Vol. 188, No. 2, 1992 October 30, 1992
BIOCHEMICALAND
BIOPHYSICALRESEARCH
COMMUNICATIONS Pages 807-812
INSULIN AND LIPOGENESIS IN RAT ADIPOCYTES. I. EFFECT OF INSULIN INCUBATION ON LIPID SYNTHESISBY ISOLATED RAT ADIPOCYTES
Luz Marina
Morales:
Gilbert0
Campos,
and Angel
Elena
Ryder
Casanova
Facultad de Ciencias, Instituto de Investigaciones Clinicas, Facultad de Medicina and Facultad de Agronomia, Universidad de1 Zulia, Maracaibo, Venezuela
Received September 11, 1992 To assessthe effect of insulin on lipid synthesis in isolated rat adipocytes, cells were preincubated for 3 h w' h high concentrations (16.6 nM) of the hormone 18 C-acetate and lipogenesis measured through incorporation into lipids, analyzing at the same time the activity of some lipogenic enzymes. It was found that insulin induced not only a decrease in the number of insulin receptors but a 30% loss in basal and insulin-stimulated acetate incorporation intototal lipids as well as a decrease in the activities of enzymes related to the novo fatty acid synthesis pathway as malic enzyme and glucose-6-phosphate dehydrogenase. a 1992 Academic Press, Inc.
Hyperinsulinemic, tes
mellitus
tion
(l-4).
tance
to
are
insulin-resistant characterized
In vitro, the
and post-receptor One of the the
stimulation
reduced cently
sinsulinemic
of
lipid
adipocytes. for
of
found into
that total
Similarly, a period
*To whom correspondence Facultad de Medicina,
of
exposure
not of
obese
have
reported
from
this
Kobayashi
after
15 days
triglycerides et showed
of
treatment
and fatty al
in
greater
acid
glucose
should be addressed at Instituto Universidad de1 Zulia. Apartado
of
receptor
view,is
that
lipogenesis
mice
(6).
However,
is
associated
(7) with were
1984 (8) reported
resisat
point
hyperinsulinemia
and Olefsky
ac-
cellular
alterations
hyperglycemic that
Thus,
8 days,
explored
in insulin
progressive
sequential
II diabe-
and Type defects
induces
with
fully
tissue
Trimble
as obesity
In 1962 it was reported
synthesis. (7-10)
such
and post-receptor
insulin,
insulin,
adipose synthesis.
rats
se incorporation insulin
actions
authors
anincreasedlipid
rat
(5).
in epididymal several
insulin
actions
site
of
by receptor
prolonged
biological
states
was rewith
in experimentally insulin,
rates
increased that
incorporation
hyper-
in
rats, into
de Investigaciones 1151, Maracaibo,
of
gluco-
isolated
infused total
with li-
ClFnicas, Venezuela.
0006-291X/92 $4.00 807
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188, No. 2, 1992
pids.
Moreover,
linism
itself
pose sis
other with
lipid
hand, insulin
found
that
in rats
synthesis
Haysted for
AND BIOPHYSICAL
treated
in vivo,
and Hardie
15 min
showed
RESEARCH COMMUNICATIONS
with
with
in
fat
1986
(9)
a 15-fold
insulin,
hyperinsu-
accumulation found
that
stimulation
in adiisolated
of lipogene-
3H-glucose. is
by several
authors
the
effect
the
acetate of
(10)
total
The advantages mented
vior
On the treated
from
et al
increased
tissue.
adipocytes
Cusin
BIOCHEMICAL
of
insulin
using
EXPERIMENTAL
(11,12,13,14),
on lipid
incorporation
some lipogenic
14C-acetate
into enzymes
synthesis total involved
lipids,
for
measuring
for
that
of
isolated
lipid
reason, rat
determining
in the
novo
fatty
synthesis it
was interesting
adipocytes, at
has been docu-
the
acid
to
measured
same time,
the
study
through beha-
synthesis.
