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Glutathione peroxidase in blood of diabetic and nondiabetic BB rats
Arch. Gerontol. Geriatr. suppl. 4 (1994) 2 0 7 - 2 1 6 9 1994 Elsevier Science Ireland Ltd. All rights reserved. 0 1 6 7 - 4 9 4 3 / 9 4 / $ 0 7 , 0 0
207
GLUTATHIONE PEROXIDASE IN BLOOD OF DIABETIC AND NONDIABETIC BB RATS
O. RACZ, F. N I S T I A R , A. SIPULOVA a and M. RIEMEROV~, Institute
of
Pathological
Medical Faculty,
Physiology;
and
P.J. Saf~rik U n i v e r s i t y ,
alnstitute
of Experimental
Medicine,
Kosice, SK-04066 Slovakia
SUMMARY The a c t i v i t y of glutathione peroxidase (GPX), an antioxidant selenoenzyme ( E C . I . 1 1 . 1 . 9 . ) , was investigated in blood of BB rats developing spontaneous diabetes mellitus. The a c t i v i t y of the enzyme was significantly higher in the 4th and 5th inbred generation (G 4 5 ) of BB rats as compared with their counterparts of lower degree of inbreeding (G 1 3 ) . O v e r t diabetes (actual blood g l u cose over 10 mM) appeared only in 8 ods 25 G~ animals but oral and i n t r a venous glucose tolerance tests revealed a gradual" worsening of glucose metabolism already in rats with lower degree of inbreeding. No difference in GPX a c t i v i t y was found between diabetic and nondiabetic members of G_ g r o u p . In the whole group of 69 rats positive correlation was found betwee~ the blood GPX a c t i v i t y and the age, weight and actual blood glucose value of the rats and negative correlation between GPX and the P/F value (ratio of peak blood g l u cose after intravenous tolerance test and fasting blood glucose). In the diabetic animals the enzyme a c t i v i t y showed inverse relationships whith e v e r y measured or calculated parameter of glucose metabolism. Our findings indicate a relationship between age and blood GPX a c t i v i t y of BB rats and suggest the possib i l i t y of deleterious effect of elevated blood glucose level on the blood GPX a c t i v i t y after development of o v e r t diabetes. Keywords: glutathione peroxidase, diabetes mellitus,
BB rats
I NTRODUCTIOH Nonenzymatic glycation,
(i.e.,
the Maillard reaction) and oxidative damage,
the best-known forms of random postsynthetic modifications of biological macromolecules, probably interact in a s y n e r g i s t i c manner in the pathogenesis of both micro- and macrovascular diabetic complications and in the aging lery
et a l . ,
1988;
1988; Wolf et a l . ,
Hunt
et a l . ,
1991; Baynes,
1988;
Oberley,
1988;
1991; Kristal and Y u ,
Sakurai
process (Giland T s u c h i y a ,
1992; Ruderman et a l . ,
1992). On the other hand cells and tissues posses powerful systems designed to prevent and repair such damage. Clinically manifest consequences of the random postsynthetic
modifications
probably
arise only in the case when defense s y s -
tems are not able to cope with the damaging forces. Glutathione peroxidase (GPX, E C . I . 1 1 . 1 . 9 ) ,
a selenoenzyme,
key antioxidant enzymes of the blood and other tissues.
is one of the
It catalyzes the break-
down of H20 2 and various organic peroxides at the expense of reduced glutathione. The results of studies on GPX a c t i v i t y in clinical and experimental diabetes are controversial al.,
1985; Watala et a l . ,
(~,~atkovics et a l . , 1986; Uzel et a l . ,
1982; HagglOff et a l . , 1987; Dohi et a l . ,
1983;
Kaji et
1988; Sklodowska
208
et al.,
1989; T h o m p s o n et a l . ,
t i s s u e GPX a c t i v i t y
vated enzyme activity T h e aim o f o u r
study
spontaneously
In some cases d e c r e a s e d e r y t h r o c y t e
b u t in o t h e r
studies
and
no c h a n g e or even ele-
was f o u n d .
b e t w e e n t h e GPX a c t i v i t y which
1992).
was o b s e r v e d ,
was to g a i n
basic
information
about
the connections
and t h e p a r a m e t e r s o f g l u c o s e metabolism
develop
insulin-dependent
diabetes
mellitus
in BB r a t s ,
on an a u t o i m -
mune basis.
M A T E R I A L S A N D METHODS BB r a t s tap
water
second week.
bred
and
week
in o u r l a b o r a t o r y
a standard
and
their
non-fasting
In t h e p r e s e n t s t u d y
were studied
(Table
I).
Larsen
(brother/sister
rat
chow.
The
(actual)
mating) animals
blood
had f r e e access to
were
weighed
every
was
checked
every
glucose
69 r a t s o f v a r i o u s d e g r e e o f i n b r e e d i n g
None of t h e animals were t r e a t e d
(G I - G 5)
with insulin.
Glucose
t o l e r a n c e t e s t s and blood g l u c o s e m e a s u r e m e n t s were c a r r i e d o u t a f t e r o v e r n i g h t f a s t i n g one d a y b e f o r e t h e GPX a s s a y .
Table I AGE AND BODY WEIGHTS OF T H E BB R A T S GIVEN AS RANGE A N D
Group
n
Age (days)
GI
12
118
150 - 260 (212)
G2
9
109 - 130 (118)
210 - 290 (251)
G3
12
115 - 122 (119)
150 - 360 (236)
G4
11
128 - 129 (129)
165 - 300 (237)
G5
25
108 - 153 (134)
160 - 330 (238)
Males
39
108 - 153 ( 1 2 5 )
150 - 360 (263)
Females
30
108 - 153 (126)
150 - 255 (197)
All
69
108 - 153 (125)
150 - 360 (234)
Intravenous
Body weight
(mean)
glucose tolerance test (IVGTT)
venous injection of I g glucoselkg
was p e r f o r m e d
by
(g)
rapid
intra-
b o d y w e i g h t { I 0 g / 1 0 0 ml s o l u t i o n ) and blood
samples w e r e t a k e n 15, 30, 60 and 120 m i n u t e s a f t e r t h e i n j e c t i o n .
According
to
K o r e c (1991) the peak v a l u e in h e a l t h y animals s h o u l d n o t s u r p a s s 11 mM and in t h e second h o u r a f t e r t h e g l u c o s e load t h e c o n c e n t r a t i o n of blood g l u c o s e s h o u l d return
to t h e f a s t i n g
level.
