Pokaż cały numer - FPN - Farmaceutyczny PrzeglÄ d Naukowy

Farm Przegl Nauk, 2009,8
incubation time up to 24 h in low glucose DMEM resulted in
an appearance of slight band corresponding to ORP150 (lane
4). The prolongation of incubation in low glucose DMEM
up to 48 h resulted in intensification of ORP150 whereas the
expression of GRP170 was slightly reduced (lane 6).
Fig. 4 shows the presence of gelatinolytic activity in
the cell culture lysates. The cells cultured for 12 h in high
glucose DMEM (lane 1) as well as in low glucose DMEM
(lane 3) have shown an enzyme which corresponds to the
zymogen form of MMP-2 this being activated under the action
of SDS. The treatment with APMA did not evoke any
change in electrophoretic activity of this enzyme (lanes: 2
and 4). No MMP-9 has been detected.
Extending the incubation up to 24 h in high glucose did
not evoke any change in electrophoretic activity of this enzyme
(lanes: 5 and 6). In contrast to them in the cells cultured
in low glucose an intensification of pro-MMP-2 bands and
appearance of an MMP-2 active form (lane 7 and 8) have
been observed. Furthermore, a slight expression of both
pro-MMP-9 and active MMP-9 was detected. The action
of APMA did not evoke further increase in MMP-2 activity
(lane 8). Extending the incubation of cells in high glucose
up to 48 h resulted in a slight intensification of pro-MMP-2
bands (lane 9 and 10). In contrast to them in the cells cultured
in low glucose DMEM for 48 h further increase of
MMP-2 expression has been observed. Bands corresponding
to both pro-MMP-2 and active MMP-2 became much
more intensive (lanes 11 and 12). Furthermore, an intensification
of bands corresponding to pro-MMP-9 and active
MMP-9 was apparent (Fig. 4).
This study suggests that
glucose may exert additional
effect. It stimulates
collagen biosynthesis,
whereas glucose
deprivation evokes up
regulation of chaperones,
which protect
newly synthesised collagen
against intracellular
degradation.
It is well known that
collagen is the major human
protein which contains
hydroxyproline.
In contrast to other proteins
it is specifically digested
by bacterial collagenase.
For these reasons
the incorporation
of radiolabeled proline
into collagenase-sensitive and hydroxyproline-containing
protein is used as an index of collagen synthesis, whereas
pulse-chase technique allows evaluating the degradation of
newly synthesised protein [19].
Fig. 4. Gelatinolytic activity of fibroblasts cultured in high glucose (H) and low glucose (L) DMEM for 12,
24 and 48 h, without APMA (-), with APMA (+). All samples submitted to zymography contained 30 μg of
protein.
APMA - p-aminophenylmercuric acetate.
We decided to study the effect of glucose deprivation
on total protein/ collagen synthesis and degradation in fibroblast
cultures, and correlation of these processes with the
expression of oxygen/glucose-regulated proteins (ORP150/
GRP170) and gelatinolytic activity. It was demonstrated that
fibroblasts incubated in high glucose DMEM synthesised
detectable amounts of collagenous proteins. They constituted
a few per cent of total radioactive proteins. The shortage
of glucose resulted in about 50% decrease in total protein
and about 30% reduction in collagenase-sensitive and
hydroxyproline-containing protein synthesis. The pulsechase
experiment demonstrated that degradation rate of
newly synthesised total protein did not change in response to
glucose deprivation. Both in control and glucose-deprived
cultures above 40% of radioactive proline incorporated into proteins
in the pulse period were degraded during the chase phase.
In contrast to that, the degradation of collagen in chase
period depended on the presence of glucose in culture medium.
Despite the cells incubated in low glucose medium
synthesised less collagen in pulse period, they protected
newly synthesised collagen against degradation in chase
phase. Proportionally less collagen was degraded in cultures
incubated in low glucose than in high glucose media despite
of increased gelatinolytic activity after 24 or 48 h. These
phenomena were accompanied by an increase in expression
of ORP150 in cultures growing in low glucose medium.
Discussion
Glucose is commonly used therapeutic agent. In common
opinion, intravenous administration of this sugar supplements
the pool of energetic substrates in human body.
The mechanism of decreased collagen synthesis at the
conditions of glucose deprivation is easy to explain. No
doubt, the shortage of the main energetic substrate in culture
medium decreases ATP-formation and reduces all energyrequiring
anabolic processes within the cells. It is more dif-
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