Exp Toxic Pathol 1999; 51: 189-198

URBAN & FISCHER

http://www.urbanfischer.de/joumals/exptoxpath

'Institute of Anatomy, 2Institute of Clinical Chemistry, and 3Institute of Biochemistry, University Leipzig, Germany

Protective effects of Gingko biloba extract EGb 761 on myocardium

of experimentally diabetic rats
I: Ultrastructural and biochemical investigation on cardiomyocytes

G. FITZL], R. MARTIN2, D. DETTMER3 , v. HERMSDORF3, H. DREWS], and K. WELT]

With 7 figures and 4 tables

Received: February 4, 1998; Accepted: February 20, 1998

Address for correspondence: Dr. G. FITZL, Liebigstr. 13, Institut fUr Anatomie der UniversiHit Leipzig, D - 04103 Leipzig,
Germany.

Key words: Ginkgo biloba extract; Diabetes, myocardium; Myocardium, diabetes; Cardiomyocytes; Myocytes, cardiac.

Summary
Chronic diabetes in man and animal models develops
cardiomyopathic alterations which cannot be absolutely
avoided by insuline therapy. Since diabetic damage is part-
ly attributed to oxidative stress antioxidative treatment
could be able to reduce the alterations. Aim of this study was
to investigate the cardioprotective effects of EGb 761,
known as a radical scavenger, against diabetic alterations
in rats.
The diabetes was induced by i.p. injection of 60 mglkg
body weight streptozotocin. Duration of diabetes was
4 months, the protected group received 100 mglkg body
weight EGb 761 with the drinking water over 3 months.
Electron and light microscopic morphometry of left-
ventricular samples revealed typical diabetic alterations
consisting in decrease of volume fraction of myofibrils, SR
and t-tubules and diminishing of cardiomyocyte diameter,
increase of interstitial volume, mitochondrial size and vo-
lume fraction, and of vacuoles and of lipid drops. EGb treat-
ment could gradually prevent the loss of myofibrils and re-
duction of myocyte diameter but has only little influence on
interstitial and mitochondria volume. The diabetic-induced
increase of lipid and vacuoles and the decrease of SR and
t-tubules were not influenced.
Biochemical parameters of oxidative stress: malondial-
dehyde (MDA) was only insignificantly altered by diabetes
and EGb. The superoxide dismutase (SOD) activity was in-
creased by diabetes and more increased by EGb treatment.
Creatine kinase (CK) activity was diminished by diabetes
but slightly increased by EGb. The polymerase chain re-
action (PCR) of i-NOS was not different between the dia-
betic and protected diabetic groups.
Introduction
It becomes increasingly accepted that chronic diabetes
leads to different structural and functional alterations of
myocardium which can be characterized by the term diabe-
tic cardiomyopathy (13 , 30, 34). The cardiomyopathic dis-
turbances regarding cardiomyocytes, interstitium and capil-
laries independent of hypertension and coronary sclerosis
are responsible for the increased cardial risk in diabetic
patients. Several studies pointed out that some of late
complications in chronic diabetes cannot be completely
avoided by insuline substitution (9, 31, 39, 40). Conse-
quently attemps were made to find additive therapeutic
possibilities. On the assumption that diabetic damage is
partly caused by increased oxidative stress with the oc-
curence of oxygen radicals the application of radical sca-
vengers seemed to be hopeful and indeed some diabetic
alterations could be mildered (35, 43). This study was
made to investigate the protective effects of EGb 761
known as radical scavenger and stabilisator of membra-
nes on the ultrastructure of cardiomyocytes in strepto-
zotocin-diabetic rats. This model although not absolutely
identical with IDDM diabetes develops cardiomyopathic
alterations similar to those of human Type I diabetes and
the spontaneously diabetic BB rats (32, 34).
Material and methods
Animals and experimental procedure
The experiments were in accordance with animal protec-
tion recommendations, approved by the Regierungsprasi-
dium Leipzig (No. 15/95).

