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Effect of glycerol on triglyceride metabolism in the rat
Metabolism Clinical and Experimental FEBRUARY 1979
VOL. XXVIII, NO. 2
PRELIMINARY
REPORT:
Effect of Glycerol Ryuzo
Abe,
on Triglyceride
Ian Macdonald,
Yoshisuke
Lipid metabolic studies were carried out on the male Wistar rats fed on glycerol-rich diet in order to elucidate the mechanism of glycerol-induced hypertriglyceridemia. No difference was found between the glycerol fed rats and the control rats in the rate of triglyceride secretion from the liver measured by the Triton WR-1339 method as well as in the rate of incorporation of labeled glycerol into liver triglyceride. The facts that the half-life of the intravenously injected Intralipid” in the blood was significantly delayed in the glycerol fed rats and that the lipoprotein lipase activity released from epididymal adipose tissue of the glycerol fed rats was markedly decreased to 19% of that of the control rats seem to account for the serum triglyceride elevation induced by the glycerol feeding.
Metabolism Maruhama,
Merabolrsm, Vol.
28. No. 2
(February), 1979
Goto
MATERIALS
AND
METHODS
Male rats of the Wistar strain, aged 6 wk and weighing 152 2 I.3 g at the start, were used in the present experiments. The rats were given with the normal
laboratory
(carbohydrate,
20.0%; vitamins,
minerals,
45.6%:
unsaturated
enriched
with
fats, 2.6%
protein,
fatty
tiber.
laboratory
group, The diet enriched diet and the control food intake f
group
different.
There
IS)
for 7
diet.
3.8 Kcal/g
The amounts
in 15 rats of the control i_ SE)
were not significantly
was no significant
and the glycerol
group
of
group
and in 15 rats of the
3 g/rat/day)
difference
gain between the rats of the control n =
contained
3.9 Kcal/g
mean f
31.87~)
ad libitum
chow was given to the control
recorded
(30
chow
and water,
(by calorie)
with glycerol
diet
3 g/rat/day;
glycerol
acids,
20% of glycerol
days. The normal
(27
I
and Yoshio
triglyceride secretion rate from the liver as well as serum triglyceride removal in animals fed on a glycerol-rich diet.
daily
T HAS BEEN REPORTED that serum triglyceride concentration is increased significantly by the administration of glycerol to man’.’ and animals.3 However, the mechanism of the glycerol-induced hypertriglyceridemia has not been elucidated. In animals, Narayan et aL3 suggested that the ingestion of glycerol might stimulate hepatic triglyceride synthesis, while Petit et al.’ indicated that, in a perfusion system, the production of very low density lipoprotein derived from hepatic triglyceride was decreased by glycerol. Thus, it remains unclear whether or not the overproduction of hepatic triglyceride could contribute the serum triglyceride elevation caused by the glycerol administration. The turnover rate of serum triglyceride that is one of the important determinants of serum triglyceride level has not been studied either in animals or in man after glycerol administration. These experiments, therefore, were carried out in order to see whether there is any change in
in the Rat
in the weight
group (40 i- 3 g/rat/wk, (36
t
4 g/rat/wk.
n =
15). After
7 days, a specimen
serum
triglyceride
fasted rats under sodium were,
then,
WR-1339.
injected Blood
determination
of blood
was obtained
pentobarbital
intravenously samples
were
secretion
the
School
obtained
oJ‘ Inrrrnal
Medicine,
Sendai,
Physiology. England
Medicine,
Sendai.
publication
.4ddress requests ment
for
Guy’s and
Tohoku
and
admm-
Hospital the
Third
University
Japan. April
6. 1978.
to R_vu:o ,4br. M.D., Medicine, Miyagi
trig!yceride injection
of intravenously
S.E.I.
Internal
of Medicine,
Received for
of
London,
of
The rats
120 mg of Triton
rate calculated.’
Department
School.
