids in the structure of the peroxidase
inhibitor, discernible throughout the purification process,
was confirmed by the fact that all the inhibitory activity

remained in the aqueous phase after emulsifying the
105 000 g supernatant fraction with an equal volume of
chloroform.
DNA and RNA were ruled out as possible components
of the inhibitor, as DNAse and RNAse did not affect the
inhibitory capacity (data not shown).
Finally, as can be seen in Table 1, peroxidase inhibition
disappeared when the isolated fractions were preincubated
with sodium metaperiodate, which produces oxidative
ruptures of carbohydrates. Furthermore, the presence of
carbohydrates in the inhibitor structure was confirmed by
TLC.
In order to achieve a more accurate identification of
the peroxidase inhibitor, fraction F4 was submitted to
the method of inositol phosphoglycans (IPG) isolation.
Figure 1 shows the inhibition on TPO elicited by the
different fractions successively isolated from the original
cytosolic supernatant. Both fractions IPG-like: type P and
type A significantly inhibited TPO activity. Fraction A was
more powerful (100%) than fraction P (61%), as indicated
by comparing the inhibition percentages of equal volumes
eluted of both fractions.
The disappearance of the inhibitory effect on the
peroxidase activity, when fraction F4 was submitted to
alkaline phosphatase (Table 1), or to a-mannosidase
(not shown) suggested that its chemical structure would
Table 1. Effects of the different treatments on the physicochemical
properties of the TPO (thyroid peroxidase) inhibition. The peroxidase
inhibitor (PI) was isolated from monolayer thyroid cell cultures per-
formed in the presence of insulin. The remnant inhibitory activity, after

the treatments is the measure of stability. Other details are given in the
text. TPO, thyroid peroxidase; PI, peroxidase inhibitor. Each value is
the average ± SEM of four samples from four different experiments.
*P<0.01.
Assay
TPO activity
(pmol IÆmin
)1
Æ
mg protein
)1
)
Sodium
metaperiodate
TPO 208 ± 12
TPO + PI 113 ± 6*
TPO + (PI + NaO
4
I) 187 ± 13
Alkaline
phosphatase
TPO 383 ± 24
TPO + PI 116 ± 15*
TPO + (PI +
alkaline phosphatase)
394 ± 32
Trypsin TPO 391 ± 35
TPO + PI 34 ± 2*
TPO + (PI + trypsin) 55 ± 4*
Chymotrypsin TPO 168 ± 14

TPO + PI 3 ± 05*
TPO + (PI + chymotrypsin) 1 ± 02*
Proteinase K TPO 325 ± 28
TPO + PI 96 ± 12*
TPO + (PI + proteinase K) 78 ± 20*
Fig. 1. TPO inhibition caused by the insulin mediator with different
purity grades. Comparison of the inhibitory effect of: supernatant from
bovine primary thyroid cell cultures (B-MLS); upper phase isolated by
the method of Folch et al. [19] (UP); fraction 4 of liquid–liquid partition
(F4); inositol phosphoglycan-like P and A (IPG P and IPGA) on the
tyrosine iodinating activity of fresh thyroid peroxidase (TPO). Each
value is the average of four samples from four different experiments.
2610 L. Krawiec et al. (Eur. J. Biochem. 271) Ó FEBS 2004
correspond to an inositol phosphoglycan (Table 1). The
similarity between the TPO inhibitor and IPGs was
confirmed by TLC, which demonstrated the presence of
inositol, glucose and mannose. Finally, it is important to
mention that the total inhibition caused by the peroxidase
inhibitor on the cyclic AMP-dependent protein kinase
(Table 2) is one of the specific characteristics of IPG as was
demonstrated by Villalba et al.[16].
To determine whether the cytosolic inhibitor, obtained
from the monolayer thyroid cell culture, has a selective
effect on TPO, we assayed its action on LPO, HRPO and
GPX activities. As shown in Table 2 all the enzymes were
inhibited by the F4 preparation. Conversely, catalase
activity was not affected at all.
Table 3 shows the total inhibition on the GPX activity
caused by both IPGs-like products as compared to the
semipurified F4. The significant increase of the hydrogen