0940-2993/99/51/03-189 $ 12.00/0 189

 Sixteen 6 months old Wi star rats, strain Crl:(Wi)Br from
Charles River GmbH Salzfeld, Germany) kept seperately
under standard conditions were divided into 3 experimental
groups:
group V (control): 5 rats without any treatment
group I (diabetes): Diabetes was induced by intraperitoneal
injection of 60 mg/kg body mass of streptozotocin (Boeh-
ringer Mannheim, Germany), dissolved in 0.1 m citrate buf-
fer, pH 4,5 immediately before use. 6 rats at the age of
2 months were injected in the morning following a night of
starvation. 12 days after injection blood glucose levels were
detennined to be 27-33 mM and were> 33,3 mM after
4 months when the rats were sacrificed.
group II (diabetes with EGb 761 treatment): 6 rats were
made diabetical according to group I; after 1 month of dia-
betes (blood glucose level> 33 mM) the rats were treated
daily with 100 mg/kg body mass Ginkgo biloba extract
(EGb 761, IPSEN, Paris, France) given in the night dis-
solved in a limited amount of drinking water (over the day
free access to drinking water). After 4 months of diabetes
the rats were sacrificed, being EGb-protected 3 months.
Tissue processing for electron microscopy
The animals were anaesthetized with ether and sacrificed
by cervical vertebral dislocation. After thoracotomy small
samples from the left ventricular myocardium near the apex
were fixed in cold KARNOVSKY solution (buffered 2 %
glutardialdehyde, 2 % paraformaldehyde, pH 7,4 and pro-
cessed in usual manner for electron microscopy. Five tissue
blocks per animal were embedded in Durcupan (FLUKA).
From each tissue block semi thin sections were taken and
stained with toluidine blue for histological analysis and to
select interesting areas for electron microscopy.
Ultrathin sections were cut using Ultracut (Reichert-lung),
electron micrographs according to the requirements of mor-
phometry were taken with the electron microscope EM 900
(ZEISS).
Morphometric analysis
Per animal 25 electron micrographs at 10 000- and
20000-fold primary magnification obtained from five tis-
sue blocks were morphometrically analysed. Classic point
counting and intersection point counting techniques were
used for calculation of volume density (VV), surface den-
Table 1. Data of animals (mean and SD) (* =p ::; 0.05; *** =P ::; 0.001).
control diabetic
mortality 12 % of all diabetic rats
body weight (g) 573 34 * 279 73
heart weight (g) 1.76 0.2 *** 1.30 0.3
relative heart weight (%) 0.31 0.47
blood glucose level (mM) "" 10.5 >33.3
190 Exp Toxic Pathol51 (1999) 3
sity (SV), mean volume (V) and numeric density (NV) of
different subcellular structures of cardiomyocytes. Measure-
ments were partly carried out using the SIS-image analysing
system. The results were statistically controlled using T-test
and WILCOXON-test.
Biochemical investigations
Parameters of oxidative stress: Tissue samples were homo-
genized by an Ultra-Turrax-homogenizer, centrifuged for
10 min at 2000 x g and the following parameters detennined
in the supernatant.
Superoxide dismutase (SOD): The activity of the CuJZn-
SOD was determined by the SOD Assey Kit of CALBIO-
CHEM (Cat. No. 574600). The kit takes advantage of a
proprietary reagent. This reagent undergoes an alkaline
autoxidation, which is accelerated by SOD and yields a
chromophore with an absorption maximum at 525 nm.
Creatine-kinase (CK): CK was determined by an optimized
standard method, in which phospate of creatine phosphate
is transferred on ADP, catalyzed by CK. The resulting ATP
was determined in the combined optical test, using hexo-
kinase as assistant enzyme and G-6-P-dehydrogenase as indi-
cator enzyme (37).
Malonic dialdehyde (MDA): MDA reacts with thiobarbi-
turic acid reagent yielding a red coloring matter, which was
extracted by butanol and measured at 535 nm (41).
Estimation of inducible Nitric oxide synthase (i-NOS)
activity by reverse transcriptase-PCR (RT-PCR): The
tissue samples were stored in liquid nitrogen until use. Total
RNA was extracted according to the method by CHOM-
CZYNSKl und SACCHI (6). RNA was reverse-transcribed into
first strand cDNA using the Superscript Preamplification
System by GIBCO. Six III of cDNA reaction were applied
as a template for PCR amplification. The upper primer
(5'-TCAGATCCCGAAACGCTACAC) and the lower pri-
mer (5' -GCAGGA AGGCAGCAGGCACAC) specific for
rat iNOS generated a 614 bp product. A rat GAPDH primer
set (5'-GCCCCTTCCGCTGATGCCCCC and 5'-AGGG-
ATGATGTTCTGGGCTGC) generating a 258 bp product
was used as control. PCR run (Gene Amp 2400, Perkin
Elmer) consisted of 40 cycles of denaturation (10 s, 94 DC),
annealing (30 s, 58 DC) and extension (60 s, 72 DC) with
1,5 U AmpliTaq DNA polymerase (Perkin Elmer). PCR
products were electrophoretically characterized on 1,5 %
agarose gels stained with ethidium bromide and visualized
with UV light.
diabetic + Egb 761
247 48
1.04 0.2
0.43
>33.3
 Table 2. Morphometrical parameters of myocardium of control, protected and unprotected diabetic rats
(mean SD) (* = p :::;; 0.05).