Department
anesthesia.
with
In the second series, the half-life
From
of
the tail vein of S-hr
50 and 100 min after the Triton
the triglyceride
Medical
for measurements
from
Tohoku
the Third
University
Depart-
School
oJ
980. Japan.
0 I979 by Grune & Stratton,
Inc.
0026-049S/79/2802-0001$01.00/0
97
ABE ET AL.
98
istered
1.75 ml/kg
nyralen
vitrum,
fasted control
turnover. from
anesthesia
the
Intralipid”
A
blood vein
specimen
under
mined
a
by
sodium
Furnishing
LTD.
England) plot,
The
Intralipid@
Intralipid@ using
for
nephelometric
is hardly
influenced
rate of serum Intralipidm In the third
the
activity
Krebs-Ringer
heparin
(20
U/ml),
fat pad
saline, buffer
released in the incubation of
glyceryl-tri
Huttunen
(l-‘%J)
as detergent.
emulsion
incubated
at 2PC
a total (pH
60 min
value
by subtracting
sample free glycerol, overestimated Kreutz“‘and
of
was measured
free fatty comparison
emulsion
sample
corrected
triglyceride of glycerol
were
lipase activity
Serum triglyceride
since serum triglyceride
in the presence
Serum glycerol
in a lipase by
and 20 ~1 of the medium
was
for true
equivalent is known by this
of to be
method.
by the method of Eggstein
and
acids by the method of ltaya and Ui.” was made according
to Student’s
t
test. RESULTS
AND
Fig. 1. (A) Serum triglyceride, (6) triglyceride secretion rate from the liver, (C) half-life of Intralipid” in blood and (D) lipoprotein lipase activity released from epididymal adipose tissue. Mean values + SE are shown between the control rats (shaded bars) and the glycerol fed rats (open bars).
the
for 60 min. The lipoprotein
5
7.4)
Gum arabic was used
by the method of Fletcher’and
triglyceride
of
lipase assay, 500 ,ul of the
was expressed as nmole FFA/g/min. determined
and
was measured
using
oleate as substrate.
In the lipoprotein
substrate
medium
et al.*
15
0
and then cut into
for
n=lO
released
The epididymal
at 37°C
n=lO
f0 10
turnover
rats of both control
bicarbonate
0
.E: E 5
triglyceride.’
in physiologic
n:lO
with that
shaker with air as gas phase. The lipoprotein
method
Statistical
serum
500 mg of the fat pad pieces were incubated
of
containing
washing
of
correlated
groups under light ether anesthesia
pieces. After
approximately
the
the 5-hr fasted
n=lO
be
serum triglyc-
lipase activity
from adipose tissue was investigated. from
line, on
the fractional
lipoprotein
0
Scientific
k2 could
determination that
the
as standard.
slope
by endogenous
series, the lipoprotein
was removed
236,
gave a straight
is significantly
of serum very low density
metabolic
was
pentobarbital
Intralipidm
which
eride.b It has been also shown
3 ml
serum ml
levels were deter-
(Type
of serum Intralipid@
a semilogarithmic calculated.
Serum
micro-nephelometer
The elimination
in
of 0.3
before and 3, 6, IO, 15, 20, and 30 min after injection.
