Atherosclerosis
Elsevier Publishing
EFFECT
Company,
Amsterdam
- Printed
OF PYRIDINOLCARBAMATE
FATTY
185
in The Netherlands
ON RAT
ADIPOSE
TISSUE
FREE
ACIDS
D. GRAFNETTER*,
T. SHIMAMOTO**
AND
F. NUMANO**
+ Institute of Clinical and Experimental Medicine, Division of Cardiovascular Diseases, Budejovickd 800, Prague - 4 - Krc’ (Czechoslovakia), and ** Institute for Cardiovascular Diseases and Department of Medicine, University, 1 - Yushima, Bunkyo-Ku, Tokyo (Japan)
(Revised, received January
Tokyo Ika-Shika
Natiolaal
24th, 1972)
SUMMARY
A relatively high concentration (1 mg/ml) of pyridinolcarbamate (PCC) Krebs-Ringer phosphate buffer containing no albumin provoked accumulation free fatty
acids (FFA) in epididymal
was approximately panied
than
that of adrenalin,
by the release of FFA into the incubation
Serum FFA, raised by injection pretreated with PDC. It is assumed that lipolysis trations
fat tissue of rat during
4000 times weaker
ilz vivo. A working
of adrenalin
medium
in controls,
incubation.
in of
This effect
and was not accom-
which contained
albumin.
were less raised in animals
is not affected by PDC in fat tissue at low concen-
theory
is raised
that
PDC, by affecting
permeability
change, could diminish the FFA output. This mechanism, if verified, might contribute to the explanation of the earlier observed decrease of blood FFA and accelerated recession
of experimentally
Key words:
Pyridilzolcarbamate
induced
atheromatosis
- Atherosclerosis
as due to PDC treatment.
- Free
fatty
acids
- Fat
tisszre -
Permeability
INTRODUCTION
Pyridinolcarbamate (PDC) was synthesized by SHIMAMOTO AND ISHIKAWA in 19621, and it has since become evident that the drug is non-toxic, that it possesses important antisclerotic psoperties and that it has an influence on the permeability in tissues. Its antisclerotic effect is accompanied by a decrease in aorta1 cholesterol levell-is. Atherosclerosis,
1972, 16: 185-191
186
D.
GRAFNETTER, T. SHIMAMOTO, F. NUMANO
In another paper we described19 the influence of PDC on blood and tissue lipids and on some other parameters in rats fed both on a standard laboratory diet and on a synthetic diet with a high content of fat and cholesterol, as well as in rats intoxicated with Calciferoli9. The observed changes were accompanied by favourable changes in the activity of some enzymes of the Krebs and glycolytic
cycle in the
vascular wall, as was demonstrated by parallel experiments of MRHOVAet al.20. GOTO AND NAKAMURA~~ described the blood free fatty acids (FFA)-lowering effect of PDC in man and we found the same in rats fed the normal diet after repeated administration of the drug 19. The blood and tissue FFA are a metabolic key fraction. Therefore, the following study was undertaken to see whether or not, and to what extent, would PDC be able to influence the accumulation and/or release of FFA in fat tissue in relation to its antisclerotic effect. MATERIALAND METHODS White male rats fed on a standard laboratory diet were used and sacrificed nonanaesthetized by decapitation using a small guillotine. Weighed pieces (around500mg) of epididymal fat were incubated under constant slow agitation in a metabolic bath with 5 ml of incubation medium at 37°C for 1 h. Krebs-Ringer phosphate buffer of pH 7.4, which contained no albumitz and which was free of calcium and magnesium ions, was used as incubation medium so that FFA were not released and had to accumulate in fat tissue. Where it was necessary, the buffer was enriched with the given concentration of PDC. Adrenalin (SPOFA, Czechoslovakia, for injection use) was diluted respectively so that 10 ,ul, added just at the beginning of incubation, provided for the terminal concentration as referred to in the text. At the end of the incubation, the pieces of fat tissue were removed, rinsed with 0.9%
solution of