PROCEDURE
Receptor grade m noiodinated porcine A-14 (lz51)-insulin, specific activity e 2200 mCi/mmol and l-l1 C-acetate, 57.7 mCi/rmnol were purchased fron New England Nuclear Research Products. Boston, MA. Cristalline porcine insulin was from Eli Lilly and Co. Collagenase was from Worthington Biochemical Corp., Freehold, NJ and bovine serum albumin (BSA) fraction V and all reagents used for enzyme assays, were supplied by Sigma Chemical Co., St. Louis, MO. Preparations of isolated adipocytes. After a 12-h fast, male Sprague Dawley rats, weighing 150-250 g were killed by decapitation and epididymal fat pads removed and weighed. Isolated fat cells were prepared by shaking at 37°C for 60 min in Krebs-Ringer-bicarbonate (KRB) buffer containing 7 mg/g collagenase and 10 mg/ml albumin, according to the method of Rodbell (15). Cells were filtered through nylon mesh (250 uM), centrifuged at 400 rpm for 15 set and washed three times in a collagenase-free buffer. The adipocytes were finally resuspended in the original volume with the same buffer. Insulin pretreatment and dissociation procedures. Adipocytes suspended in pH 7.4 KRB buffer containing 11 mM dextrose and 1% BSA were incubated with 16.6 nM insulin in 25 ml sterile polypropylene containers. Cells were then gently agitated in a shaking water bath for 3 h at 37°C. At the end of the incubation period, cells were transferred to sterile polypropylene tubes, centrifuged at 2000 rpm for 15 set and washed with insulin-free KRB buffer pH 7.0. Adipocytes were washed again two times, resuspended in the same buffer, transferred to sterile polypropylene containers and receptor-bound insulin allowed to dissociate at pH 7.0 for 1 h at 37'C. After the l-h dissociation period, cells were washed and resuspended in KRB buffer pH 7.4. Control cells without insulin added underwent similar washing, dissociation and centrifugation procedures as insulin-treated cells. Insulin
binding studies. Isolated adipocytes were incubated in a total volume of 1 'nger-phosphate (KRP) buffer pH 7.8 with trace amounts (0.16 nM; 0.76 uCi) I-insulin, in the absence and presence of an excess of unlabelled insulin (833 nM). Incubations were performed for 2 h at 16°C as in Marshall and Olefsky (16) and adipocytes associated radioactivity determined in triplicate from each incubation tube. All data were corrected for non-specific binding and expressed as percentaje of binding per mg of cell DNA. Acetate incorporation studies. A 0.5 ml aliquot of control and insulin-treated adiocytes were added to 0 5 ml of KRB with glucose (final concentration 5 mM) and 2 uCi P4C-acetate. Insulin wa; added to cell samples at concentrations of 0.0, 0.03, 0.08 and 4 nM. All determinations were made in duplicate. Following 1 h incubation at 37oC, the reaction was terminated by acidification with sulfuric and 14C-incorporation into lipids was determined by extraction with chloroform-methanol 2:l. Enzyme activities assays. Control and 3-hour-insulin-treated adipocytes in KRB buffer pH 7.4 were homogenized in a Polytron for 15 set, centrifuged at 20.000 g for 2 h and
$ :$$!t
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the cytosolic fraction used for the enzyme assays following the procedure of Ochoa (17) for malic enzyme, of de Chatelet et al (18) for glucose-6-phosphate dehydrogenase and of Srere (19) for citrate lyase. DNA determination. Adipocyte DNA content was determined as in Burton (20). Statistical analysis. Data are presented as means significance by the SAS-82 Computer Program, using Least Square Means.
* S.E. and analyzed for statistical the General Model Procedure and the
RESULTS The data Fig.
insulin
1, where
be seen, bind
of
binding
the
insulin
for
mean binding
the
control
parameters
preincubation
led
and treated from
adipocytes
10 preparations
to a 46.9%
decrease
are are
in the
illustrated
shown.
ability
As it
in can
of adipocytes
to
insulin. The effect
lipid
synthesis
basal
lipid
on l4 C-acetate is
revealed
synthesis.
stimulation
of
synthesis
In treated
cells
only
33% at
highest
the
this
in the
treated
Concomitantly,
the
synthesis
pathway
mcmmo~
in Fig.
Increasing
lipid
lin.
synthesis
incorporation
were
2.
insulin
levels
in the
treated
EZZITREATED 0
Fig. 2. cytes. absence 14C-acetate Results
a significant
by one half,
cells
relevant
to
(Fig.
3).
treatmenF(16.6 nM) expressed as the mean experiments (p -= 0.01).
on insulin percentage
In the
it
reached the
fatty
of malic
-TREATED
binding of of binding
isolated rat per mg of cell
Dose response of insulin stimulated lipid synthesis by isolated rat Control (0) or 16.6 nM insulin treated (*) adipocytes were incubated (basal) or presence of increasing concentrations of insulin 0.03 to incorporation into total lipids are expressed as DPM/mg of cell are mean f SE of 10 separate experiments (p< 0.0001).