(We c o n s i d e r e d
the 120 m i n u t e v a l u e p a t h o l o g i c a l
i t e x c e e d e d t h e f a s t i n g v a l u e b y I mM or m o r e . ) e v a l u a t e d as f o l l o w s :
The ratio
b e t w e e n t h e peak
The IVGTT
if
data w e r e f u r t h e r
( 1 5 t h rain v a l u e )
and f a s t i n g
209
blood g l u c o s e ( P / F r a t i o ) ,
a n d the h a l f - t i m e
r a n c e o f t h e e x c e s s g l u c o s e ( C o n a r d et a l . , Oral
glucose
test giving
tolerance
test
2 g glucose/kg
and 120 m i n u t e s .
by
(OGTT) a gastric
From t h e r e s u l t s
(ti12)
of the exponential disappea-
1953) w e r e c a l c u l a t e d . was
performed
tube,
after
samples
t h e area u n d e r
the
were
the c u r v e
intravenous
taken
at 30,
(AUC)
60,
was c a l c u -
lated a c c o r d i n g t h e f o r m u l a : AUC (mmole/hr)
= (G o + 2G30 + 3G30 + 3G60 + G120)14
w h e r e G o _ 120 a r e t h e r e s p e c t i v e blood g l u c o s e v a l u e s .
Blood g l u c o s e m e a s u r e -
ments w e r e p e r f o r m e d w i t h e n z y m a t i c ( G O D ) m e t h o d . GPX was measured in 20 ~I o f w h o l e blood w i t h d r a w n The spectrophotometric
assay,
Paglia and V a l e n t i n e (1967) p e r f o r m e d at 37~ lyzer
a m o d i f i c a t i o n o f the o r i g i n a l method p r o p o s e d b y
(RANSEL kit,
Rar, d o x L a b s . ,
with cumene h y d r e p e r o x i d e
(Hoffman-La
Roche,
from the t a i l o f r a t s .
Switzerland).
Northern
Ireland)
was
s u b s t r a t e on a Cobas M i r a S a n a -
The
plasma
was less t h a n 5 % of t h e w h o l e blood enzyme a c t i v i t y
GPX-like
activity
and t h e r e f o r e
were c o n s i d e r e d as red cell a c t i v i t i e s and e x p r e s s e d in p k a t a l / g
in
rats
the results
Hb.
RESULTS Diabetes
development
a b o v e 10 mM) a p p e a r e d neration.
in
BB
in 8 r a t s
rats.
Overt
diabetes
(6 males and 2 females)
(actual
blood
glucose
o f t h e 5th i n b r e d g e -
F a s t i n g blood g l u c o s e a b o v e 7 mM w e r e f o u n d in 7 a n i m a l s , 5 from the
g r o u p o f 8 w i t h o v e r t d i a b e t e s and t w o G 3 males ( T a b l e I I ) .
T a b l e II ACTUAL
A N D F A S T I N G BLOOD GLUCOSE OF BB R A T S IN raM,
mean + S . D .
(range)
Group
Actual
GI
5.2 + 0.8
Fasting
(4.6 -
7.8)
4.3 + 0.6
(3.9 -
6.0)
G2
4.9 + 0.4
(4.3 -
5.8)
4.0 + 0.7
(3.5 -
5.7)
G3
5.9 + 1.4
(4.6 -
9.4)
4.8 + 1.4
(3.8 -
7.7) b
G4
7.0 + 0.7 w
(5.9 -
8.2)
4.5 + 0.4
(3.9 -
5.2)
G5
9.8 + 3.0 w167
( 7 . 0 - 19.8) a
6.2 + 1.7 w167
(4.2 - I0.8) c
Males
7.5 + 3.0
( 4 . 4 - 19.8)
5.2 + 1.6
(3.6 - I0.8)
Females
6.9 + 2.6
( 4 . 3 - 16.9)
4.9 + 1.3
( 3 . 5 - 10.4)
All
7.2 + 2.8
( 4 . 3 - 19.8)
5.1 + 1.5
( 3 . 5 - 10.8)
w and
w167i n d i c a t e
previous group;
significance
at
p
< 0.05
a8 v a l u e s a b o v e 10 mM; b ' c 2 ,
or
0.01,
respectively,
versus
or 5 v a l u e s a b o v e 7 raM, r e s p .
the
210 Tolerance metabolism
tests,
however,
in animals with
cose challenge almost half
disclosed
a gradual
worsening
lower d e g r e e of i n b r e e d i n g .
After
of
the
glucose
intravenous glu-
(42 %) of the rats had peak blood glucose above 11
mM and in 15 out of the 69 rats the b l o o d glucose did not r e t u r n to the fasting value + I mM a f t e r 2 hours (Table I I I ) . The ratios between the peak and fasting blood glucose ( P / F ) , of the excess glucose disappearance ( t l / 2 )
the half-time
as well as the area u n d e r the blood
glucose c u r v e a f t e r oral glucose load are summarized in Table IV. Table III ABNORMAL I V C T T VALUES II'~ BB RATS Group
Peak (> 11 mM)
D i f f e r e n c e (> I raM)
n
%
n
%
GI
I
(8.3)
0
G2
3
(33.3)
I
(11.1)
G3
5
(41.7)
2
(16.7)
G4
4
(36.4)
2
(18.2)
G5
16
(64.9)
10
(40.0)
Males
18
(46.2)
9
(23.1)
Females
11
(36.7)
6
(20.0)
All
29
(42.0)
15
(21.7)
D i f f e r e n c e meant between 120 min and fasting value l a r g e r than I mM. GPX a c t i v i t y .
The a c t i v i t y of GPX was s i g n i f i c a n t l y
h i g h e r in the blood of
the 4th and 5th generation of BB rats than in t h e i r G I - G 3 c o u n t e r p a r t s
b u t no
d i f f e r e n c e was found between the enzyme a c t i v i t y of diabetic (actual blood g l u cose > 10 raM) and nondiabetic members of the 5th generation ( T a b l e V ) . No s i g n i f i c a n t divided
according
differences to t h e i r
in GPX a c t i v i t y
fasting
were found
in g r o u p s
blood glucose concentration
of rats
(dividing
line 7
mM) and abnormal peak or r e t u r n time values of the I V G T T . Correlation tight
analysis.
b u t statistically
In
the
significant
whole g r o u p
of 69 rats
positive correlations
and age, w e i g h t and actual blood glucose (Table V l , glucose corelated neither with the GPX a c t i v i t y rats.
we found
not v e r y
between the GPX a c t i v i t y Figure I ) .
Fasting blood
nor with age and w e i g h t of the
The various parameters calculated from the glucose tolerance tests did not
reveal any s i g n i f i c a n t correlations
with blood GPX a c t i v i t y .
The only exception
211
was t h e P I F r a t i o , -0.36,
but
its a s s o c i a t i o n w i t h
the enzyme activity
was i n v e r s e
(r =
p < 0.01). Contrary
to t h e
whole group
overt
diabetes,
every
measured or calculated
(tl/2)
and AUC even
of the rats
inverse
o f 69 IDI3 r a t s
associations were found
reached
in t h i s g r o u p
parameter
in t h e g r o u p
G5 rats
between the activity
of glucose metabolism.
the limit of significance
(Table
of
o f GPX a n d
Two of them,
despite
with
the
t h e small n u m b e r
VII).
