Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo-CEU, E-28668, Madrid, Spain
Submitted 22 October 2002 ; accepted in final form 4 April 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
adipose tissue; 3-adrenoceptor; lipolysis; lipogenesis
Insulin and catecholamines are of major importance for the endocrine
control of lipid mobilization from fat cells. In rats, catecholamines
stimulate lipolysis by acting via the 3-adrenoceptors,
activating adenylate cyclase, cAMP-dependent protein kinase (PKA), and
hormone-sensitive lipase (HSL), resulting in the hydrolysis of
triacylglycerols (for review, see Ref.
34). In contrast, the
antilipolytic action of insulin is a multi-step process, so acute effects of
insulin are related to activation of cGMP-inhibited phosphodiesterase (PDE3)
(12), activation of a
phosphatase, and/or sequestration of
3-adrenoceptors from the
cell surface (15). The
mechanisms by which long-term insulin treatment reduces
-adrenergic
sensitivity are less documented, although it is known that long-term exposure
to insulin induces a decrease of mRNA
3-adrenoceptor
expression in both isolated adipocytes
(14) and 3T3-F442A
preadipocytes (18).
Enhanced maternal adipose tissue lipolytic activity during late gestation
has been related to autonomic nervous system activation, secondary to
hypoglycemia. However, a decreased response of adipocytes to catecholamines
has been reported during late gestation in the rat
(1,
23) and in the sheep
(22,
58). Furthermore, this effect
could be a consequence of decreased adrenoceptor activity secondary to
maternal hyperinsulinemia, since when nonpregnant animals become
hyperinsulinemic by means of the euglycemic hyperinsulinemic clamp the
-adrenoceptor activity is impaired
(23).
Because fat accumulation is the result of a balance between lipid synthesis
and breakdown, the present study was designed to clarify the modulation of
lipogenic and lipolytic responses to insulin in the "anabolic" and
"catabolic" phases of pregnancy in the rat. To address this issue,
adipocytes from virgin and pregnant rats (7, 14, and 20 days of gestation)
were exposed to different concentrations of the -agonists forskolin or
isobutylmethylxanthine, and lipolysis was quantified as glycerol release into
the incubation medium. Furthermore, to investigate the molecular mechanism
underlying the observed changes in the responsiveness of the tissue at
different stages of pregnancy, the study was extended to determine by means of
immunodetection studies whether
3-adrenoceptor expression is
affected during pregnancy, and insulin sensitivity of lipolysis was assessed
by isoproterenol-stimulated lipolysis in the presence of various
concentrations of insulin. In another set of animals, the dose response of
insulin stimulation of lipogenesis was studied by quantifying glucose
incorporation into fatty acids. These experiments revealed that adipose tissue
lipolysis and lipogenesis become highly responsive to insulin during early
pregnancy, whereas a decreased responsiveness to insulin of these pathways
emerges at late pregnancy.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasma analysis. Plasma aliquots were used to measure glucose by an enzymatic colorimetric test (glucose oxidase, GOD/PAP method, Roche Diagnostics, Barcelona, Spain) (3). Insulin was determined in plasma samples with a specific ELISA kit for rats (Mercodia, Uppsala, Sweden), the values within the detection range of the assay being 0.075.5 µg insulin/ml (1.8% intra-assay variation, 3.8% interassay variation). Leptin was assayed by ELISA in diluted plasma samples as indicated by the manufacturer, by use of a commercially available kit specific for rat leptin (Assay Designs, Ann Arbor, MI). Leptin concentrations were within the detection range of the kit, i.e., 0.063.6 ng leptin/ml (11.6% intra-assay variation, 11.0% interassay variation). Glycerol was determined in plasma samples by an enzymatic colorimetric test (GPO-Trinder; Sigma-Aldrich, Madrid, Spain), and nonesterified fatty acids (NEFA) were analyzed in EDTA-plasma samples by a colorimetric method (Wako Chemicals, Neuss, Germany).
Oral glucose tolerance test. Glucose tolerance tests were performed in 14-h-fasted rats. After a basal blood sample from the tail was drawn, a bolus of glucose (2 g/kg) was administered orally to the animals. Subsequently, blood samples were collected into heparinized tubes at 2.5, 5, 10, 15, 20, 30, 45, 60, and 90 min after glucose dose and placed on ice. Samples were centrifuged, and plasma was stored at -20°C until processed.