control diabetes diabetes + EGb

average diameter of cardiomyocytes 12.4 1.4 * 10.4 1.1 11.7 1.9

number of myocyte cross sections per mm2 2272.3 305 2871.4 385 2270.9 357
I * I
volume fraction ofmyocytes 0.75 0.02 0.74 0.02 0.73 0.02
volume fraction of interstitium 0.13 0.01 * 0.17 0.01 0.17 0.03
(without vessels)
interstitium to myocyte ratio 0.18 0.23 0.22
number of capillaries per mm 2 2741.8 360 2810.1 396 2492.6 486
capillaries to myocytes ratio 1.21 0.98 1.10

Results

Light microscopic results

There were no obvious differences between myocar-

dium of control-, diabetic- and EGb-protected diabetic
rats at the histological level. The cardiomyocytes of the
diabetic groups appeared normally, the diameter of muscle
cells seemed to be somewhat smaller in the diabetic groups
and the interstitium was partly slightly extended (table 2).

Qualitative electron microscopic results

Qualitative comparison of cardiomyocytes did not ex-

hibit obvious differences between the control and the pro-
tected group and unprotected diabetic groups.
In the control group myofibrils and mitochondria were
of normal appearence. Cardiomyocytes of the diabetic
group showed some irregularities of sarcomeres and accu-
mulation of glycogen granules in many cells, partly se-
questered by membranes (fig. 1,2). Mitochondria showed
slight clearing of the matrix and slight irregularities in
structure and arrangement of cristae (fig. 1). Lipid drop-
lets, partly related to mitochondria seemed to be more fre-
quent in the diabetic group. Sarcoplasmic reticulum and
t-tubules did not show abnormalities. In the protected dia-
betic group the structure of sarcomeres and of mitochon-
dria seemed to be somewhat better, glycogen content of
the cells was somewhat less increased. (fig. 3) Fig. 1. Cardiomyocyte of the unprotected diabetic group with
irregularities of sarcomeres and mitochondria; glycogen
distributed between the myofibrils (x 24 000).
Quantitative electron microscopic results

The quantitative analysis of some myocytic compart-
ments revealed differences between the control and non-
protected and protected diabetic groups (table 3).
The volume fraction (VV) of myofibrils was signifi-
cantly reduced in the unprotected diabetic group but not
in the protected one (fig. 4; table 3). The complementary
increase of the volume fraction of sarcoplasm was signi-
ficant only between the control and diabetic group (fig. 4;
table 3). The volume fraction of mitochondria showed a
slight elevation in the diabetic but increased significantly