sodium chloride, immersed into a test tube with 5 ml DOLE'S extraction mixturez2, and then crushed by the revolving movements of a glass rod, until they dissolved completely. Then 3 ml of heptane and 3 ml of water were added rinsing the rod at the same time. FFA were determined basically according to DoLE~~. An amount of 1 ml of the indicator prepared from thymol blue dissolved in absolute ethanol and a titration agent prepared dissolving metallic sodium in absolute ethanol, were used for titration. Pure nitrogen was allowed to bubble through the titration mixture, in order to get it stirred and to prevent carbon dioxide from becoming admixed. In one experimental series, both the post-incubational FFA content in fat tissue and the release of FFA into the medium had to be determined. Only in this series were 3 o/o bovine albumin (Armour Comp., fraction V) added to the buffer, and the pH re-adjusted to 7.4. The FFA were measured in the incubation medium at the beginning and the end of incubation which, in this case, was extended to 3 h at 37°C. An amount of 3 ml of the medium was used for incubation. For the experiments in vivo, the same type of white male rats fed on normal diet were used. Half their number received PDC dissolved in drinking water for 3 or 4 days. Each animal thus received approximately 30 mg/kg PDC daily. The other AtheroscZevosis, 1972, 16: 185-191
EFFECT OF PYRIDINOLCARBAMATE
half served as control,
ON RAT ADIPOSE TISSUE
and the animals
received
drinking
FFA
187
water without
PDC apart
from food. In one experiment 100 pug, in the other 20 ,ug of adrenalin were injected subcutaneously to each animal of either group 1 h prior to sacrifice. The FFA were determined
in blood serum by the method
tion and titration
of water solutions
the determination
of FFA.
of TROUT et a1.23. It was found by extrac-
of PDC that
the drug does not interfere
with
RESULTS
FFA
In one of the first experiments in vitro, it became evident that the content of in fat tissue incubated with the addition of adrenalin increased far more if
PDC was also added to the medium at a concentration of 1 mg/ml. The question arose, therefore, as to whether this increase could be the result of the potentiated lipolytic effect of adrenalin, or whether PDC was also capable of accumulating and the resulting value thus equaled the sum total of both effects.
FFA,
In the following experiment, therefore, the influence of PDC concentration was investigated. Tissue specimens from fed and starved (72 h) rats were used; the animals were of an older age, in order to ensure a sufficient
amount
of the same epididymal
fat. Accumulation of FFA increased accordingly with the increase in concentration of PDC (Fig. 1). At a concentration of 1 mg/ml the increase was already statistically significant
both in fed and starved
rats. In another
experiment
with younger
weight 240 g) fasted (24 h) rats a statistically significant increase place already at a lower concentration of 0.5 mg/ml of PDC.
(mean
(~5 < 0.025) took
The effect of adrenalin and PDC was investigated in fed rats (Fig. 2), separately and both together. Adrenalin and PDC, as well as each of them separately, affected accumulation of FFA in fat tissue. When both drugs were added to the medium, the resulting effect was approximately the sum total of the two effects. Figs. 1 and 2 demonstrate that the effect of PDC leading to accumulation of FFA was much weaker
I
pmWg
10-
p
-
1
0.01
0.10
1.00
Fig. 1. Effect of increasing PDC concentrations on the contents of FFA in fat tissue (means &S.D. are given). ?? , rats starved 72 h (weight = 390 g); 0, fed rats (weight = 390 g). Atherosclerosis,
1972, 16: 185-191
D. GRAFNETTER, T. SHIMAMOTO, F. NUMANO
188
Adrenalin PDC
I
-
1 -
Fig. 2. Accuqmlation of FFA in fat tissue under the effect of PDC and adrenalin. Fed rats (weight = 390 g). 100% = 2.13 f 0.45 pmole/g.
than
the strong lipolytic
had to be present
activity
of adrenalin.