Glucose tolerance (Kg) was estimated as the rate of
plasma glucose disappearance
(59) corrected by the maximal
increase in plasma insulin. Plasma glucose disappearance was calculated as the
slope of the plasma glucose concentration vs. time, using the samples obtained
from the 2.5- to 15-min period after glucose administration
[glucose/
time (mg · µg-1 ·
min-1)] (30).
Isolation of adipocytes and determination of cell size and number.
Adipocytes were prepared from white adipose tissue according to the method of
Rodbell (50) with minor
modifications. Briefly, freshly isolated adipose pads were cut into small
pieces and digested by collagenase A (1 mg/ml; Roche Diagnostics; activity
0.21 U/mg) in Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) containing 4%
(wt/vol) BSA (fatty acid free, fraction V; Sigma-Aldrich) and 5.5. mM glucose
(KRB-Buffer), for 30 min at 37°C in an O2-CO2
atmosphere (19:1, vol/vol) with shaking (60 cycles/min). Subsequently, fat
cells were dispersed and filtered through a silk screen, washed three times
with KRB-Buffer to eliminate collagenase, and resuspended in the same buffer
at a concentration of 0.4 x 106 cells/ml. The size of a
fat cell was measured by direct microscopic determination, and the mean
adipocyte diameter was calculated from measurements of 100 cells. Because
adipocytes have 95% lipid content and are spherical, their volume and weight
can be estimated from their diameters
(26). Total cell lipid content
was determined gravimetrically after organic extraction
(13), and the number of fat
cells was calculated by dividing the total lipid weight by the mean cell
weight.
Lipolysis measurements. Freshly isolated adipocytes were incubated at 37°C in KRB-Buffer containing 1 U/ml of adenosine deaminase (Sigma-Aldrich) in the absence and presence of the indicated concentrations of (±)-isoproterenol hydrochloride, BRL-37344 (Sigma-RBI), forskolin, or 3-isobutyl-1-methylxanthine (all from Sigma-Aldrich). After 90 min of incubation, the tubes were placed on ice, and aliquots of 50 µl of the infranatant were removed for the enzymatic determination of glycerol (GPO-Trinder). Glycerol released into the incubation medium was taken as an index of lipolytic activity and expressed as nanomoles of glycerol released per minute per 100 milligrams of cell lipid (23).
Concentration-effect curves of glycerol release as a function of either agonist were calculated as a percentage of basal lipolysis (i.e., without agonist). Half-maximal effective agonist concentration (EC50) and maximum effect (Emax) values were obtained by computer fitting of concentration-effect curves of the agonists to Hill's model (5, 11) by using the Sigma-Plot program (version 4.0; SPSS Jandel Scientific, Erkrath, Germany).
Insulin sensitivity of adipose tissue lipolysis. The antilipolytic effect of insulin was measured in adipocytes isolated from lumbar adipose tissue from nonpregnant and pregnant rats. Cells were incubated in KRB-Buffer for 5 min at 37°C with varying concentrations of insulin (0 to 100 nM) (insulin from bovine pancreas, Sigma-Aldrich) before addition of isoproterenol (100 nM) and incubation for a further 90 min. Subsequently, glycerol release into the incubation medium was quantified.
Insulin sensitivity of adipose tissue lipogenesis. Rat adipocytes freshly isolated from lumbar white adipose tissue of virgin and 7-, 14-, and 20-day pregnant rats were incubated in KRB buffer containing 5 mM glucose in the presence of increasing concentrations of insulin (0 to 1,000 nM). After 30 min, 0.5 µCi/tube of D-[U-14C]glucose (specific activity 12.5 mCi/mmol, New England Nuclear, Madrid, Spain) was added and incubated for a further 90 min at 37°C with shaking. Lipids were extracted from adipocytes in chloroform-methanol (2:1) by the method of Folch et al. (20) with modifications (24). Aliquots of total lipids were saponified in 5 N ethanolic KOH for 1 h at 100°C, and, after acidification with H2SO4, fatty acids were extracted with heptane. Radioactivity measurements were expressed as nanomoles of glucose incorporated into fatty acids per 100 milligrams of cellular lipid.
Protein extraction and immunoblotting. To extract protein from the tissues, 200 mg of frozen lumbar adipose tissue were powdered in liquid nitrogen in a mortar precooled to -80°C and disrupted in an ice-cold hypotonic lysis buffer (10 mM Tris · HCl, 2.5 mM EDTA, pH 7.4). Cellular debris was pelleted and discarded after centrifugation at 1,500 g for 5 min at 4°C. Supernatants, containing cellular proteins, were centrifuged at 17,000 g for 30 min at 4°C. The resulting pellets, containing the crude membranes, were pooled, and after protein determination by the Bradford method (6), they were resuspended in the appropriate volume of Laemmli buffer to give a final protein concentration of 1 mg/ml. After being boiled for 3 min, the insoluble material was pelleted at 15,000 g for 20 min. The resulting supernatant was stored at -20°C until use for Western blot analysis.