Exp Toxic Pathol 51 (1999) 3 191


in the protected diabetic group (fig. 4; tab. 3). Concerning
the mean mitochondrial volume (V) there was a moderate
significant increase in both diabetic groups (fig. 5; table 3).
The numeric density (NV) of mitochondria was signifi-
cantly diminished in the diabetic and non-significantly di-
minished in the protected diabetic group (fig. 5; table 3).
The surface/volume ratio SVNV of mitochondria was
decreased in both diabetic groups without statistical sig-
nificance. The ratio of hit points on mitochondria to hit
points of myofibrils (PmlPmyo) rose in the diabetic and
somewhat more in the protected diabetic group (table 3).
The internal compartments of mitochondria (volume frac-
tion of cristae and matrix) exhibited no differences be-
tween the control and both diabetic groups, only the vo-
192 Exp Toxic Patho151 (1999) 3
Fig. 2. Cardiomyocytes of unprotected
diabetic group with sequestered gly-
cogen accumulations. (Inset: glycogen
sequester containing glycogen a-gra-
nules) (x 24 000).
Fig. 3. Cardiomyocytes of the EGb-
protected diabetic group show better
structured sarcomeres and organelles
than in those of the unprotected group
(x 24 000).
lume fraction of degenerated areas increased significantly
from 0.004 (control) to 0.025 (diabetes) rsp. 0.021 (Egb-
protected diabetes). The number of ATPase particles per
100 nm internal mitochondrial membrane slightly but sig-
nificantly decreased between the control (14.9 ) and the
diabetic groups (unprotected: 13.9; EGb-protected: 14.1)
without significant difference between the latter.
Sarcoplasmic reticulum and t-tubules both showed
decrease of their voluminal fraction in diabetic and pro-
tected diabetic condition; only when protected the diffe-
rence to the control was significant (fig. 6; table 3).
Concerning the volume fraction of vacuoles there was
a significant increase in the diabetic and a highly signifi-
cant increase in the protected diabetic group. The volume
 Table 3. Morphometrical parameters of components of cardiomyocytes in the experimental groups
(mean SD) (statistic see fig. 4-5).

control diabetic diabetic with

EGb 761 treatment

myofibrils 0.449 0.05 0.399 0.04 0.405 0.03

mitochondria
volume density (VV) 0.375 0.03 0.392 0.01 0.423 0.02
numeric density (NV) [/lm-3] 0.354 0.06 0.282 0.03 0.275 0.06
volume (V) [/lm3] 1.124 0.13 1.626 0.28 1.681 0.26
surface to volume ratio 4.648 0.47 4.098 0.35 3.557 0.24
(SVNVm) [/lm- I ]
ratio pm/pmyo 0.932 0.20 1.204 0.13 1.279 0.29
sarcoplasm volume density (VV) 0.118 0.05 0.274 0.137 0.127 0.027
sarcoplasmic reticulum
volume density (VV) 0.034 0.004 0.021 0.006 0.02 0.003
t-tubules volume density (VV) 0.002 0.002 0.0012 0.0006 0.0009 0.0004
vacuoles volume density (VV) 0.0055 0.0022 0.0113 0.0006 0.0143 0.0141
lipid drops volume density (VV) 0.002 0.003 0.014 0.006 0.018 0.014