To achieve a comparable
in a much higher concentration
This comparison
led to the assumption
effect, PDC
than adrenalin.
that PDC might effect accumulation
of
FFA in vivo and reduction of blood FFA by mechanisms different from those which act upon the lipolytic system of fat tissue. Among the other mechanisms which should be taken into account the possibility exists that PDC, by effecting permeability changes, might reduce the transport of FFA from fat stores. An experiment was performed in which the liberation of FFA into the medium containing albumin on the one hand and the residual content of FFA in fat tissue on the other, were investigated. It can be seen from Table 1 that FFA liberation remained on the control level when 1 mg/ml of PDC was added to the incubation medium, although of FFA was clearly higher inside the fat tissue after incubation. TABLE
1
RELEASE
AND
FAT
TISSUE
CONTENT
OF
FFA
ON
INCUBATION
OF
CARBAMATE
System
Release of FFA into the incubation medium pmole/ml/g
No PDC added
0.278 f
0.043
1 mg/ml PDC added
0.290 f
0.220
Sign@cance
n.s.
Post-incubation content of FFA in fat tissue pmolelg No PDC added
0.84 f
0.34
1 mg/ml PDC added
1.35 f
0.51
Atherosclerosis,
1972, 16: 185-191
P < 0.05
FAT
TISSUE
WITH
the content
PYRIDINOL-
EFFECT OF PYRIDINOLCARBAMATE
TABLE
ON RAT ADIPOSE TISSUE
189
FFA
2
SERUM FFA
AFTER
THE
ADMINISTRATION
Number of animals in brackets.
Experiment
ADRENALIN,AND PDC
OF
For details see MATERIALS
Treatment
FFA
Adrenalin injection (100 pg) 1 h prior to sacrifice
720 f
AND
AND METHODS.
(pmoleslml)
129
ADRENALIN Means
TOGETHER
f SD. are
given.
P
(10) ll.S.
I.
PDC feeding for 5 days + adrenalininjection (100,ug) 1 h prior to sacrifice
709 + 162
(10)
Adrenalin injection (20 pg) 1 h prior to sacrifice
881 *
(10)
141
< 0.025 PDC feeding for 3 days + Adrenalin injection (20 ,ug) 1 h prior to sacrifice
II.
In order to furnish
748 f
more evidence
84
(IO)
on the action
were carried out (Table 2) in which adrenalin
in. viva of PDC, experiments
was applied both to the control animals
and to the rats which had been kept on PDC. The injection sacrifice was used to stimulate
lipolysis
of adrenalin
in fat tissue and to bring about
blood FFA level. With the lower dose (20 pg per rat) of adrenalin,
1 h prior to an elevated
serum FFA level
was significantly lower in animals which were pretreated with PDC. This result, in accordance with the preceding one, supports the possibility that FFA output might be diminished
by PDC.
DISCUSSION
As mentioned an influence ably
also in other
The blood
in the
INTRODUCTION,
on the permeability tissues),
and tissue
FFA
and
PDC has an antisclerotic
and on the enzymes interferes
represent
of the vascular
most likely
an important
secondarily
metabolic
effect, exercises wall (and probwith lipidsi -so.
fraction
which
also
constitutes a link between the lipid and carbohydrate metabolism. This fraction is of great importance to the mechanism of deposition, mobilization and utilization of lipids. Confirmation of the influence of PDC on FFArs*sr might be of importance for explaining the favourable effect which this drug has shown on blood lipids and first of all on vessel wall lipids. It seemed plausible to suspect that PDC might exert an inhibitory action on fat tissue lipolysis as it lowers serum FFArs~sr. However, the above experiments furnished evidence of the capacity of PDC to lead, in higher concentrations in vitro, to accumulation of FFA in fat tissue of rat. This effect was approximately 4000 times weaker than that of adrenalin as results from the comparison of concentrations producing a comparable effect. The term accumulation is used here intentionally because our present experiments have not unequivocally explained whether the Atherosclerosis,
1972, 16: 185-191
190
D. GRAFNETTER, T. SHIMAMOTO,F. NUMANO
increase
of fat tissue FFA,
the activation
as caused by OS-l.0
of lipolysis.