peroxide release into the culture medium, 30 min after the
addition of the inhibitor is depicted in Table 4. Table 5
shows the significant increase in concentration of the
peroxidase inhibitor by the action of insulin, during 48 h,
in thyroid primary cultures with 0.1% fetal bovine serum,
totally depleted of the hormone, 48 h earlier. Table 6
depicts the effect produced on cell proliferation of the
primary thyroid culture by the addition of insulin and both
IPGs-like compounds, under equal conditions to those
described for experiments of Table 5. In all cases, thyroid
cell cultures underwent a significant increase in cell number
and [
3
H]thymidine incorporation into DNA compared to
the controls.
Discussion
It has been demonstrated that insulin and IGF I have
similar effects as growth promoting factors [23]. Even
though insulin is 1000 times less active on the IGF I
receptor than on its own, the effects of high levels of insulin
may be explained by a dual action on both [24]. Besides, it
has been identified hybrids formed by subunits of the insulin
and the IGF I receptors [23].
Table 2. Comparative effects of the cytosolic inhibitor on thyroid per-
oxidase (TPO), lactoperoxidase (LPO), horseradish peroxidase
(HRPO), glutathione peroxidase (GPX), cAMP-dependent protein
kinase (PKA) and catalase (CAT) activities. The cytosolic inhibitor,
obtained from monolayer thyroid cell cultures, in the presence of
insulin, was purified, as fraction F4 or IPG-like, by the methods of
liquid–liquid partition and anionic interchange. Other details are given

in the text. TPO, 0.5 mg of protein; lactoperoxidase, 50 mU; horse-
radish peroxidase, 100 mU; glutathione peroxidase, 600 mU; catalase,
60 U and F4 equivalent to 15 lg of protein. PKA was totally inhibited
by F4 and by the purified IPG-like fraction (not shown). Each value is
the average of four samples from four different experiments.
Assay % of inhibition
TPO + F4 50
LPO + F4 82
HRPO + F4 100
GPX + F4 100
CAT + F4 0
PKA + F4 100
Table 3. Inhibition of glutathione peroxidase (GPX) activity by the
cytosolic inhibitor. Technical details are given in the text. Each value is
the average of four samples from four different experiments. F4,
fraction 4 of the liquid–liquid partition, equivalent to 30 lgofprotein.
IPGP-like and IPGA-like: inositol phosphoglycan-like P and A,
equivalent to 6 lgofprotein.
Assay GPX activity (mUÆmL
)1
)
GPx control 77.5 ± 8.5
GPx + F4 0
GPx + IPGP-like 0
GPx + IPGA-like 0
GPx + H
2
O milli q 75.2 ± 6.7
Table 4. H
2

O
2
released by monolayer primary cultures of bovine thyroid
cells in confluence, treated with IPGP-like or IPGA-like. H
2
O
2
was
measured 30 min after the medium renewal in the presence or absence
of IPG-like P or A (equivalent to 20 lg of protein in the extract). Each
value is the average of four samples from four different experiments.
*P < 0.01 vs. control.
Assay
H
2
O
2
released to the medium
(nmolÆmin
)1
Æmg
)1
of cell protein) %
Control 28.8 ± 4.7 100
IPGP-like 52.8 ± 7.4* 183
IPGA-like 99.2 ± 14.2* 344
Table 5. Effect of insulin on the peroxidase inhibitor production.
Comparison of the inhibitory effect, on TPO activity, of the super-
natants (80 lg protein) obtained from primary monolayer cell cultures
of bovine thyroid (B-MLS), in the absence or presence of 3.4 l

M
insulin during 48 h. Each value is the average ± SEM of six samples
from four different experiments. *P<0.05 compared to fresh TPO.
**P<0.01 compared to fresh TPO.
Assay
TPO activity
(pmol IÆmin
)1
Æmg protein
)1
)
%
Inhibition
Fresh bovine
TPO control
439 ± 56
Fresh bovine TPO +
BMLS
241 ± 42* 45
Fresh bovine TPO +
(B MLS + Insulin)
87 ± 37** 80
Table 6. Effect of insulin and its mediators on the proliferation of bovine
thyroid cells. Technical details are described in the text. Aliquot’s
protein: Insulin, 3.4 l
M
; IPG-like P and A, 1 lgÆmL
)1
. The treatments
were carried on during 48 h. Each value is the average ± SEM of six