Table IV GLUCOSE
TOLERANCE
TEST
Test
RESULTS
IN B B R A T S
(mean + S.D. and range)
IVGTT
Group
GI
PIF ratio
ti/2,
2.37 + 0.19
33.2 +
(2.00 - 2.63) G2
(2.51
- 3.26)
2.65 + 0.67 (2.04 - 4.44)
G4
(1.70 - 3.53) 2.47 + 0.35
71 I )
43.0 +
24 9
40.4 + ( 7.9 -
2.43 + 0.55
49.7 +
(I .70 - 4 . 4 4 ) All
50 7)
(14.1
( 7.9 -
2.45 + 0.45
44.4 +
2.2
- 21.3)
17.2 +
1.9
(14.7 - 21.3) 22.0 +
89 7)
(14.2
29 9
6.6 b
- 33.4)
18.7 +
4.1
(15.9 - 30.2) 2 9 . 5 + 18.0 b
40 0
( 6 . 6 - 203 0)
(1.70 - 3.53) Females
p
( 7.9 -
55.4 +
mmolelh
16.4 +
( 2 2 . 0 - 123 3)
2.29 + 0.45
Males
10 6
18 0
39.5 +
(2.13 - 2.90)
AUC,
36.9 +
(19.3 -
2.40 + 0.24
G5
min
(17.4 -
2 . 8 0 + 0.21 a
G3
Peak of OGTT
(15.5 - 89.7)
22 9
2 3 . 0 + 13 0
98 2)
( 1 4 . 7 - 89 7)
38 0
22.1 + 11 9
98 2)
( 1 4 . 7 - 89 7)
30 5
2 2 . 6 + 12 5 w
{1.70 - 4.44)
Significant
difference:
( 6 . 6 - 203 0)
a a t p < 0.01
versus
G1,4,5;
(14.1 b
- 89 7)
P < 0.05 versus
GI
DISCUSSION The activity
o f GPX in b l o o d o f BID r a t s i n c r e a s e d
gain of the animals. neration,
i.e.,
The enzyme activity
in t h e
same w h e r e
overt
with
was t h e h i g h e s t diabetes
(with
the age and weight
in t h e 5 t h permanent
inbred
ge-
hyperglyce-
mia) appeared. The view of
antioxidant
that
aging
systems
is
is a s s o c i a t e d w i t h an
an o v e r a l l
oversimplification
and
decrease of the capacity
surely
does
not
hold
for
212
Table V GPX A C T I V I T Y
IN THE BLOOD OF BB RATS (mean + S . D . and r a n g e )
Group
GPX,
] J k a t a l / g Hb
GI
3.76 + 0 21
(3.39 - 4.10)
G2
3.65 + 0 78
(2.91 - 5.42)
G3
3.34 + 0 38
(2.89 - 4.05)
G4
4.05 + 1 03 w167
(2.59 - 5.58)
G5 G5, o v e r t diabetes
5.02 + 1 25 w167
(3.33 - 7.29)
4.90 + 1 22
(3.34 - 6.61)
G5, n o n d i a b e t i c s
5.10 + 1 30
(3.33 - 7.29)
Males
4.48 + 1 16
(3.02 - 7.29)
Females
3.79 + 0 96
(2.59 - 7.12)
All
4.18 + 1 12
(2.59 - 7.29)
m
w167i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s at p < 0.01 v e r s u s p r e v i o u s g r o u p .
T a b l e Vl SIMPLE
CORRELATIONS
BETWEEN
BLOOD
GPX
ACTIVITY,
AGE
AND
BODY
WEIGHT OF BB RATS ( r )
Parameter
GPX
Age
Age
0.65 w167
Body weight
0.41 w167
0.26 w
A c t u a l blood g l u c .
0.34 w167
n.s.
Body weight
n.s.
w and w167i n d i c a t e s i g n i f i c a n t c o r r e l a t i o n s at p < 0.05 and 0.01, GPX. vity
Vertechy
et al.
(1989) and Sohal et al.
(1990)
found
respectively.
e l e v a t e d GPX a c t i -
in h e a r t and muscle, as well as in l i v e r of old male r a t s ,
compared w i t h t h e i r y o u n g c o u n t e r p a r t s . various
parts
of
the
same organ
respectively,
A c c o r d i n g to C i r i o l o et al.
reveal
different
changes of t h e a n t i o x i d a n t enzyme a c t i v i t i e s .
patterns
They observed
of GPX in mesencephalon and p a r i e t a l c o r t e x ,
of
as
(1991) even
age-dependent
increased activity
decreased a c t i v i t y
in b r a i n
and no change o f enzyme a c t i v i t y in t h e p r e f r o n t a l c o r t e x o f a g i n g r a t s .
stem
GPX is
a selenoenzyme and its a c t i v i t y d e p e n d s on t h e a v i a l a b i l i t y of t h i s t r a c e element (Perona tween
et a l . ,
1978).
selenium c o n t e n t
G r o m a d z i n s k a et al. of various
organs
(1988) of
the
found rat
and
close r e l a t i o n s h i p their
GPX
be-
activity.
213
10 O
O o
9"r
o
8
0 O
0
O,
0
000
0
0
o/0
m
6
~oe
D.
? 0
0
o
ov
_
O0
0
0
0
I
i
i
~
4
I
~
~
L
8
I
~
~
12
~
I
~
,
~
I
20
16
Blood glucose, mM Figure I. Correlation between blood g l u c o s e in BB r a t s .
blood
glutathione
peroxidase
activity
and a c t u a l
Table VII CORRELATION (r)
BETWEEN
GPX
ACTIVITY
AND
BLOOD
GLUCOSE
PARAMETERS
IN BB R A T S WITH O V E R T D I A B E T E S AND W I T H O U T D I A B E T E S
Group
Diabetics
Nondiabetics
Correlation with
G 5 (8)
G 5 (17)
A c t u a l B G , mM
- 0.53
< 0.20
+ 0.43 w167
F a s t i n g BG, mM
-0.53
< 0.20
< 0.2
IVGTT
OGTT,
peak,
mM
A l l (61)
-0.53
< 0.20
-0.38 w
PIF ratio
- 0.66
- 0.21
< 0.2
TI12,
- 0.77 w
+ 0.39
< 0.2
- 0.71 w
< 0.20
+ 0.28 w
min
AUC,
mmolelhr
amultiplicative correlations; 0.05 and 0 . 0 1 , r e s p e c t i v e l y .
w and
According
al.
to
Yamaguchi
et
raises w i t h age in h e a l t h y selenium c o n c e n t r a t i o n
(1992)
both
men and t h i s
in plasma and
s t a t u s o f t h e BB r a t s in t h i s s t u d y age-dependent
w167i n d i c a t e
significant
red
cell
increase
red cells.
and
correlations
at
plasma
activity
is p a r a l l e l
We d i d
GPX
to the
<
i n c r e a s e of
n o t f o l l o w the s e l e n i u m
and t h u s we can o n l y h y p o t h e s i z e
i n c r e a s e o f t h e GPX a c t i v i t y
p
t h a t the
is r e l a t e d to s e l e n i u m a c c u m u l a t i o n .