809
4 nM insu-
de novo case
in
(p
BIOCHEMICALAND
BIOPHYSICALRESEARCH
COMMUNICATIONS Pages 807-812
INSULIN AND LIPOGENESIS IN RAT ADIPOCYTES. I. EFFECT OF INSULIN INCUBATION ON LIPID SYNTHESISBY ISOLATED RAT ADIPOCYTES
Luz Marina
Morales:
Gilbert0
Campos,
and Angel
Elena
Ryder
Casanova
Facultad de Ciencias, Instituto de Investigaciones Clinicas, Facultad de Medicina and Facultad de Agronomia, Universidad de1 Zulia, Maracaibo, Venezuela
Received September 11, 1992 To assessthe effect of insulin on lipid synthesis in isolated rat adipocytes, cells were preincubated for 3 h w' h high concentrations (16.6 nM) of the hormone 18 C-acetate and lipogenesis measured through incorporation into lipids, analyzing at the same time the activity of some lipogenic enzymes. It was found that insulin induced not only a decrease in the number of insulin receptors but a 30% loss in basal and insulin-stimulated acetate incorporation intototal lipids as well as a decrease in the activities of enzymes related to the novo fatty acid synthesis pathway as malic enzyme and glucose-6-phosphate dehydrogenase. a 1992 Academic Press, Inc.
Hyperinsulinemic, tes
mellitus
tion
(l-4).
tance
to
are
insulin-resistant characterized
In vitro, the
and post-receptor One of the the
stimulation
reduced cently
sinsulinemic
of
lipid
adipocytes. for
of
found into
that total
Similarly, a period
*To whom correspondence Facultad de Medicina,
of
exposure
not of
obese
have
reported
from
this
Kobayashi
after
15 days
triglycerides et showed
of
treatment
and fatty al
in
greater
acid
glucose
should be addressed at Instituto Universidad de1 Zulia. Apartado
of
receptor
view,is
that
lipogenesis
mice
(6).
However,
is
associated
(7) with were
1984 (8) reported
resisat
point
hyperinsulinemia
and Olefsky
ac-
cellular
alterations
hyperglycemic that
Thus,
8 days,
explored
in insulin
progressive
sequential
II diabe-
and Type defects
induces
with
fully
tissue
Trimble
as obesity
In 1962 it was reported
synthesis. (7-10)
such
and post-receptor
insulin,
insulin,
adipose synthesis.
rats
se incorporation insulin
actions
authors
anincreasedlipid
rat
(5).
in epididymal several
insulin
actions
site
of
by receptor
prolonged
biological
states
was rewith
in experimentally insulin,
rates
increased that
incorporation
hyper-
in
rats, into
de Investigaciones 1151, Maracaibo,
of
gluco-
isolated
infused total
with li-
ClFnicas, Venezuela.
0006-291X/92 $4.00 807
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188, No. 2, 1992
pids.
Moreover,
linism
itself
pose sis
other with
lipid
hand, insulin
found
that
in rats
synthesis
Haysted for
AND BIOPHYSICAL
treated
in vivo,
and Hardie
15 min
showed
RESEARCH COMMUNICATIONS
with
with
in
fat
1986
(9)
a 15-fold
insulin,
hyperinsu-
accumulation found
that
stimulation
in adiisolated
of lipogene-
3H-glucose. is
by several
authors
the
effect
the
acetate of
(10)
total
The advantages mented
vior
On the treated
from
et al
increased
tissue.
adipocytes
Cusin
BIOCHEMICAL
of
insulin
using
EXPERIMENTAL
(11,12,13,14),
on lipid
incorporation
some lipogenic
14C-acetate
into enzymes
synthesis total involved
lipids,
for
measuring
for
that
of
isolated
lipid
reason, rat
determining
in the
novo
fatty
synthesis it
was interesting
adipocytes, at
has been docu-
the
acid
to
measured
same time,
the
study
through beha-
synthesis.