small
in the 5-hr
groups in order to estimate
tail
Intralipid@
glycerol
(Apolesksvaruce-
in blood was determined
and glycerol
triglyceride obtained
body weight
Sweden)
DISCUSSION
Serum glycerol levels in the rats of the control and glycerol groups, after a 5-hr fast, were 2.20 t- 0.20 mg/lOO ml and 3.49 f 0.45 mg/lOO ml, respectively; the latter value being significantly higher than the former (p < 0.005). Serum free fatty acid level in the control rats was 0.87 2 0.11 mEq/l and that in the glycerol fed rats 1.13 + 0.13 mEq/l; the difference being not statistically significant. As shown in the left upper panel of Fig. 1, serum triglyceride concentration in the glycerol fed group (63 f 5 mg/lOO ml) was significantly higher (p < 0.005) than in the control group (33 k 5 mg/lOO ml). No significant difference was found in the triglyceride secretion rate between
the glycerol fed group (0.130 f 0.009 mg/min) and the control group (0.148 f 0.012 mg/min) as shown in the right upper panel. The incorporation of orally administered “C-U-glycerol into the liver triglyceride was 55 k 6 (X 1000) cpm/whole liver in the glycerol group (n = 8) and 46 + 5 (X 1000) cpm/whole liver in the control group (n = 8); no significant difference was noted between the above values. On the other hand, as shown in the left lower panel, the half-life of the intravenously injected Intralipidm in the blood was significantly delayed in the glycerol group (10.8 + 0.7 min, p < 0.02) as compared to that in the control group (8.4 k 0.5 min). As shown in the right lower panel, the lipoprotein lipase activity released from epididyma1 adipose tissue of the glycerol fed rats was 11.6 + 1.6 nmole FFA/g/min, whereas that in the control rats was 62.0 ? 15.2 nmole FFA/g/min. There is a highly significant difference in the lipoprotein lipase activity between the two groups (p < 0.005). Thus, the elevation of serum triglyceride observed in the glycerol fed rats does not seem to be attributed to the accelerated triglyceride secretion from the liver, but to the blunted lipoprotein lipase activity in adipose tissue, which accounts for the delay in serum triglyceride turnover suggested in the Intralipidm experiment.
GLYCEROL
EFFECT
ON
TRIGLYCERIDE
99
METABOLISM
REFERENCES I. Nikkila
EA. Pelkonen
hypertriglyccridemia Sot Exp Biol Med 2. Macdonald glyceride
R: Enhancement
by fructose 123:91-94,
I: Effect
intravenous
of alimentary in man. Proc
Stand
glycerol
on the serum
between
Br J Nutr
24:537-543,
and glycerol
fat
J Clin
tolerance
level of men and women.
fractional
triglyceride
and of
3. Narayan
KA.
glycerol-induced Reports
Mcmullen.
fat
accumulation
International
4. Petit
l2:2l
D, Raisonnier
glycerol
on VLDL
Biophys
Acta
secretion
transport:
rate.
lipase
lipoprotein
6. <.arlson
25:533-542, IA.
DP,
in rat
et al: Dietary
livers.
Nutrition
1975
Bound
ME,
et al: Etfect
of
by the isolated rat liver. Biochim I976
JD. Yee E. Albers
and triglyceride Metabolism
I-219, A.
43 I :48 I --492,
5. Bagdade
Butter
Erect
and plasma
8. Huttunen plasma
on triglyceride lipoprotein
secretion in the rat.
Rossncr S: A methodological
study of an
et al: Comparison endogenous
(Intravenous
Invest 4:109-l
lipase
and
fat
14, 1974
hepatic
lipase
tion
various
to sex,
metabolism.
age and
Clin Sci Mol
9. Fletcher
MJ:
IO. Eggstein Neutralfette 441262-267, fatty
parameters
Clin
Chim
M, Kreutz
im Blutserum
of
Mcd 50:249-260.
A calorimetric FH:
in
normal Correla-
triglyceride
1976
method
Acta
plasma tolerance
M, et al: Post heparin
with hypcrtriglyceridemia:
11. Iraya
1976
LA, of
C. Kekki
emulsion.
1972
subjects and in patients
serum triglycerides. J, et al: Glucocorticoids
rate
lntralipida
JK, Ehnholm
lipoprotein
IntralipidR
J, Carlson
turnover
test) in man. Eur J Clin
I970
with
29: 271.-280.
7. Rossncr S, Boberg
1966
of dietary
test
Lab Invest
for
estimating
22:393-397,
1968
Eine neue Bestimmung
und Gewebe.
Klin
der
Wochenschr
I966 K,
Ui
M: Calorimetric
acids in biological
fluids.