214
A s l i g h t positive c o r r e l a t i o n between the actual blood glucose and the GPX activity
was
found
in
the
whole g r o u p
somewhat s t r o n g e r in 61 n o n - d i a b e t i c diabetes
in BB rats
of 69 13B rats.
rats (Table V I I ) .
is age d e p e n d e n t
(Marliss,
1983).
from the correlation analysis of the whole g r o u p diabetic rats there was a loose r e l a t i o n s h i p glucose ( r = 0.36, enzyme a c t i v i t y
p < 0.01).
This
association
was
The manifestaton of the This
(Table V l )
was not a p p a r e n t b u t in the 61 non-
between t h e i r age and actual blood
It is possible,
that the association
between the
and the actual blood glucose in nondiabetic rats is only an i n -
d i r e c t one due to the age dependence of the GPX a c t i v i t y and the glucose metabolism d e t e r i o r a t i o n . The ratio between the peak and fasting
blood glucose value a f t e r
nous glucose load is not a classical parameter of glucose metabolism. related to the glucose d i s t r i b u t i o n of the body
in the e x t r a c e l l u l a r
to metabolize glucose.
intraveIt may be
space and to the a b i l i t y
Between the PIF ratio and the blood GPX
a c t i v i t y of the rat we found a n e g a t i v e correlation
(which is a little s t r o n g e r in
rats with fasting blood glucose below 7 raM; - r = 0.43, p < 0.01) b u t we have no plausible explanation for this phenomenon at p r e s e n t . In the diabetic rats negative relationships were found between glucose metabolism parameters and blood GPX a c t i v i t y .
The correlations
regarding
sociation of the enzyme a c t i v i t y with the measured variables (actual, IVGTT
peak blood glucose)
On the o t h e r integrated
hand,
did
not reach the level of statistical
the associations
variables calculated
fasting and significance.
between the blood GPX a c t i v i t y
from fasting
and post-load
values
the as-
and the
(glucose d i s -
appearance a f t e r IVGTT and AUC) are s i g n i f i c a n t . O x i d a t i v e damage may play an important role in the biochemical b a c k g r o u n d of diabetic complications oxidative
stress
and
when at least one of its basic conditions
diminished
r e f e r s to the second condition.
antioxidant
Ruderman
compensatory
et a l . ,
is
fulfilled.
(exaggerated Our
study
The available data on the a n t i o x i d a n t status in
diabetes mellitus ( f o r reviews see O b e r l e y , 1992;
capacity)
1988; Baynes, 1991; Kristal and Y u ,
1992) are c o n t r a d i c t i v e .
Oxidative
stress
can t r i g g e r
reactions which lead to increased a n t i o x i d a n t enzyme a c t i v i t y .
On
the other side reactive o x y g e n species and high ambient glucose c o n c e n t r a t i o n can damage the same enzymes.
The deletorious effect of in v i t r o
s u p e r o x i d e dismutase and g l u t a t h i o n e et a l . , BB
1987; B l a k y t n y and H a r d i n g , rats
have
the
advantage
reductase was already demonstrated
of
(Arai
1992). of
spontaneous
diabetes
development
o t h e r experimental models of this disease. No arteficial i n t e r v e n t i o n and o v e r t diabetes is preceded with a long p r e d i a b e t i c state. rats the main d e t e r m i n a n t of blood GPX a c t i v i t y of diabetes hyperglycemia
glycation
over
is necessary
In nondiabetic BB
is t h e i r age. A f t e r development
seems to have a deleterious effect on the a c t i v i t y
of
215
this important antioxidant enzyme. This fits well in the above outlined oxidative damage hypothesis of biochemical pathogenesis of diabetic complications but f u r ther experimental and clinical studies are necessary to b r i n g strong evidence about its r e l i a b i l i t y . ACKNOWLEDGEMENT This s t u d y was supported by the Slovak Grant Agency. 1/259/92.
Grant number:
REFERENCES Arai, K . , lizuka, S., Tada, Y . , Oikawa, K. and T a n i g u c h i , N. (1987): Increase in the glucosylated form of e r y t h r o c y t e C z - Z n - s u p e r o x i d e dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme a c t i v i t y . Biochem. Biophys. Acta 924, 292-296. Baynes, J.W. (1991): Role of oxidative stress in development of complications in diabetes. Diabetes 40, 405-412. B l a k y t n y , R. and Harding, J . J . (1992): Glycation (non-enzymic glycosylation) inactivates glutathione reductase. Biochem. J . , 288, 303-307. Ciriolo, M . R . , Fiskin, K . , Corasaniti, M . T . , Nistico, G. and Rotilio, G. (1991): Age-related changes in Cu, Zn superoxide dismutase, Se-dependent and independent glutathione peroxidase and catalase activities in specific areas of rat b r a i n . Mech. Ageing D e v . , 61, 287-297. Conard, V . , Ranckson, J . R . M . , Bastenie, P . A . , Kestens, J. and Kovacs, L. (1953): l~tude c r i t i q u e du t r i a n g l e d'hyperglyc@mie i n t r a v e i n e u x chez I ' homme normal et determinatior, d "un "coefficient" d 'assimilation g l u c i d i q u e " . A r c h . I n t . Pharmacodyn., 93, 277-292 (in French). Dohi, T . , Kawamura, K . , Morita, K . , Okamoto, H. and Tsujimoto, A. (1988): Alterations of the plasma selenium concentrations and the activities of tissue peroxide metabolism enzymes in streptozotocin-induced diabetic rats. Horm. Metab. Res., 20, 671-675. G i l l e r y , P., Monboisse, J . C . , Maquart, F.X. and Borel, J . P . (1988): Glycation of proteins as a source of superoxide. Diab. Metab., 14, 25-30. Gromadzinska, J . , Sklodowska, M. and Wasowicz, W. (1988): Glutathione peroxidase a c t i v i t y , lipid peroxides and selenium concentration in various rat organs. Biomed. Biochem. Acta, 47, 19-24. Haggl6ff, 13., Marklund, S.L. and Holmgren, G. {1983): CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in lymphocytes and e r y t h r o c y t e s in i n s u l i n - d e p e n d e n t diabetic c h i l d r e n . Acta Endocrinol., 102, 235-239. Hunt, J . V . , Dean, R . T . and Wolff, S.P. (1988): H y d r o x y l radical production and autooxidative glycosylation. Biochem. J . , 256, 205-212. Kaji, H., Kurasaki, M., Ito, K . , Saito, T . , Saito, K . , Niioka, T . , Kojima, Y . , Ohsaki, Y . , Ide, H. and T s u j i , M. (1985): Increased lipoperoxide value and glutathione peroxidase a c t i v i t y in blood plasma of type 2 ( n o n - i n sulin-dependent) diabetic women. Klin. Wschr., 63, 765-768. Korec, R. (1991): Experimental and Spontaneous Diabetes Mellitus in the Rat and Mouse. 2nd edn. pp. 31-37. Edition Center of Saf~rik U n i v e r s i t y , Kosice, Kristal, B.S. and Yu, B.P. (1992): An emerging hypothesis: Synergistic i n d u c tion of aging by free radicals and Maillard reactions. J. Gerontol. Biol. Sci., 47, B107-B114. Marliss, E.B. (1983): The Juvenile Diabetes Foundation Workshop on the spontaneously diabetic BD rat: its potential for i n s i g h t into human juvenile diabetes. Metab. Clin. E x p . , 32, Suppl. I , 1-166. Matkovics, B . , Varga, S., Szab6, L. and Witas, FI. (1982): The effect of d i a betes on the activities of the peroxide metabolizing enzymes. Horm. Met. Res., 14, 77-79.