W
0.5

0.4

0.3

0.2

0.1

0
myollbrils ureoplasrna

I
~~~~~~~~~I

W
0.04
I .I
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
Fig. 4. Volume densities of selected organelles of
cardiomyocytes in the three experimental groups
(* = p:S; 0.05; ** = p:S; 0.01; *** = p:S; 0.001).

fraction of lipid drops was elevated in the diabetic and
considerably more elevated in the protected diabetic
group (fig. 6; table 3).
Biochemical results
Creatine kinase (CK) activity related to moist weight
was significantly diminished in the diabetic and protected
diabetic group, however slightly increased in the pro-
tected diabetic group, related to protein content (fig. 6;
table 4).
The malondialdehyde (MDA) content of the myo-
cardium was slightly increased in the protected diabetic
group. Related to moist weight there was a significant
decrease in the diabetic, and only slight decrease in the
protected diabetic groups (fig. 6; table 4).

Exp Toxic Pathol51 (1999) 3 193

 2

15

05

o Fig. 5. Selected morphometrical pa-

rameters of mitochondria in the three
experimental groups (* = p :::; 0.05).

I I

MDA (nmoIIgl SOD (U/gl CK tumollg)

rl
-c-
~ --~~~~~~~~~~I

related to moist _",ht

1~ ~------------------~~~------------------,

0.8

0.15

0.4

02

o
DA (nmollmg) SOD (U/g) CK (UmoIImg)
Fig. 6. Biochemical results of myocardial tissue
~1.~C~~~~~.~~~~~~+~E@G~bJI of the three experimental groups a: related to moist
weight; b: related to protein content (* =p:::; 0.05;
related to protein content ** =p :::; 0.01).

The superoxide dismutase activity (SOD) was increa-
sed moderately in the diabetic- and significantly increa-
sed in the protected diabetic group, with significant dif-
ference between the diabetic and protected diabetic group
(fig 6; table 4).
RT peR (Reverse transcriptase polymerase
chain reaction) of i-NOS
The transcription of inducible nitric oxid synthase
(i-NOS) was investigated in the control, diabetic, and

194 Exp Toxic Pathol 51 (1999) 3

 Table 4. Biochemical parameters of myocardium (mean and SD) (statistic see fig. 6).

control diabetic diabetic with

Egb 761 treatment

MDA moist weight (nmol/g) 76.88 25.4 49.6 12.6 57.68 6.8
protein content (nmol/mg) 0.51 0.1 0.47 0.1 0.65 0.2
SOD moist weight (U/g) 3.11 2.9 4.32 1.9 5.423.1
protein content (U/g) 0.431 0.27 0.766 0.25 1.166 0.34
CK moist weight (flmol/g) 37.3 6.4 23.7 4.6 21.7 1.9
protein content (flmol/g) 0.236 0.017 0.227 0.023 0.286 0.08

Bp
2000
1500
1000
700 iNOS
500
400
300
200
100

II II II II v V V V
34 28 31 33 t31 17 2 3 13 11 62 60 59 208
300
GAPDH
200

Fig. 7. RT -peR of myocardial tissue of the control- (V); unprotected diabetic- (I) and EGb- protected diabetic group (II).
peR products were analyzed by agarose gel electrophoresis.

EGb 761-protected diabetic groups with the mean ofRT
peR.
Amplificates specific for i-NOS could be demonstrated
in all animals of the control, and in one of five diabetic
animals but not in the animals of the protected diabetic
group (fig. 7).
Discussion
The actual knowledge about diabetic cardiomyopathy
indicates that most but not all of the metabolic, functional
and structural alterations of the myocardium can be avoided
or become reversible under the adequate insulin substitu-
tion (9, 40). The cardiac risk of diabetic patients remained
significantly increased (34). To avoid late complications
of treated chronical diabetes it appears logical to apply
supportive therapeutics which are able to scavenge free
oxygen radicals occurring during diabetic metabolism.
Different substances were successfully tested, e.g. toco-
pherol (vit. E) (35) and Ginseng extract (43).
In our opinion Ginkgo biloba Extract EGb 761 appeared
as a hopefull candidate for supporting treatment of chro-
nic diabetes and was never before tested with respects to
ultrastructural protection of the diabetic myocardium.
EGb 761 is a highly standardized extract of Ginkgo bilo-
ba leafes containing 24 % flavone glycosides and 6 % ter-
pene lactones. The flavone glycosides and partly quercitine
are responsible for radical scavenger properties (23, 25).
Besides inhibition of lipid peroxidation of biological
membranes EGb 761 has anta-gonistic effects on P AF, in-
activates the superoxide anion O 2- and stimulates the
EDRF (= NO) liberation which is depressed by oxygen
radicals. It improves glucose- and O 2 uptake, the reestab-
lishment of mitochondrial metabolism and A TP synthe-
sis, and stabilizes lysosomal membranes (29).
Therefore some protective effects of EGb in diabetic
cardiomyopathy could be expected.

Exp Toxic Pathol 51 (1999) 3 195

 According to observations of FRENZEL et al. (8) we
found no obvious qualitative differences between the
myocardial ultrastructure of control and chronically dia-
betic rats in contrast to other investigators who described
myofibrillar damage and interstitial and perivascular fi-
brosis (39) and severe mitochondrial damage (35). In