Future
mg/ml
experiments,
of PDC in vitro, was due to
using,
glycerol release, would bring more light into this question. ical conditions negligible
the low concentrations
- if any - influence
in fat tissue.
PDC on serum FFA in vivo should be due to another fat tissue during might
incubation.
and thus diminish not brought favoured
Therefore,
mechanism.
to exercise the effect of
One of our experi-
in the FFA release while FFA accumulated
This result seems to point to the possibility
affect the transport
of FFA from fat stores by causing
the natural
FFA output.
direct proof in this respect.
by our experiment
in which adrenalin
the assumption injections
in the
that PDC
a permeability
change
We are aware that our experiments
However,
of
However, under physiolog-
of PDC irt vivo may be expected
on the lipolysis
ments in vitro showed no increase
e.g., also measurement
have
made seems to be
were used to accentuate
fat tissue lipolysis and to elevate the level of serum FFA in viva. No difference was found when the control and PDC-pretreated rats were injected a high dose of adrenalin. This dose probably bined with a dramatic PDC reached This result under
drastically
a significantly
also points
affected
glucose increase.
fat tissue lipolysis
However,
and was also com-
serum FFA of rats pretreated
lower level when a lower dose of adrenalin
to the possibility
with
was used.
that less FFA could enter the blood stream
the effect of PDC. Since a lower blood level of FFA may lead to a decreased
output of glycerides from the liver, to a drop in their blood level, to a better utilization of fat substances in the tissues, and thus to an impediment of their deposition, the observed
lowering
effect of PDC on blood FFA19121 can be regarded
as a beneficial
one. KRITCHEVSKY AND TEPPER~~ observed a drop of the glyceride level in blood and liver and an increase in cholesterol oxidation in mitochondrial liver fractions after the administration of PDC to rats. With regard to vascular wall, a decreased “pressure” of lipids from the blood stream would constitute a lesser danger of lipid deposition and a higher dependence of the vascular wall on utilization of stores within it. The accelerated disappearance of experimentally produced atheromatosis in rabbits and the drip in aorta1 cholesterol level, as shown in experiments with PDC by SHIMAMOTO and othersIs, could in part be explained in this way. However, the evidence furnished hitherto does not exclude other possible interpretations. For example, some favourable changes of enzyme activities were found in the vascular wall under the action of PDV. Expression of the FFA-lowering effect of PDC will certainly be dependent from many factors in human therapy. Among them the choice of the dose could be of great importance. Dependance of described effects of PDC on the dose has been shown in animal experiments3. As far as the mechanism of the effect of PDC on FFA is concerned, we are aware that further work is needed to bring an unequivocal explanation, particularly with respect to the possible participation of the permeability-affecting properties of the drug.