Membrane protein (20 µg) was subjected to 7.5% SDS-PAGE and
electrophoretically transferred to PVDF membranes (Amersham Pharmacia Biotech,
Barcelona, Spain). The membranes were incubated with an
anti-3 polyclonal antibody raised in goat (Santa Cruz
Biotechnology, Santa Cruz, CA) at a 1:200 dilution for 35 min and subsequently
with a mouse anti-goat polyclonal antibody conjugated to horseradish
peroxidase (Sigma-Aldrich) at a 1:30,000 dilution. Immunoreactive bands were
visualized using the enhanced chemiluminescence (ECL) system (Amersham
Pharmacia Biotech) and quantified by densitometry (Bio-Rad, Madrid, Spain). In
each gel, samples of adipose tissue from the four experimental groups (0, 7,
14, and 20 days of pregnancy) were always run in parallel.
Statistical analysis. Results are expressed as means ± SE of 410 animals per group. Data were analyzed for homogeneity of variance with a Levene test. Values were log transformed to equalize the variance between conditions. Statistical comparisons were made by analysis of variance followed by a Bonferroni test with 95% confidence limits (51), using the SPSS program (version 9.0.1). With respect to the log normal distribution of the EC50 values, the statistical analyses were done on the logarithm of these parameters (5).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental animals and fat depot characteristics. All the pregnant animals included in the present study exhibited hyperphagia, as total daily food intake in nonpregnant rats (19.3 ± 0.6 g/day) was significantly lower than in the pregnant animals (26.0 ± 1.1, 29.5 ± 1.8, and 30.4 ± 1.0 g/day for 7-, 14-, and 20-day pregnant rats, respectively).
As shown in Table 1, maternal body weight progressively increased with gestational time. Although this change partially corresponds to the increase in conceptus mass (mean number of fetuses was 14 ± 0.34 fetuses/mother), the value of this parameter was almost negligible on day 7, and most of the increase at this gestational time corresponded to conceptus-free maternal weight (Table 1). The conceptus-free body weight progressively increased until day 14 of pregnancy, whereas from 14 to 20 days of gestation, corresponding to maximal fetal growth, there was no further increase.
|
Changes in the weight of lumbar adipose tissue are summarized in
Table 1, showing that lumbar
fat pad weight increases from the beginning of pregnancy. This increase in fat
content was also observed when the values were expressed as percent net body
weight (0.38 ± 0.02, 0.40 ± 0.04, 0.54 ± 0.03, and 0.73
± 0.07% for 0-, 7-, 14-, and 20-day pregnant rats, respectively). The
augmented adipose tissue weight parallels an increase in the mean size of the
cells but not in adipocyte number (3 x 106 cells/lumbar
fat pads), indicating that fat deposition during gestation is the result of
the observed hypertrophy of the tissue.
Plasma glycerol and NEFA during pregnancy. Glycerol and NEFA were determined in plasma samples of rats on different days of gestation. Values for glycerol were 279 ± 18, 342 ± 38, 408 ± 57, and 640 ± 79 µM for 0-, 7-, 14-, and 20-day pregnant rats, respectively. NEFA values were 486 ± 63, 515 ± 59, 662 ± 109, and 1,120 ± 148 µM for 0-, 7-, 14-, and 20-day pregnant animals, respectively. Plasma levels of both glycerol and NEFA in the 20-day pregnant rats were significantly higher than those of rats at 0, 7, and 14 days of gestation, confirming the overall higher lipolytic activity in late pregnancy.