216
Oberley, L.W. (1988): Free radicals and diabetes. Free Rad. Biol. Med., 5, 113-124. Paglia, D.E. and Valentine, W.N. (1967): Studies on the q u a n t i t a t i v e and qualitative characterization of e r y t h r o c y t e glutathione peroxidase. J. Lab. Clin. Med., 70, 158-169. Perona, G., Guidi, G . C . , Piga, A . , Cellerino, R., Menna, R. and Zatti, M. (1978): In vivo and in v i t r o variations of human e r y t h r o c y t e glutathione peroxidase a c t i v i t y as r e s u l t of cells ageing, selenium availability and peroxide activation. B r i t . J. Haematol., 39, 399-407. Ruderman, N . B . , Williamson, J . R . and Brownlee, M. (1992): Glucose and diabetic vascular disease. FASEB J . , 6, 2905-2914. Sakurai, T. and T s u c h i y a , S. (1988): Superoxide production from nonenzymatically glycated protein. FEBS L e t t . , 236, 406-410. Sklodowska, M., Gromadzinska, J. and Wasowicz, W. (1989): Lipid peroxide concentration, selenium level and glutathione peroxidase a c t i v i t y in blood of type II ( n o n - i n s u l i n - d e p e n d e n t ) diabetic elderly people. J. Clin. Biochem. N u t r . , 7, 35-41. Sohal, R . S . , A r n o l d , L . A . and Sohal, 13.H. (1990): Age-related changes in a n t i o x i d a n t enzymes and prooxidant generation in tissues of the rat with special reference to parameters in two insect species. Free Rad. Biol. Med., 10, 495-500. Thompson, K . H . , Godin, D.V. and Lee, M. (1992): Tissue a n t i o x i d a n t status in streptozotocin-induced diabetes in rats. Effects of dietary manganese deficiency. Biol. Trace Elem. Res., 35, 213-224. Uzel, N., Sivas, A . , Uysal, M. and Oz, H. (1987): E r y t h r o c y t e lipid peroxidation and glutathione peroxidase a c t i v i t y in patients with diabetes mellitus. Horm. Met. Res., 19, 89-90. Vertechy, M., Cooper, M . B . , G h i r a r d i , O. and Ramacci, M.T. (1989): A n t i o x i dant enzyme activities in heart and skeletal muscle of rats of d i f f e r e n t ages. Exper. Gerontol., 24, 211-218. Watala, C . , Bryszewska, M., Stefaniak, B. and Nowak, S. (1986): Peroxide metabolism enzymes in diabetic c h i l d r e n relationship to duration and control of diabetes. Cytobios 47, 101-105. Wolf, S . P . , Jiang, Z . Y . and H u n t , J . V . (1991): Protein glycation and oxidative stress in diabetes mellitus and aging. Free Rad. Biol. Med., 10, 339-552. Yamaguchi, T . , Uchimura, K . , Kurokoawa, Y . , Hamada, M., Inoue, K. and Shibuya, N. (1992): Selenium concentration and glutathione peroxidase a c t i v i t y in plasma and e r y t h r o c y t e s from human blood. J. Clin. Biochem. N u t r . , 12, 41-50.
207
GLUTATHIONE PEROXIDASE IN BLOOD OF DIABETIC AND NONDIABETIC BB RATS
O. RACZ, F. N I S T I A R , A. SIPULOVA a and M. RIEMEROV~, Institute
of
Pathological
Medical Faculty,
Physiology;
and
P.J. Saf~rik U n i v e r s i t y ,
alnstitute
of Experimental
Medicine,
Kosice, SK-04066 Slovakia
SUMMARY The a c t i v i t y of glutathione peroxidase (GPX), an antioxidant selenoenzyme ( E C . I . 1 1 . 1 . 9 . ) , was investigated in blood of BB rats developing spontaneous diabetes mellitus. The a c t i v i t y of the enzyme was significantly higher in the 4th and 5th inbred generation (G 4 5 ) of BB rats as compared with their counterparts of lower degree of inbreeding (G 1 3 ) . O v e r t diabetes (actual blood g l u cose over 10 mM) appeared only in 8 ods 25 G~ animals but oral and i n t r a venous glucose tolerance tests revealed a gradual" worsening of glucose metabolism already in rats with lower degree of inbreeding. No difference in GPX a c t i v i t y was found between diabetic and nondiabetic members of G_ g r o u p . In the whole group of 69 rats positive correlation was found betwee~ the blood GPX a c t i v i t y and the age, weight and actual blood glucose value of the rats and negative correlation between GPX and the P/F value (ratio of peak blood g l u cose after intravenous tolerance test and fasting blood glucose). In the diabetic animals the enzyme a c t i v i t y showed inverse relationships whith e v e r y measured or calculated parameter of glucose metabolism. Our findings indicate a relationship between age and blood GPX a c t i v i t y of BB rats and suggest the possib i l i t y of deleterious effect of elevated blood glucose level on the blood GPX a c t i v i t y after development of o v e r t diabetes. Keywords: glutathione peroxidase, diabetes mellitus,
BB rats
I NTRODUCTIOH Nonenzymatic glycation,
(i.e.,
the Maillard reaction) and oxidative damage,
the best-known forms of random postsynthetic modifications of biological macromolecules, probably interact in a s y n e r g i s t i c manner in the pathogenesis of both micro- and macrovascular diabetic complications and in the aging lery
et a l . ,
1988;
1988; Wolf et a l . ,
Hunt
et a l . ,
1991; Baynes,
1988;
Oberley,
1988;
1991; Kristal and Y u ,
Sakurai
process (Giland T s u c h i y a ,
1992; Ruderman et a l . ,
1992). On the other hand cells and tissues posses powerful systems designed to prevent and repair such damage. Clinically manifest consequences of the random postsynthetic
modifications
probably
arise only in the case when defense s y s -
tems are not able to cope with the damaging forces. Glutathione peroxidase (GPX, E C . I . 1 1 . 1 . 9 ) ,
a selenoenzyme,
key antioxidant enzymes of the blood and other tissues.
is one of the
It catalyzes the break-
down of H20 2 and various organic peroxides at the expense of reduced glutathione. The results of studies on GPX a c t i v i t y in clinical and experimental diabetes are controversial al.,
1985; Watala et a l . ,
(~,~atkovics et a l . , 1986; Uzel et a l . ,
1982; HagglOff et a l . , 1987; Dohi et a l . ,
1983;
Kaji et
1988; Sklodowska
208
et al.,
1989; T h o m p s o n et a l . ,
t i s s u e GPX a c t i v i t y
vated enzyme activity T h e aim o f o u r
study
spontaneously
In some cases d e c r e a s e d e r y t h r o c y t e
b u t in o t h e r
studies
and
no c h a n g e or even ele-
was f o u n d .
b e t w e e n t h e GPX a c t i v i t y which
1992).
was o b s e r v e d ,
was to g a i n
basic
information
about
the connections
and t h e p a r a m e t e r s o f g l u c o s e metabolism
develop
insulin-dependent
diabetes
mellitus
in BB r a t s ,
on an a u t o i m -
mune basis.