J Lipid
determination Rcs 6:16-20.
of free 1965
VOL. XXVIII, NO. 2
PRELIMINARY
REPORT:
Effect of Glycerol Ryuzo
Abe,
on Triglyceride
Ian Macdonald,
Yoshisuke
Lipid metabolic studies were carried out on the male Wistar rats fed on glycerol-rich diet in order to elucidate the mechanism of glycerol-induced hypertriglyceridemia. No difference was found between the glycerol fed rats and the control rats in the rate of triglyceride secretion from the liver measured by the Triton WR-1339 method as well as in the rate of incorporation of labeled glycerol into liver triglyceride. The facts that the half-life of the intravenously injected Intralipid” in the blood was significantly delayed in the glycerol fed rats and that the lipoprotein lipase activity released from epididymal adipose tissue of the glycerol fed rats was markedly decreased to 19% of that of the control rats seem to account for the serum triglyceride elevation induced by the glycerol feeding.
Metabolism Maruhama,
Merabolrsm, Vol.
28. No. 2
(February), 1979
Goto
MATERIALS
AND
METHODS
Male rats of the Wistar strain, aged 6 wk and weighing 152 2 I.3 g at the start, were used in the present experiments. The rats were given with the normal
laboratory
(carbohydrate,
20.0%; vitamins,
minerals,
45.6%:
unsaturated
enriched
with
fats, 2.6%
protein,
fatty
tiber.
laboratory
group, The diet enriched diet and the control food intake f
group
different.
There
IS)
for 7
diet.
3.8 Kcal/g
The amounts
in 15 rats of the control i_ SE)
were not significantly
was no significant
and the glycerol
group
of
group
and in 15 rats of the
3 g/rat/day)
difference
gain between the rats of the control n =
contained
3.9 Kcal/g
mean f
31.87~)
ad libitum
chow was given to the control
recorded
(30
chow
and water,
(by calorie)
with glycerol
diet
3 g/rat/day;
glycerol
acids,
20% of glycerol
days. The normal
(27
I
and Yoshio
triglyceride secretion rate from the liver as well as serum triglyceride removal in animals fed on a glycerol-rich diet.
daily
T HAS BEEN REPORTED that serum triglyceride concentration is increased significantly by the administration of glycerol to man’.’ and animals.3 However, the mechanism of the glycerol-induced hypertriglyceridemia has not been elucidated. In animals, Narayan et aL3 suggested that the ingestion of glycerol might stimulate hepatic triglyceride synthesis, while Petit et al.’ indicated that, in a perfusion system, the production of very low density lipoprotein derived from hepatic triglyceride was decreased by glycerol. Thus, it remains unclear whether or not the overproduction of hepatic triglyceride could contribute the serum triglyceride elevation caused by the glycerol administration. The turnover rate of serum triglyceride that is one of the important determinants of serum triglyceride level has not been studied either in animals or in man after glycerol administration. These experiments, therefore, were carried out in order to see whether there is any change in
in the Rat
in the weight
group (40 i- 3 g/rat/wk, (36
t
4 g/rat/wk.
n =
15). After
7 days, a specimen
serum
triglyceride
fasted rats under sodium were,
then,
WR-1339.
injected Blood
determination
of blood
was obtained
pentobarbital
intravenously samples
were
secretion
the
School
obtained
oJ‘ Inrrrnal
Medicine,
Sendai,
Physiology. England
Medicine,
Sendai.
publication
.4ddress requests ment
for
Guy’s and
Tohoku
and
admm-
Hospital the
Third
University
Japan. April
6. 1978.
to R_vu:o ,4br. M.D., Medicine, Miyagi
trig!yceride injection
of intravenously
S.E.I.
Internal
of Medicine,
Received for
of
London,
of
The rats
120 mg of Triton
rate calculated.’
Department
School.