samples from six different experiments. *P < 0.01 vs. control.
Treatment Cells · 10
3
per dish
[
3
H]Thymidine
(c.p.m.Æwell
)1
)
Control 470 ± 29 4518 ± 309
Control + insulin 1070 ± 37* 7023 ± 234*
Control + IPGP-like 970 ± 50* 6546 ± 315*
Control + IPGA-like 810 ± 50* 6420 ± 129*
Ó FEBS 2004 Peroxidase inhibition and proliferation (Eur. J. Biochem. 271) 2611
Our previous studies [1] demonstrated that monolayer
primary cultures of bovine thyroid cells, maintained in the
presence of insulin, produced a cytosolic inhibitor of TPO
activity. Its molecular mass of less than 2 kDa coincides
with that reported previously for the insulin mediators [10].
Under our experimental conditions the TPO inhibitor
was neither detected in fresh tissue nor in free follicle
cultures. The insulin concentration in the culture medium,
which is 1000- to 10 000-fold higher than that of calf
plasma, explains the undetectable levels of the TPO
inhibitor in fresh thyroid. The fact that insulin mediators
were isolated from 1 kg of fresh liver [11] supports this
assumption. On the other hand, the absence of the inhibitor
in free follicle cultures, may be due to the lack of cellular
proliferation, absent in this type of culture. These facts

support the hypothesis that insulin regulates negatively the
activity of the thyroid peroxidase.
The similar effects observed on TPO, LPO, HRPO and
GPX show that the inhibition has not enzyme specificity,
suggesting that insulin regulates the different peroxidase
activities by means of a cytosolic inhibitor as mediator of its
action.
To rule out the possibility that the cytosolic inhibition of
peroxidase, instead of being specific for the enzyme activity,
could be part of a generalized action on the cellular
metabolism, we assayed the influence of the cytosol on the
glucose uptake by free thyroid follicles. There were no
differences, when the cytosol was added, discarding this
mechanism for the effect of insulin on glucose uptake
(results not shown). These results agree with those obtained
when the action of phospho-oligosaccharides on glucose
uptake, by isolated rat adipocytes, was studied [25].
The purification procedure demonstrated the hydro-
phylic nature of the peroxidase inhibitor and the absence
of lipid constituents in its structure. The compound has
no protein residues, as the activity persisted after the
action of proteinases and this was confirmed by the
absence of inhibitory activity in protein fractions isolated
by HPLC (not shown). The loss of biological activity by
treatment with sodium metaperiodate and the identifica-
tion of carbohydrate residues after the inhibitor purifica-
tion by TLC would suggest that the compound is an
oligosaccharide.
Purification of the cytosolic material by liquid–liquid
partition and anionic interchange shows that the peroxidase

inhibitory activity corresponds to two similar products to
IPG (P and A) isolated and characterized as signal
transducers of insulin action in bovine [11] and human liver
[12]. This similarity was demonstrated by analytical meth-
ods and confirmed by their characteristic inhibition caused
on the cyclic AMP-dependent protein kinase, which is
specific of IPG as was demonstrated by Villalba et al.[16].
Jones and Varela-Nieto [9] reported a long list of
metabolic pathways and enzymatic activities that are
affected by the insulin mimetic action of IPG P and IPG
A. The observation that both compounds isolated by us
inhibit the different peroxidase activities is a new finding
that must be added to the previously mentioned list.
Our results demonstrate that insulin enhances the
peroxidase inhibitory action in bovine thyroid cell cultures,
suggesting that the hormone modulates the peroxidase
activities through a mediator such as the one reported for
other enzyme activities [9]. IPG mimics the action of insulin
in placenta [11], liver [12] and adipocytes [25].
Vasta et al. [26] working in cultures of human fibroblasts
and Leo
´
n et al. [23] in organotypic cultures of chicken otic
vesicles, reported the mitogenic effects of insulin. These
effects were attributed to IPG, acting as its second
messenger. In addition, anti-IPG Ig block the mitogenic
effects of IGF-I on the otic vesicle, suggesting that the
hydrolysis of GPI to produce IPG is an important pathway
in the mechanism of action on cell proliferation by insulin
and IGF- I [23].