M A T E R I A L S A N D METHODS BB r a t s tap
water
second week.
bred
and
week
in o u r l a b o r a t o r y
a standard
and
their
non-fasting
In t h e p r e s e n t s t u d y
were studied
(Table
I).
Larsen
(brother/sister
rat
chow.
The
(actual)
mating) animals
blood
had f r e e access to
were
weighed
every
was
checked
every
glucose
69 r a t s o f v a r i o u s d e g r e e o f i n b r e e d i n g
None of t h e animals were t r e a t e d
(G I - G 5)
with insulin.
Glucose
t o l e r a n c e t e s t s and blood g l u c o s e m e a s u r e m e n t s were c a r r i e d o u t a f t e r o v e r n i g h t f a s t i n g one d a y b e f o r e t h e GPX a s s a y .
Table I AGE AND BODY WEIGHTS OF T H E BB R A T S GIVEN AS RANGE A N D
Group
n
Age (days)
GI
12
118
150 - 260 (212)
G2
9
109 - 130 (118)
210 - 290 (251)
G3
12
115 - 122 (119)
150 - 360 (236)
G4
11
128 - 129 (129)
165 - 300 (237)
G5
25
108 - 153 (134)
160 - 330 (238)
Males
39
108 - 153 ( 1 2 5 )
150 - 360 (263)
Females
30
108 - 153 (126)
150 - 255 (197)
All
69
108 - 153 (125)
150 - 360 (234)
Intravenous
Body weight
(mean)
glucose tolerance test (IVGTT)
venous injection of I g glucoselkg
was p e r f o r m e d
by
(g)
rapid
intra-
b o d y w e i g h t { I 0 g / 1 0 0 ml s o l u t i o n ) and blood
samples w e r e t a k e n 15, 30, 60 and 120 m i n u t e s a f t e r t h e i n j e c t i o n .
According
to
K o r e c (1991) the peak v a l u e in h e a l t h y animals s h o u l d n o t s u r p a s s 11 mM and in t h e second h o u r a f t e r t h e g l u c o s e load t h e c o n c e n t r a t i o n of blood g l u c o s e s h o u l d return
to t h e f a s t i n g
level.
(We c o n s i d e r e d
the 120 m i n u t e v a l u e p a t h o l o g i c a l
i t e x c e e d e d t h e f a s t i n g v a l u e b y I mM or m o r e . ) e v a l u a t e d as f o l l o w s :
The ratio
b e t w e e n t h e peak
The IVGTT
if
data w e r e f u r t h e r
( 1 5 t h rain v a l u e )
and f a s t i n g
209
blood g l u c o s e ( P / F r a t i o ) ,
a n d the h a l f - t i m e
r a n c e o f t h e e x c e s s g l u c o s e ( C o n a r d et a l . , Oral
glucose
test giving
tolerance
test
2 g glucose/kg
and 120 m i n u t e s .
by
(OGTT) a gastric
From t h e r e s u l t s
(ti12)
of the exponential disappea-
1953) w e r e c a l c u l a t e d . was
performed
tube,
after
samples
t h e area u n d e r
the
were
the c u r v e
intravenous
taken
at 30,
(AUC)
60,
was c a l c u -
lated a c c o r d i n g t h e f o r m u l a : AUC (mmole/hr)
= (G o + 2G30 + 3G30 + 3G60 + G120)14
w h e r e G o _ 120 a r e t h e r e s p e c t i v e blood g l u c o s e v a l u e s .
Blood g l u c o s e m e a s u r e -
ments w e r e p e r f o r m e d w i t h e n z y m a t i c ( G O D ) m e t h o d . GPX was measured in 20 ~I o f w h o l e blood w i t h d r a w n The spectrophotometric
assay,
Paglia and V a l e n t i n e (1967) p e r f o r m e d at 37~ lyzer
a m o d i f i c a t i o n o f the o r i g i n a l method p r o p o s e d b y
(RANSEL kit,
Rar, d o x L a b s . ,
with cumene h y d r e p e r o x i d e
(Hoffman-La
Roche,
from the t a i l o f r a t s .
Switzerland).
Northern
Ireland)
was
s u b s t r a t e on a Cobas M i r a S a n a -
The
plasma
was less t h a n 5 % of t h e w h o l e blood enzyme a c t i v i t y
GPX-like
activity
and t h e r e f o r e
were c o n s i d e r e d as red cell a c t i v i t i e s and e x p r e s s e d in p k a t a l / g
in
rats
the results
Hb.
RESULTS Diabetes
development
a b o v e 10 mM) a p p e a r e d neration.
in
BB
in 8 r a t s
rats.
Overt
diabetes
(6 males and 2 females)
(actual
blood
glucose
o f t h e 5th i n b r e d g e -
F a s t i n g blood g l u c o s e a b o v e 7 mM w e r e f o u n d in 7 a n i m a l s , 5 from the
g r o u p o f 8 w i t h o v e r t d i a b e t e s and t w o G 3 males ( T a b l e I I ) .
T a b l e II ACTUAL
A N D F A S T I N G BLOOD GLUCOSE OF BB R A T S IN raM,
mean + S . D .
(range)
Group
Actual
GI
5.2 + 0.8
Fasting
(4.6 -
7.8)
4.3 + 0.6
(3.9 -
6.0)
G2
4.9 + 0.4
(4.3 -
5.8)
4.0 + 0.7
(3.5 -
5.7)
G3
5.9 + 1.4
(4.6 -
9.4)
4.8 + 1.4
(3.8 -
7.7) b
G4
7.0 + 0.7 w
(5.9 -
8.2)
4.5 + 0.4
(3.9 -
5.2)
G5
9.8 + 3.0 w167
( 7 . 0 - 19.8) a
6.2 + 1.7 w167
(4.2 - I0.8) c
Males
7.5 + 3.0
( 4 . 4 - 19.8)
5.2 + 1.6
(3.6 - I0.8)
Females
6.9 + 2.6
( 4 . 3 - 16.9)
4.9 + 1.3
( 3 . 5 - 10.4)
All
7.2 + 2.8
( 4 . 3 - 19.8)
5.1 + 1.5
( 3 . 5 - 10.8)
w and
w167i n d i c a t e
previous group;
significance
at
p
< 0.05
a8 v a l u e s a b o v e 10 mM; b ' c 2 ,
or
0.01,
respectively,
versus
or 5 v a l u e s a b o v e 7 raM, r e s p .
the
210 Tolerance metabolism
tests,
however,
in animals with
cose challenge almost half
disclosed
a gradual
worsening
lower d e g r e e of i n b r e e d i n g .