Department
anesthesia.
with
In the second series, the half-life
From
of
the tail vein of S-hr
50 and 100 min after the Triton
the triglyceride
Medical
for measurements
from
Tohoku
the Third
University
Depart-
School
oJ
980. Japan.
0 I979 by Grune & Stratton,
Inc.
0026-049S/79/2802-0001$01.00/0
97
ABE ET AL.
98
istered
1.75 ml/kg
nyralen
vitrum,
fasted control
turnover. from
anesthesia
the
Intralipid”
A
blood vein
specimen
under
mined
a
by
sodium
Furnishing
LTD.
England) plot,
The
Intralipid@
Intralipid@ using
for
nephelometric
is hardly
influenced
rate of serum Intralipidm In the third
the
activity
Krebs-Ringer
heparin
(20
U/ml),
fat pad
saline, buffer
released in the incubation of
glyceryl-tri
Huttunen
(l-‘%J)
as detergent.
emulsion
incubated
at 2PC
a total (pH
60 min
value
by subtracting
sample free glycerol, overestimated Kreutz“‘and
of
was measured
free fatty comparison
emulsion
sample
corrected
triglyceride of glycerol
were
lipase activity
Serum triglyceride
since serum triglyceride
in the presence
Serum glycerol
in a lipase by
and 20 ~1 of the medium
was
for true
equivalent is known by this
of to be
method.
by the method of Eggstein
and
acids by the method of ltaya and Ui.” was made according
to Student’s
t
test. RESULTS
AND
Fig. 1. (A) Serum triglyceride, (6) triglyceride secretion rate from the liver, (C) half-life of Intralipid” in blood and (D) lipoprotein lipase activity released from epididymal adipose tissue. Mean values + SE are shown between the control rats (shaded bars) and the glycerol fed rats (open bars).
the
for 60 min. The lipoprotein
5
7.4)
Gum arabic was used
by the method of Fletcher’and
triglyceride
of
lipase assay, 500 ,ul of the
was expressed as nmole FFA/g/min. determined
and
was measured
using
oleate as substrate.
In the lipoprotein
substrate
medium
et al.*
15
0
and then cut into
for
n=lO
released
The epididymal
at 37°C
n=lO
f0 10
turnover
rats of both control
bicarbonate
0
.E: E 5
triglyceride.’
in physiologic
n:lO
with that
shaker with air as gas phase. The lipoprotein
method
Statistical
serum
500 mg of the fat pad pieces were incubated
of
containing
washing
of
correlated
groups under light ether anesthesia
pieces. After
approximately
the
the 5-hr fasted
n=lO
be
serum triglyc-
lipase activity
from adipose tissue was investigated. from
line, on
the fractional
lipoprotein
0
Scientific
k2 could
determination that
the
as standard.
slope
by endogenous
series, the lipoprotein
was removed
236,
gave a straight
is significantly
of serum very low density
metabolic
was
pentobarbital
Intralipidm
which
eride.b It has been also shown
3 ml
serum ml
levels were deter-
(Type
of serum Intralipid@
a semilogarithmic calculated.
Serum
micro-nephelometer
The elimination
in
of 0.3
before and 3, 6, IO, 15, 20, and 30 min after injection.