Insulin acts through the interaction with its membrane
receptors, producing long and short-term metabolic effects
[27]. The long-term process involves, as mediators, phos-
phorylated proteins produced by the tyrosine kinase acti-
vation. On the other hand, the short-term effects are
developed by the action of IPG stimulating a phosphopro-
tein phosphatase [27].
Considering that both mechanisms mediate mitogenic
effects of insulin, it may be licit to postulate that IPG could
act as the transducer of the short-term mechanism, without
discarding the long-term effect on the same process.
Taking into account that IPG is a potent mitogen for a
variety of tissues [28,29] it seems important to determine
whether the same phenomenon occurs in thyroid, with the
peroxidase inhibitor purified as IPG, verifying the possible
relationship between the peroxidase inhibition and the
proliferation process(Fig. 2).
Our results, without discarding the anti-apoptotic effect
of insulin [24], demonstrate that both insulin and the
peroxidase inhibitor, in the absence of TSH, stimulate cell
growth of thyroid cultures. Conversely, Petitfrere et al.[5]
and Deleu et al. [30] considered the presence of insulin and
TSH necessary to stimulate cell growth, ruling out the
individual effects of both hormones. Petitfrere et al.’s [5]
conclusions are not unequivocal, as they did not assay
insulin and TSH separately. On the other hand, Deleu et al.
[30] utilized bovine hormones for dog thyroid cultures but
Leo
´
n et al. [23] stressed the importance of using homolog-

ous species factors in in vitro studies, as we have carried out
in the present studies. Finally, Deleu et al. [30] point out
that thyroid cell regulation varies from one species to
another, thus explaining our discrepancy with their results.
Fig. 2. Proposed mechanism for the relationship between peroxidase
inhibition and cell proliferation.
2612 L. Krawiec et al. (Eur. J. Biochem. 271) Ó FEBS 2004
The stimulation of cell growth by insulin was ascribed to the
induction, mediated by IPG, of the nuclear proto-oncogene
c-jun expression [23].
Our results show a clear relationship among the enhance-
ment of cellular proliferation, the increase in the hydrogen
peroxide concentration and the GPX inhibition caused by
the addition of the insulin mediator. Hydrogen peroxide, at
noncytotoxic concentrations, has been pointed out as a cell
transducing signal for insulin and other hormones [31]. The
normal level of hydrogen peroxide, in different tissues,
ranges between 10
)9
and 10
)7
M
[32] and an extracellular
concentration of 10 n
M
H
2
O
2
promotes fibroblast growth

[33]. Hydrogen peroxide has been postulated as the signal
transduction for the induction of protooncogene c-jun
expression [34]. The intracellular levels of H
2
O
2
are main-
tained as a result of its metabolism by GPX and catalase [35].
In conclusion, the present results propose that the insulin
mediator, as inhibitor of TPO activity, is responsible for
the decrease of thyroid hormone biosynthesis in primary
cultures in the presence of insulin. In addition, our data
suggest that insulin stimulates cell division, in bovine
thyroid cultures, promoting the hydrolysis of a membrane
glycosyl-phosphtidyl inositol (GPI) which generates an
inositol phosphoglycan-like substance as insulin’s second
messenger, which mimics the insulin’s effect on cell prolif-
eration by inhibiting glutathione peroxidase (GPX). This
inhibition positively modulates the H
2
O
2
level and its
derivative compounds, the reactive oxygen species. These
changes would be the signal for the induction of a mitogenic
mechanism.
Acknowledgements
The authors thank Dr Amanda Schwint for reviewing the English
version of the manuscript, Dr R.M. de Lederkremer, and Dr Oscar
J. Opezzo for the discussion of results. The contribution of Dr Marı

´
a.
A. Dagrosa and Dr Silvia Moreno in some experiments and the excellent
technical assistance of Ms. Gabriela Beraldi are acknowledged.
These studies were supported by grants from the National Secretary
of Science and Technology (SEPCYT-ANPCYT) and CONICET to
M.A.P.
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