aggreement with FRENZEL (8) we stated several diabetic-
induced alterations with the mean of morphometrical ana-
lysis. Thus we observed only a slight increase of the volume
fraction of interstitium and a significant decrease of car-
diomyocyte diameter in aggreement with GIACOMELLI and
WEINER (11) and THOMPSON (40), which was less ex-
pressed in the EGb-protected diabetic group. This beha-
viour was also reflected in the number of muscle cell cross
sections per mm2 , which was more increased in the dia-
betic than in the protected diabetic group. This protective
effect of EGb 761 was also present in the relative heart
weights, which were more increased in the diabetic than
in the protected diabetic group despite considerable de-
crease of absolute heart weights in both diabetic groups.
A moderate diabetes-induced atrophy of the cardio-
myocytes was confirmed by a significant decrease of vo-
lume fraction of myofibrils which was less expressed in
the protected diabetic group and accompanied by a com-
plementary increase of the sarcoplasmic fraction. The
latter may be due partly to a real loss of contractile mate-
rial but to a certain extent also to local edemas of cardio-
myocytes which were also observed by GAIL et al. (9).
Edema localized predominantly near the SR tubules may
be due to depression of SR Ca++ binding and Ca++ - Mg++
ATPase activity leading to the disturbed Ca++ transport
into the SR (2) resulting Ca++ overload of the cytoplasm
and finally in a disturbed relaxion (24). The mebrane-
stabylizing effect of EGb 761 (5) may counteract this
process. Although according to PIERCE and DHALLA (26)
the oxidative capacity, Mg-ATPase activity, and Ca++-
accumulation capacity of mitochondria was diminished
we observed only negligable alterations of internal
mitochondrial structure except a slight but statistically
significant increase of degenerated intramitochondrial
areas, which was less expressed in the protected group.
Counting of ATPase particles at the inner mitochon-
drial membranes, performed after negative-staining tech-
nique revealed slight but significant decrease of particles
in both diabetic groups without a clear protective effect of
EGb.
A pronounced loss of myofibrils after 26 weeks dura-
tion of diabetes in BB rats was also reported by ZHOU et
al. (44). MALL et al. (17) found direct correlation between
loss of myofibrils in biopsies of human diabetic hearts and
depressions of ejection fraction. In our experiment the vo-
lume fraction and average volume of mitochondria were
significantly increased in the diabetic groups accompa-
nied by decrease of their number per unit volume without
obvious alteration of structure. ZHOU et al. (44) observed
also increase of mitochondrial volume in diabetes con-
nected with structural degeneration, which is not to be ob-
served after perfusion fixation (9).
196 Exp Toxic Pathol51 (1999) 3
Concomitantly performed cytophotometrically measu-
red quantifications of some enzyme activities in our ex-
periment revealed significant alterations of oxidative and
glycolytic mitochondrial enzymes (28) which were partly
normalized by EGb-treatment.
Reduction of the myofibrillar compartment may lead to
concomitant reduction of SR and t-tubules as described
by THOMPSON (39) and was also present in our material.
In contrast ZHOU et al. (44) observed swelling of SR and
t-tubules in cardiomyocytes of BB rats after 26 weeks of
diabetes, explained by disturbed Ca++ binding capacity of
SR in diabetes. Surprisingly in our experiment the volume
fractions of SR and t-tubules were more depressed when
the rats were treated with EGb. Probably this is due to the
membrane-stabylising effects of EGb (5) preventing Ca++
flux into these compartments and thereby preventing also
hydration. Otherwise the diabetes-induced increase of
vacuoles in the sarcoplasm was not prevented by EGb.
Possibly a part of the SR-tubules degenerate under dia-
betic condition forming vacuoles which is not influenced
by EGb. Vacuolization of the sarcoplasm was also ob-
served by JACKSON et al. (12) and related to swelling
of the sarcoplasmic reticulum. Vacuoles and swollen
parts of degenerated SR may be hardly to distinguish. As
manyfold mentioned in the literature (9, 12, 45) we ob-
served a considerable diabetic induced increase of lipid
drops (volume fraction) which was not prevented by EGb.
This phenomenon is caused by the disturbance of the glu-
cose- and fatty acid metabolism in diabetes leading to
high levels of free fatty acids in the blood and to in-
creasing synthesis of triglicerides accompanied by in-
hibition of lipolysis in the myocardium (36). Toxic meta-
bolites generated in these processes (acylcarnitine, long-
chain acyl CoA) disturb many subcellular membrane
structures (14), further the Ca-ATPase of the SR (1) and
also the Na+-K+- ATPase of the sarcolemma (7). In mito-
chondria the ATP synthesis is diminished by inhibition of