After
of
the
glucose
intravenous glu-
(42 %) of the rats had peak blood glucose above 11
mM and in 15 out of the 69 rats the b l o o d glucose did not r e t u r n to the fasting value + I mM a f t e r 2 hours (Table I I I ) . The ratios between the peak and fasting blood glucose ( P / F ) , of the excess glucose disappearance ( t l / 2 )
the half-time
as well as the area u n d e r the blood
glucose c u r v e a f t e r oral glucose load are summarized in Table IV. Table III ABNORMAL I V C T T VALUES II'~ BB RATS Group
Peak (> 11 mM)
D i f f e r e n c e (> I raM)
n
%
n
%
GI
I
(8.3)
0
G2
3
(33.3)
I
(11.1)
G3
5
(41.7)
2
(16.7)
G4
4
(36.4)
2
(18.2)
G5
16
(64.9)
10
(40.0)
Males
18
(46.2)
9
(23.1)
Females
11
(36.7)
6
(20.0)
All
29
(42.0)
15
(21.7)
D i f f e r e n c e meant between 120 min and fasting value l a r g e r than I mM. GPX a c t i v i t y .
The a c t i v i t y of GPX was s i g n i f i c a n t l y
h i g h e r in the blood of
the 4th and 5th generation of BB rats than in t h e i r G I - G 3 c o u n t e r p a r t s
b u t no
d i f f e r e n c e was found between the enzyme a c t i v i t y of diabetic (actual blood g l u cose > 10 raM) and nondiabetic members of the 5th generation ( T a b l e V ) . No s i g n i f i c a n t divided
according
differences to t h e i r
in GPX a c t i v i t y
fasting
were found
in g r o u p s
blood glucose concentration
of rats
(dividing
line 7
mM) and abnormal peak or r e t u r n time values of the I V G T T . Correlation tight
analysis.
b u t statistically
In
the
significant
whole g r o u p
of 69 rats
positive correlations
and age, w e i g h t and actual blood glucose (Table V l , glucose corelated neither with the GPX a c t i v i t y rats.
we found
not v e r y
between the GPX a c t i v i t y Figure I ) .
Fasting blood
nor with age and w e i g h t of the
The various parameters calculated from the glucose tolerance tests did not
reveal any s i g n i f i c a n t correlations
with blood GPX a c t i v i t y .
The only exception
211
was t h e P I F r a t i o , -0.36,
but
its a s s o c i a t i o n w i t h
the enzyme activity
was i n v e r s e
(r =
p < 0.01). Contrary
to t h e
whole group
overt
diabetes,
every
measured or calculated
(tl/2)
and AUC even
of the rats
inverse
o f 69 IDI3 r a t s
associations were found
reached
in t h i s g r o u p
parameter
in t h e g r o u p
G5 rats
between the activity
of glucose metabolism.
the limit of significance
(Table
of
o f GPX a n d
Two of them,
despite
with
the
t h e small n u m b e r
VII).
Table IV GLUCOSE
TOLERANCE
TEST
Test
RESULTS
IN B B R A T S
(mean + S.D. and range)
IVGTT
Group
GI
PIF ratio
ti/2,
2.37 + 0.19
33.2 +
(2.00 - 2.63) G2
(2.51
- 3.26)
2.65 + 0.67 (2.04 - 4.44)
G4
(1.70 - 3.53) 2.47 + 0.35
71 I )
43.0 +
24 9
40.4 + ( 7.9 -
2.43 + 0.55
49.7 +
(I .70 - 4 . 4 4 ) All
50 7)
(14.1
( 7.9 -
2.45 + 0.45
44.4 +
2.2
- 21.3)
17.2 +
1.9
(14.7 - 21.3) 22.0 +
89 7)
(14.2
29 9
6.6 b
- 33.4)
18.7 +
4.1
(15.9 - 30.2) 2 9 . 5 + 18.0 b
40 0
( 6 . 6 - 203 0)
(1.70 - 3.53) Females
p
( 7.9 -
55.4 +
mmolelh
16.4 +
( 2 2 . 0 - 123 3)
2.29 + 0.45
Males
10 6
18 0
39.5 +
(2.13 - 2.90)
AUC,
36.9 +
(19.3 -
2.40 + 0.24
G5
min
(17.4 -
2 . 8 0 + 0.21 a
G3
Peak of OGTT
(15.5 - 89.7)
22 9
2 3 . 0 + 13 0
98 2)
( 1 4 . 7 - 89 7)
38 0
22.1 + 11 9
98 2)
( 1 4 . 7 - 89 7)
30 5
2 2 . 6 + 12 5 w
{1.70 - 4.44)
Significant
difference:
( 6 . 6 - 203 0)
a a t p < 0.01
versus
G1,4,5;
(14.1 b
- 89 7)
P < 0.05 versus
GI
DISCUSSION The activity
o f GPX in b l o o d o f BID r a t s i n c r e a s e d
gain of the animals. neration,
i.e.,
The enzyme activity
in t h e
same w h e r e
overt
with
was t h e h i g h e s t diabetes
(with
the age and weight
in t h e 5 t h permanent
inbred
ge-
hyperglyce-
mia) appeared. The view of
antioxidant
that
aging
systems
is
is a s s o c i a t e d w i t h an
an o v e r a l l
oversimplification
and
decrease of the capacity
surely
does
not
hold
for
212
Table V GPX A C T I V I T Y
IN THE BLOOD OF BB RATS (mean + S . D . and r a n g e )
Group
GPX,
] J k a t a l / g Hb
GI
3.76 + 0 21
(3.39 - 4.10)
G2
3.65 + 0 78
(2.91 - 5.42)
G3
3.34 + 0 38
(2.89 - 4.05)
G4
4.05 + 1 03 w167
(2.59 - 5.58)
G5 G5, o v e r t diabetes
5.02 + 1 25 w167
(3.33 - 7.29)
4.90 + 1 22
(3.34 - 6.61)
G5, n o n d i a b e t i c s
5.10 + 1 30
(3.33 - 7.29)
Males
4.48 + 1 16
(3.02 - 7.29)
Females
3.79 + 0 96
(2.59 - 7.12)
All
4.18 + 1 12
(2.59 - 7.29)
m
w167i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s at p < 0.01 v e r s u s p r e v i o u s g r o u p .
T a b l e Vl SIMPLE
CORRELATIONS
BETWEEN
BLOOD
GPX
ACTIVITY,
AGE
AND
BODY
WEIGHT OF BB RATS ( r )
Parameter
GPX
Age
Age
0.65 w167
Body weight
0.41 w167
0.26 w
A c t u a l blood g l u c .
0.34 w167
n.s.
Body weight
n.s.
w and w167i n d i c a t e s i g n i f i c a n t c o r r e l a t i o n s at p < 0.05 and 0.01, GPX. vity
Vertechy
et al.
(1989) and Sohal et al.