PROCEDURE
Receptor grade m noiodinated porcine A-14 (lz51)-insulin, specific activity e 2200 mCi/mmol and l-l1 C-acetate, 57.7 mCi/rmnol were purchased fron New England Nuclear Research Products. Boston, MA. Cristalline porcine insulin was from Eli Lilly and Co. Collagenase was from Worthington Biochemical Corp., Freehold, NJ and bovine serum albumin (BSA) fraction V and all reagents used for enzyme assays, were supplied by Sigma Chemical Co., St. Louis, MO. Preparations of isolated adipocytes. After a 12-h fast, male Sprague Dawley rats, weighing 150-250 g were killed by decapitation and epididymal fat pads removed and weighed. Isolated fat cells were prepared by shaking at 37°C for 60 min in Krebs-Ringer-bicarbonate (KRB) buffer containing 7 mg/g collagenase and 10 mg/ml albumin, according to the method of Rodbell (15). Cells were filtered through nylon mesh (250 uM), centrifuged at 400 rpm for 15 set and washed three times in a collagenase-free buffer. The adipocytes were finally resuspended in the original volume with the same buffer. Insulin pretreatment and dissociation procedures. Adipocytes suspended in pH 7.4 KRB buffer containing 11 mM dextrose and 1% BSA were incubated with 16.6 nM insulin in 25 ml sterile polypropylene containers. Cells were then gently agitated in a shaking water bath for 3 h at 37°C. At the end of the incubation period, cells were transferred to sterile polypropylene tubes, centrifuged at 2000 rpm for 15 set and washed with insulin-free KRB buffer pH 7.0. Adipocytes were washed again two times, resuspended in the same buffer, transferred to sterile polypropylene containers and receptor-bound insulin allowed to dissociate at pH 7.0 for 1 h at 37'C. After the l-h dissociation period, cells were washed and resuspended in KRB buffer pH 7.4. Control cells without insulin added underwent similar washing, dissociation and centrifugation procedures as insulin-treated cells. Insulin
binding studies. Isolated adipocytes were incubated in a total volume of 1 'nger-phosphate (KRP) buffer pH 7.8 with trace amounts (0.16 nM; 0.76 uCi) I-insulin, in the absence and presence of an excess of unlabelled insulin (833 nM). Incubations were performed for 2 h at 16°C as in Marshall and Olefsky (16) and adipocytes associated radioactivity determined in triplicate from each incubation tube. All data were corrected for non-specific binding and expressed as percentaje of binding per mg of cell DNA. Acetate incorporation studies. A 0.5 ml aliquot of control and insulin-treated adiocytes were added to 0 5 ml of KRB with glucose (final concentration 5 mM) and 2 uCi P4C-acetate. Insulin wa; added to cell samples at concentrations of 0.0, 0.03, 0.08 and 4 nM. All determinations were made in duplicate. Following 1 h incubation at 37oC, the reaction was terminated by acidification with sulfuric and 14C-incorporation into lipids was determined by extraction with chloroform-methanol 2:l. Enzyme activities assays. Control and 3-hour-insulin-treated adipocytes in KRB buffer pH 7.4 were homogenized in a Polytron for 15 set, centrifuged at 20.000 g for 2 h and
$ :$$!t
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the cytosolic fraction used for the enzyme assays following the procedure of Ochoa (17) for malic enzyme, of de Chatelet et al (18) for glucose-6-phosphate dehydrogenase and of Srere (19) for citrate lyase. DNA determination. Adipocyte DNA content was determined as in Burton (20). Statistical analysis. Data are presented as means significance by the SAS-82 Computer Program, using Least Square Means.
* S.E. and analyzed for statistical the General Model Procedure and the
RESULTS The data Fig.
insulin
1, where
be seen, bind
of
binding
the
insulin
for
mean binding
the
control
parameters
preincubation
led
and treated from
adipocytes
10 preparations
to a 46.9%
decrease
are are
in the
illustrated
shown.
ability
As it
in can
of adipocytes
to
insulin. The effect
lipid
synthesis
basal
lipid
on l4 C-acetate is
revealed
synthesis.
stimulation
of
synthesis
In treated
cells
only
33% at
highest
the
this
in the
treated
Concomitantly,
the
synthesis
pathway
mcmmo~
in Fig.
Increasing
lipid
lin.
synthesis
incorporation
were
2.
insulin
levels
in the
treated
EZZITREATED 0
Fig. 2. cytes. absence 14C-acetate Results
a significant
by one half,
cells
relevant
to
(Fig.
3).
treatmenF(16.6 nM) expressed as the mean experiments (p -= 0.01).
on insulin percentage
In the
it
reached the
fatty
of malic
-TREATED
binding of of binding
isolated rat per mg of cell
Dose response of insulin stimulated lipid synthesis by isolated rat Control (0) or 16.6 nM insulin treated (*) adipocytes were incubated (basal) or presence of increasing concentrations of insulin 0.03 to incorporation into total lipids are expressed as DPM/mg of cell are mean f SE of 10 separate experiments (p< 0.0001).
809
4 nM insu-
de novo case
in
(p