Atherosclerosis,
1972, 16: 185-191
EFFECT
OF PYRIDIDOLCARBAMATE
ON BAT ADIPOSE
TISSUE
FFA
191
REFERENCES
1 An
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(Eds.), Atherogenesis, Excerpta Medica Foundation, Amsterdam, 1969, p. 119. 6 SHIMAMOTO, T., The relation of atheromatous arterial reaction to atherosclerosis and thrombosis, J. Atheroscler. Res., 1963, 3: 87. 7 SHIMAMOTO, T., The permeability of the arterial wall, Abbotempo, Book 3, 1970, p. 8. s Wu, C. C., T. S. HUANC AND C. J. Hsu, Prevention of experimental atherosclerosis with pyriAmer. Heart J., 1969, 77: 657. dinolcarbamate, 9 DEOLIVEIRA, J. M., The effect of pyridinolcarbamate on experimental atherosclerosis, in T. SHIMAMOTO AND F. NUMANO (Eds.), Atherogenesis, Excerpta Medica Foundation, Amsterdam, 1969, p. 53. 12 NODA, K., Studies on the treatment and prophylaxis of arteriosclerosis. Effect of pyridinolcarbamate on the development of experimental atherosclerosis, Bull. Inst. Constitutional Med., Kumato Univ., 1966, 17: 136. 11 PICK R The effect of pyridinolcarbamate on the induction and regression of aortic and coronary ‘athkrosclerosis in cholesterol-fed cockerels, in T. SHIMAMOTO AND F. NUMANO (Eds.), Atherogenesis, Excerpta Medica Foundation, Amsterdam, 1969, p. 57. 12 NUMANO, F., The prevention of atherosclerosis by pyridinolcarbamate in cholesterol-fed rabbits, Jap. J. Med., 1966, 5: 307. 13 SHIMAMOTO, T. AND T. ATSUMI, Pyridinolcarbamate; bradykinin antagonist in patients suffering from arteriosclerosis obliterans and thromboangiitis obliterans (Burger’s disease), Jap. Heart J., 1965, 6: 407. 14 MOTOMIYA, T., T. ATSUMI, N. ISOKANE, S. YAMASHITA, T. SANO, T. ISHIOKA AND T. SHIMAMOTO, Pyridinolcarbamate treatment of atherosclerosis obliterans analysed by toe plethysmography, Jap. Heart J., 1970, 11: 433. 15 SHIMAMOTO, T., H. MAEZAWA, H. YAMAZAKI, T. ATSUMI, T. FUJITA, T. ISHIOKA AND T. SUNAGA, Pyridinolcarbamate, a bradykinin antagonist in veins. A preliminary report on pharmacological and clinical observations, Amer. Heart J., 1966, 71: 297. 16 SHIMAMOTO T., An antiatherosclerotic, pyridinolcarbamate a microcirculatory aspect of atherogenesis and thrombogenesis, Asian Med. J., 1967, 10: 425. 17 SHIMAMOTO, T., Le traitement de l’atherosclerose par le pvridinolcarbamate. Revue de l’historique de l’introduction du pyridinolcarbamate et des resuitats cliniques, Arch. Maladies Coeur (Revue de I’Athdroscldrose), Suppl. 3, 1968, 61: 67. 18 SHIMAMOTO, T., in T. SHIMAMOTO AND F. NUMANO Medica (Eds.), Atherogenesis, Excerpta Foundation, Amsterdam, 1969. 19 GRAFNETTER, D., T. SHIMAMOTO AND F. NUMANO, Metabolic effect of pyridinolcarbamate on rats kept on a high-fat and cholesterol diet or treated with calciferol, Cor et Vasa, in the press. 20 MRHOVA, O., T. SHIMAMOTO AND F. NUMANO, The metabolic effect of pyridinolcarbamate in rats, Acta Path. Jap., 1972, 22(2): 353. 21 GOTO, Y., AND H. NAKAMURA, Effect of pyridinolcarbamate on the plasma lipids and glucose in viva, in T. SHIMAMOTO AND F. NUMANO (Eds.), Atherogenesis, Excerpta Medica Foundation, Amsterdam, 1969, p. 119. 22 DOLE, V. P., A relation between non-esterified fatty acids in plasma and the metabolism of glucose, J. Clin. Invest., 1956, 35: 150. 22 TROUT, D. L., E. H. ESTES, JR. AND S. J. FRIEDBERG, Titration of free fatty acids in plasma: a study of current methods and a new modification, J. Lipid Res., 1960, 1: 199. 24 KRITCHEVSKY, D. AND S. A. TEPPER, Influence of pyridinolcarbamate on oxidation of cholesterol by ra+ liver mitochondria, Arzneimittelforschung (Drug Research), 1971, 21: (No. 1) 146.
Atherosclerosis,
1972, 16: 185-191