Plasma glucose, insulin, leptin, and glucose tolerance during pregnancy. Plasma glucose levels were significantly lower in late pregnancy (day 20), whereas no change in this metabolite was observed in the 7-day pregnant rats. Late pregnancy was accompanied by hyperinsulinemia with no modification of insulin concentration in early gestation (Table 2). Glucose tolerance (Kg; calculated as the slope of the plasma glucose concentration over time for 2.515 min after the oral bolus administration of glucose) was significantly higher in 7-day pregnant animals than in the other groups, suggesting an enhanced insulin responsiveness in early pregnancy. In contrast, the 20-day pregnant rats showed an impairment of glucose tolerance. Gestation was also accompanied by considerable changes in plasma leptin concentration. As shown in Table 2, plasma leptin levels gradually increased until day 14 of pregnancy and on day 20 started to decrease, the values at postpartum being similar to those of the virgin animals (data not shown).
|
Lipolytic activities stimulated by -adrenergic
agonists. To test catecholamine-stimulated lipolysis during pregnancy,
isolated adipocytes from rats at different times of gestation were incubated
with various
-adrenergic agonists. Because previous studies had reported
that some variables of catecholamine-stimulated lipolysis vary with fat cell
size and gestation itself results in increased adipocyte volume, we eliminated
the influence of this factor by expressing lipolysis per lipid content in the
preparation and corrected as a percentage of the basal value (i.e., without
agonist) (5,
23). In our study, we found no
significant differences in basal lipolysis (1.9 ± 0.18, 1.97 ±
0.25, 1.57 ± 0.18, and 2.21 ± 0.34 nmol glycerol ·
min-1 · 100 mg cell lipid-1 for 0-, 7-, 14-, and
20-day pregnant rats, respectively).
Figure 1 summarizes the
concentration-response relationships after isolated adipocytes were incubated
in the presence of various concentrations (0.01 to 10,000 nM) of isoproterenol
(nonspecific -agonist; Fig.
1A) and BRL-37344 (
3-agonist;
Fig. 1B). The
lipolytic response to both agents was substantially decreased in adipocytes
from rats at 7 and 20 days of gestation compared with those at days 0
and 14. To further analyze these activities, the lipolytic efficacies
(i.e., Emax) and EC50 values were obtained from the
concentration-response curves. Lipolytic efficacies
(Table 3) of both compounds
were higher in adipocytes from virgin and 14-day pregnant rats than in those
from rats at 7 and 20 days of gestation. The computed EC50 values
(lipolytic potency) are presented in Table
3. As shown, the lipolytic potency of both isoproterenol and
BRL-37344 showed a significant decrease in 14- and 20-day pregnant rats
compared with the nonpregnant animals.
|
|
We also studied the relationship between the maximal lipolytic effects of
isoproterenol and BRL-37344. As shown in
Fig. 2, an excellent
correlation can be observed between BRL-37344- and isoproterenol-mediated
lipolytic effects (r = 0.98, P < 0.0001). Furthermore, to
ensure that such dependence occurs independently of the reproductive state of
the animals, linear correlations were also made within each group. As
expected, isoproterenol and BRL-37344 Emax correlated significantly
in all groups (r = 0.986, 0.966, 0.89, and 0.918 for virgin and 7-,
14-, and 20-day pregnant rats, respectively) with a slope close to 1. This
result indicates that the reduction of the lipolytic effect of
isoproterenol-stimulated lipolysis during pregnancy is associated with a
reduction of the 3-mediated component.
|
Lipolytic activities stimulated by nonadrenergic agents. As the difference in lipolytic responsiveness between the groups could be localized at any step in catecholamine-induced lipolysis from adrenoceptors to the final activation of HSL, lipolysis was stimulated using different agents acting at 1) the adenylate cyclase level (forskolin, an adenylate cyclase activator) and 2) at the PDE level (isobutylmethylxanthine, a nonspecific PDE inhibitor).
Results are summarized in Table 4, where both forskolin- and isobutylmethylxanthine-stimulated lipolysis are shown. A similar lipolytic activity stimulated by forskolin or isobutylmethylxanthine was found in the adipocytes of virgin and 7-, 14-, or 20-day pregnant rats. The lack of differences in forskolin and isobutylmethylxanthine suggests the presence of modifications in the lipolytic cascade located at the receptor level.
|
Characterization of 3-adrenoceptor
protein expression in white adipocytes from pregnant rats by immunoblot.
Because in 7- and 20-day pregnant rats a decrease, not only in sensitivity but
also in the maximal response to the
-agonist, was observed
(Fig. 1), a decline in the
membrane receptors was expected. To determine whether the impairment in
catecholamine action in adipocytes from 7- and 20-day pregnant rats was
associated with changes in the
3-adrenoceptor component,
immunoblotting was performed with specific antibodies against the rat
3-adrenoceptor.