small
in the 5-hr
groups in order to estimate
tail
Intralipid@
glycerol
(Apolesksvaruce-
in blood was determined
and glycerol
triglyceride obtained
body weight
Sweden)
DISCUSSION
Serum glycerol levels in the rats of the control and glycerol groups, after a 5-hr fast, were 2.20 t- 0.20 mg/lOO ml and 3.49 f 0.45 mg/lOO ml, respectively; the latter value being significantly higher than the former (p < 0.005). Serum free fatty acid level in the control rats was 0.87 2 0.11 mEq/l and that in the glycerol fed rats 1.13 + 0.13 mEq/l; the difference being not statistically significant. As shown in the left upper panel of Fig. 1, serum triglyceride concentration in the glycerol fed group (63 f 5 mg/lOO ml) was significantly higher (p < 0.005) than in the control group (33 k 5 mg/lOO ml). No significant difference was found in the triglyceride secretion rate between
the glycerol fed group (0.130 f 0.009 mg/min) and the control group (0.148 f 0.012 mg/min) as shown in the right upper panel. The incorporation of orally administered “C-U-glycerol into the liver triglyceride was 55 k 6 (X 1000) cpm/whole liver in the glycerol group (n = 8) and 46 + 5 (X 1000) cpm/whole liver in the control group (n = 8); no significant difference was noted between the above values. On the other hand, as shown in the left lower panel, the half-life of the intravenously injected Intralipidm in the blood was significantly delayed in the glycerol group (10.8 + 0.7 min, p < 0.02) as compared to that in the control group (8.4 k 0.5 min). As shown in the right lower panel, the lipoprotein lipase activity released from epididyma1 adipose tissue of the glycerol fed rats was 11.6 + 1.6 nmole FFA/g/min, whereas that in the control rats was 62.0 ? 15.2 nmole FFA/g/min. There is a highly significant difference in the lipoprotein lipase activity between the two groups (p < 0.005). Thus, the elevation of serum triglyceride observed in the glycerol fed rats does not seem to be attributed to the accelerated triglyceride secretion from the liver, but to the blunted lipoprotein lipase activity in adipose tissue, which accounts for the delay in serum triglyceride turnover suggested in the Intralipidm experiment.
GLYCEROL
EFFECT
ON
TRIGLYCERIDE
99
METABOLISM
REFERENCES I. Nikkila
EA. Pelkonen
hypertriglyccridemia Sot Exp Biol Med 2. Macdonald glyceride
R: Enhancement
by fructose 123:91-94,
I: Effect
intravenous
of alimentary in man. Proc
Stand
glycerol
on the serum
between
Br J Nutr
24:537-543,
and glycerol
fat
J Clin
tolerance
level of men and women.
fractional
triglyceride
and of
3. Narayan
KA.
glycerol-induced Reports
Mcmullen.
fat
accumulation
International
4. Petit
l2:2l
D, Raisonnier
glycerol
on VLDL
Biophys
Acta
secretion
transport:
rate.
lipase
lipoprotein
6. <.arlson
25:533-542, IA.
DP,
in rat
et al: Dietary
livers.
Nutrition
1975
Bound
ME,
et al: Etfect
of
by the isolated rat liver. Biochim I976
JD. Yee E. Albers
and triglyceride Metabolism
I-219, A.
43 I :48 I --492,
5. Bagdade
Butter
Erect
and plasma
8. Huttunen plasma
on triglyceride lipoprotein
secretion in the rat.
Rossncr S: A methodological
study of an
et al: Comparison endogenous
(Intravenous
Invest 4:109-l
lipase
and
fat
14, 1974
hepatic
lipase
tion
various
to sex,
metabolism.
age and
Clin Sci Mol
9. Fletcher
MJ:
IO. Eggstein Neutralfette 441262-267, fatty
parameters
Clin
Chim
M, Kreutz
im Blutserum
of
Mcd 50:249-260.
A calorimetric FH:
in
normal Correla-
triglyceride
1976
method
Acta
plasma tolerance
M, et al: Post heparin
with hypcrtriglyceridemia:
11. Iraya
1976
LA, of
C. Kekki
emulsion.
1972
subjects and in patients
serum triglycerides. J, et al: Glucocorticoids
rate
lntralipida
JK, Ehnholm
lipoprotein
IntralipidR
J, Carlson
turnover
test) in man. Eur J Clin
I970
with
29: 271.-280.
7. Rossncr S, Boberg
1966
of dietary
test
Lab Invest
for
estimating
22:393-397,
1968
Eine neue Bestimmung
und Gewebe.
Klin
der
Wochenschr
I966 K,
Ui
M: Calorimetric
acids in biological
fluids.
J Lipid
determination Rcs 6:16-20.
of free 1965