the adenine nucleotid translocator; proteases, phospholi-
pases, and lysosomal enzymes were activated. The hyper-
lipidemia leads to an altered fatty acid profile of mem-
branes which decreases the activity of the glucose
transporter resulting in reduction of myocardial glucose
utilization (4).
Glycogen accumulation as seen in diabetic cardiomyo-
cytes with and without EGb protection in our experiment
was also reported by other authors. MAULIK et al. (20)
observed a two-fold increase of myocardial glycogen
content in STZ-diabetic rats already after 5 weeks. This
glycogen accumulation finally results from the increased
citrate level as a consequence of the elevated free fatty
acids leading to inhibition of phosphofructokinase (21).
The rate of glycolysis as well as glucose uptake and oxi-
dation are reduced, probably due to the cellular depletion
of glucose transporters (10) resulting in increased tissue
content of glucose-6-phosphate activating the glycogen
synthase and inhibiting the phosphorylase. Thus the low
amount of transported glucose is diverted to glycogen
production (3).
 To get insight into energy supplying systems for ATP
necessary for contraction we measured biochemically the
activity of creatine kinase in the myocardium of different
experimental groups. Related to moist weight we registe-
red a significant decrease of CK activity in the diabetic
rats wich aggrees with literature (19, 27) and also in the
protected diabetic group. Related to protein content how-
ever there was no diabetes-induced decrease but a signi-
ficant increase of CK activity in the protected diabetic
group. This latter result we would like to interprete as a
protective effect of EGb 761 leading to approved ATP
supply in the myocardium because the CKlPCr systeme
is functionally coupled with adenine nucleotide translo-
case and various A TPases in mitochondria, myofibrils,
SR, and sarcolemma (42).
MDA as an indicator for lipid peroxidation and sca-
venger induction is known to be elevated in diabetic con-
dition (18, 22). In our experiment however it was un-
changed (related to protein content) or slightly decreased,
(related to moist weigth). This behaviour can be regarded
as adaption of the relative young rats to the chronical dia-
betic stress. EGb treatment in our rats did not alter signi-
ficantly the MDA level.
In agreement with the literature reporting increase of
SOD activity (16, 22, 38) except KUMAR and MENON (15)
wich observed decrease, the myocardial SOD activity in
our diabetic rats was slightly elevated, when EGb-treated
even significantly increased. This can be interpreted as
increased antiradical capacity or reactivity of the myo-
cardium against oxidative stress by the action of EGb.
Additionally, it can not be excluded that EGb directly in-
fluences certain enzyme activities or even their synthesis.
Because expression of i-NOS demonstrated by immu-
nohistochemistry was only sparsely and very weak in the
diabetic groups wich may be due to the "burning out" of
the system during long term diabetes (unpublished results),
we tested the i-NOS presence at the stage of translation
by the mean of RT PCR. The result corresponds to that
of immunohistochemistry; only one of five diabetic ani-
mals gave i-NOS specific amplificates, all EGb-protected
diabetic animals were negative. Surprisingly all control
animals were positive. This may be explained by the stress
occuring during the ether narcosis starting the i-NOS de-
fence system in healthy animals.
Summarising our results we could demonstrate several
effects of EGb 761 on cardiomyocytes in chronically-
diabetic rats:
the diabetes-induced increase of interstitial volume was
gradually diminished, the decrease of cardiomyocyte
diameter resp. cross section area, the increase of rela-
tive heart weights, and the decrease of volume fraction
of myofibrils were attenuated. Mitochondrial parameters
were only less influenced.
CK and SOD activity ofthe diabetic myocardium were
increased improving the energetic and antioxidative
state; diabetes-induced glycogen accumulation was
somewhat decreased.
Effects on myocardial rnicrovessels and interstitial ma-
trix will be presented in the second part of this study .
Acknowledgements: We are very greatful to the IPSEN
Institut, Paris, for providing us with the Ginkgo biloba ex-
tract, to the Intersan Institute Ettlingen for supporting this
study, and to Prof. P. ROSEN (Diabetes Research Institute
Dusseldorf) and to Dr. W. BLOCH (Centre of Anatomy, Uni-
versity Koln) for valuable advises.
We wish to thank the Animal Breeding Center of the Me-
dical Faculty of the University Leipzig, especially Dr. P.
MADAJ-STERBA, for carefully keeping and therapeutical ma-
nagement of the animals. We thank Mrs. D. Muller and Mrs.
Ch. SCHNEIDER and Mrs. Chr. LUCAS for excellent technical
assistence.
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