3-Adrenoceptor protein
determined in lumbar white adipose tissue from rats at different times of
gestation (0, 7, 14, and 20 days) is shown in
Fig. 3. The inset of
this figure shows a representative autoradiogram of
3-adrenoceptor protein in a single nonpregnant and in 7-,
14-, and 20-day pregnant rats. The immunoreactive band of
3-adrenoceptor had an apparent molecular mass of 68 kDa. The
quantity of immunoreactive band was clearly decreased in the adipose tissue
membranes of 7- and 20-day pregnant animals.
|
The compiled values of each group show
(Fig. 3) that the
3-adrenoceptor was reduced by >50% in adipocytes from 7-
and 20-day pregnant rats, an effect that is on the same order of magnitude as
the observed decrease in the lipolytic efficacies of
-agonists
(Table 3). These results,
together with the absence of changes in forskolin- and
isobutylmethylxanthine-stimulated lipolysis, indicate that, during pregnancy,
catecholamine-stimulated lipolytic activity is modulated at the
3-adrenoceptor level and that the response is highly impaired
in 7- and 20-day pregnant rats but not in the 14-day pregnant animals.
Concentration-dependent effect of insulin on isoproterenol-stimulated
lipolysis. We next examined the concentration-dependent capacity of
insulin to decrease the lipolytic action of catecholamines. After stimulation
with varying concentrations of insulin, cells were treated with a submaximal
concentration of the -agonist isoproterenol (100 nM). We found a
concentration-dependent inhibition of lipolysis by insulin in adipocytes from
all groups (Fig. 4A).
However, the degree of inhibition differed among them. The maximum
antilipolytic effect of insulin was found in adipocytes from the 7-day
pregnant animals and the lowest in those from the 20-day pregnant rats. The
response of the adipocytes of the 14-day pregnant rats was similar to that
from virgin animals (Fig.
4B).
|
Concentration-dependent effect of insulin on lipogenesis. To analyze the lipogenic activity of adipose tissue during pregnancy, the formation of labeled fatty acids from [U-14C]glucose was examined. Figure 5A summarizes the results obtained during incubation of adipocytes from pregnant rats for 90 min in KRB containing [14C]glucose in the absence of insulin (basal state). In this condition, glucose incorporation into fatty acids was significantly higher at 7 days of pregnancy compared with the other experimental groups, which gave values similar to one another. To investigate the insulin sensitivity of fatty acid synthesis, dose-response experiments were performed. As shown in Fig. 5, B and C, the stimulation of fatty acid synthesis by insulin was maximal at 7 days of gestation, whereas the lowest activation of lipogenesis by insulin takes place in the late-pregnant rats.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During early gestation, both glucose and insulin concentrations in the mother remain unchanged. This happens even when already by day 6 of gestation the pancreatic content of insulin is significantly enhanced (43), indicating that the augmented release of insulin takes place only under an intense insulinotropic stimulus such as food intake. The results presented here show that 7-day pregnant rats have a higher overall response to insulin than nonpregnant animals, as deduced from the glucose tolerance test. Different clinical studies have also suggested an enhanced insulin sensitivity in early gestation in women. In a recent report, it was observed that, in normal healthy women in the first trimester of gestation, maternal glucose falls (40). Furthermore, type 1 diabetic women in the late first trimester of pregnancy have a transient decrease in their insulin requirement (28), and it has been proposed that an increased insulin sensitivity is responsible for such a decline (53). Our results show that the augmented insulin responsiveness present in early gestation completely switches to a normal condition by day 14 of pregnancy, a situation previously observed in rats on day 15 of gestation (43). This is comparable to what occurs in late second trimester of pregnancy when insulin requirements in type 1 diabetic women are similar to those before pregnancy (10). Therefore, at this stage of gestation, normal sensitivity to insulin in the presence of hyperphagia and hyperinsulinemia sets the appropriate scene for the active anabolic condition present in the mother. As pregnancy proceeds, insulin sensitivity is impaired, and the situation of insulin resistance becomes maximal in late gestation. This insulin resistance state is one of the most characteristic features of late pregnancy both in human and in rat, as with the hyperinsulinemic euglycemic clamp technique (8, 46) it has been demonstrated that insulin-mediated glucose disposal decreases as much as 4060% from nonpregnancy or early pregnancy to late gestation.