(1990)
found
respectively.
e l e v a t e d GPX a c t i -
in h e a r t and muscle, as well as in l i v e r of old male r a t s ,
compared w i t h t h e i r y o u n g c o u n t e r p a r t s . various
parts
of
the
same organ
respectively,
A c c o r d i n g to C i r i o l o et al.
reveal
different
changes of t h e a n t i o x i d a n t enzyme a c t i v i t i e s .
patterns
They observed
of GPX in mesencephalon and p a r i e t a l c o r t e x ,
of
as
(1991) even
age-dependent
increased activity
decreased a c t i v i t y
in b r a i n
and no change o f enzyme a c t i v i t y in t h e p r e f r o n t a l c o r t e x o f a g i n g r a t s .
stem
GPX is
a selenoenzyme and its a c t i v i t y d e p e n d s on t h e a v i a l a b i l i t y of t h i s t r a c e element (Perona tween
et a l . ,
1978).
selenium c o n t e n t
G r o m a d z i n s k a et al. of various
organs
(1988) of
the
found rat
and
close r e l a t i o n s h i p their
GPX
be-
activity.
213
10 O
O o
9"r
o
8
0 O
0
O,
0
000
0
0
o/0
m
6
~oe
D.
? 0
0
o
ov
_
O0
0
0
0
I
i
i
~
4
I
~
~
L
8
I
~
~
12
~
I
~
,
~
I
20
16
Blood glucose, mM Figure I. Correlation between blood g l u c o s e in BB r a t s .
blood
glutathione
peroxidase
activity
and a c t u a l
Table VII CORRELATION (r)
BETWEEN
GPX
ACTIVITY
AND
BLOOD
GLUCOSE
PARAMETERS
IN BB R A T S WITH O V E R T D I A B E T E S AND W I T H O U T D I A B E T E S
Group
Diabetics
Nondiabetics
Correlation with
G 5 (8)
G 5 (17)
A c t u a l B G , mM
- 0.53
< 0.20
+ 0.43 w167
F a s t i n g BG, mM
-0.53
< 0.20
< 0.2
IVGTT
OGTT,
peak,
mM
A l l (61)
-0.53
< 0.20
-0.38 w
PIF ratio
- 0.66
- 0.21
< 0.2
TI12,
- 0.77 w
+ 0.39
< 0.2
- 0.71 w
< 0.20
+ 0.28 w
min
AUC,
mmolelhr
amultiplicative correlations; 0.05 and 0 . 0 1 , r e s p e c t i v e l y .
w and
According
al.
to
Yamaguchi
et
raises w i t h age in h e a l t h y selenium c o n c e n t r a t i o n
(1992)
both
men and t h i s
in plasma and
s t a t u s o f t h e BB r a t s in t h i s s t u d y age-dependent
w167i n d i c a t e
significant
red
cell
increase
red cells.
and
correlations
at
plasma
activity
is p a r a l l e l
We d i d
GPX
to the
<
i n c r e a s e of
n o t f o l l o w the s e l e n i u m
and t h u s we can o n l y h y p o t h e s i z e
i n c r e a s e o f t h e GPX a c t i v i t y
p
t h a t the
is r e l a t e d to s e l e n i u m a c c u m u l a t i o n .
214
A s l i g h t positive c o r r e l a t i o n between the actual blood glucose and the GPX activity
was
found
in
the
whole g r o u p
somewhat s t r o n g e r in 61 n o n - d i a b e t i c diabetes
in BB rats
of 69 13B rats.
rats (Table V I I ) .
is age d e p e n d e n t
(Marliss,
1983).
from the correlation analysis of the whole g r o u p diabetic rats there was a loose r e l a t i o n s h i p glucose ( r = 0.36, enzyme a c t i v i t y
p < 0.01).
This
association
was
The manifestaton of the This
(Table V l )
was not a p p a r e n t b u t in the 61 non-
between t h e i r age and actual blood
It is possible,
that the association
between the
and the actual blood glucose in nondiabetic rats is only an i n -
d i r e c t one due to the age dependence of the GPX a c t i v i t y and the glucose metabolism d e t e r i o r a t i o n . The ratio between the peak and fasting
blood glucose value a f t e r
nous glucose load is not a classical parameter of glucose metabolism. related to the glucose d i s t r i b u t i o n of the body
in the e x t r a c e l l u l a r
to metabolize glucose.
intraveIt may be
space and to the a b i l i t y
Between the PIF ratio and the blood GPX
a c t i v i t y of the rat we found a n e g a t i v e correlation
(which is a little s t r o n g e r in
rats with fasting blood glucose below 7 raM; - r = 0.43, p < 0.01) b u t we have no plausible explanation for this phenomenon at p r e s e n t . In the diabetic rats negative relationships were found between glucose metabolism parameters and blood GPX a c t i v i t y .
The correlations
regarding
sociation of the enzyme a c t i v i t y with the measured variables (actual, IVGTT
peak blood glucose)
On the o t h e r integrated
hand,
did
not reach the level of statistical
the associations
variables calculated
fasting and significance.
between the blood GPX a c t i v i t y
from fasting
and post-load
values
the as-
and the
(glucose d i s -
appearance a f t e r IVGTT and AUC) are s i g n i f i c a n t . O x i d a t i v e damage may play an important role in the biochemical b a c k g r o u n d of diabetic complications oxidative
stress
and
when at least one of its basic conditions
diminished
r e f e r s to the second condition.
antioxidant
Ruderman
compensatory
et a l . ,
is
fulfilled.
(exaggerated Our
study
The available data on the a n t i o x i d a n t status in
diabetes mellitus ( f o r reviews see O b e r l e y , 1992;
capacity)
1988; Baynes, 1991; Kristal and Y u ,
1992) are c o n t r a d i c t i v e .
Oxidative
stress
can t r i g g e r
reactions which lead to increased a n t i o x i d a n t enzyme a c t i v i t y .
On
the other side reactive o x y g e n species and high ambient glucose c o n c e n t r a t i o n can damage the same enzymes.
The deletorious effect of in v i t r o
s u p e r o x i d e dismutase and g l u t a t h i o n e et a l . , BB
1987; B l a k y t n y and H a r d i n g , rats
have
the
advantage
reductase was already demonstrated
of
(Arai
1992). of
spontaneous
diabetes
development
o t h e r experimental models of this disease. No arteficial i n t e r v e n t i o n and o v e r t diabetes is preceded with a long p r e d i a b e t i c state. rats the main d e t e r m i n a n t of blood GPX a c t i v i t y of diabetes hyperglycemia
glycation
over
is necessary
In nondiabetic BB
is t h e i r age. A f t e r development
seems to have a deleterious effect on the a c t i v i t y
of
215
this important antioxidant enzyme. This fits well in the above outlined oxidative damage hypothesis of biochemical pathogenesis of diabetic complications but f u r ther experimental and clinical studies are necessary to b r i n g strong evidence about its r e l i a b i l i t y . ACKNOWLEDGEMENT This s t u d y was supported by the Slovak Grant Agency. 1/259/92.
Grant number:
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