Although the occurrence of insulin resistance in late pregnancy is well documented, the underlying mechanisms causing the changes in adipose tissue insulin responsiveness during pregnancy are still unclear. Ongoing work in our laboratory and data from other groups (19, 55) indicate that there is virtually no decrease in insulin receptor number and that an impaired insulin receptor tyrosine kinase activity could account for the insulin resistance state at late pregnancy (Ramos MP, Crespo-Solans MD, Martinez JI, Herrera E, unpublished data). In addition, preliminary data from our laboratory, investigating the largely unknown molecular mechanisms that account for enhanced insulin responsiveness at early gestation, point to changes in postreceptor protein function (Ramos MP, Crespo-Solans MD, Martinez JI, Herrera E, unpublished data). However, the molecular events responsible for both enhanced insulin responsiveness in early pregnancy and insulin resistance in late gestation remain to be established.
In agreement with previous reports (19), our results show that gestation is a stage that results in increased adipocyte volume, and it is known that adipose fat depots are the net balance of synthesis and hydrolysis of triacylglycerols via lipogenesis and lipolysis. Furthermore, the present study demonstrates that pregnancy causes a striking change in catecholamine-stimulated lipolysis in isolated rat adipocytes, with 50% decreased activity at both 7 and 20 days of gestation with a temporary normalization at midpregnancy (14 days). It has been reported previously that an enlargement in fat cell size causes an increase in lipolysis (27, 57). However, although adipose cells from 20-day pregnant rats are significantly larger than those from nonpregnant animals, their isoproterenol responsiveness is significantly lower. Thus differences in cell size cannot completely explain the perturbations in fat cell metabolism associated with pregnancy.
In rat adipocytes, catecholamines stimulate lipolysis by acting via the
3-adrenoceptors activating adenylate cyclase; then cAMP
promotes lipolytic activity by activating PKA, which phosphorylates HSL (for
review, see Ref. 34). To study
the molecular factors regulating adipose tissue lipolytic activity during
pregnancy, we performed immunodetection of the
3-adrenergic
receptor by using a specific antibody. We observed that
3-adrenoceptor protein was reduced by >50% in adipocytes
from both 7- and 20-day pregnant rats. Similarly, catecholamine-stimulated
lipolysis was reduced in these two groups by 50%. In different cell systems,
due to the existence of spare receptors, only a fraction of the receptors has
to be occupied to obtain a full biological effect after hormone stimulation.
Previous studies have observed that such a receptor reserve also exists for
-adrenergic-induced lipolysis in human fat cells
(2). According to this
hypothesis, a small reduction in receptor number is accompanied by a shift to
the right of the lipolytic dose-response curve without a change in its
amplitude (49). However, when
a larger receptor fraction is inactivated, an additional reduction of the
responsiveness should be found. Even though present results show a 50%
reduction on the isoproterenol responsiveness, we cannot exclude the
possibility that the observed decrease in the lipolytic activity to
catecholamines in the adipocytes of 7- and 20-day pregnant animals is a
postreceptor defect. In our study, forskolin and isobutylmethylxanthine
responses revealed similar lipolytic efficacies in virgin and pregnant rats,
suggesting that differences in lipolytic signals are due to adaptations at the
level of the plasma membrane with a decreased number of
3-adrenoceptors.
In adipose cells, insulin and catecholamines are of major importance for
the endocrine control of lipid mobilization. Present findings, showing an
augmented adipose tissue responsiveness to the antilipolytic action of insulin
at early pregnancy, provide an explanation for the decrease in both
3-adrenoceptor protein and activity, since these variables
are known to be interconnected
(14,
18). This is in agreement with
results showing that the lipolytic response to catecholamines is impaired in
biopsies of subcutaneous femoral adipose tissue in late first trimester
pregnant women (48). This
higher antilipolytic action of insulin in early gestation should play a very
important role in the maintenance of fat deposition. Previous studies with rat
fat cells have hypothesized that the degree of antilipolysis induced by
insulin depends on the rate of lipolysis before the addition of the hormone
(56). Thus the antilipolytic
effect is more pronounced when the lipolysis is raised. Our study does not
support this hypothesis, since the lipolytic activity in absence of insulin is
even higher in virgin than in the 7-day pregnant rats but the antilipolytic
effect of insulin is
30% higher in the latter group. Others have found a
positive correlation between the antilipolytic effect of insulin and the size
of human fat cells (44).
However, we do not find such a relationship in our study, so differences in
fat cell size cannot explain this higher response in the 7-day pregnant
rat.
Enhanced maternal adipose tissue lipolytic activity during late gestation
has been related to autonomic nervous system activation, secondary to
hypoglycemia. However, a decreased response of lumbar adipocytes to
norepinephrine or isoproterenol has been reported during late gestation in the
rat (1) and in the sheep
(22,
58). In addition, it is known
that lipolysis varies to a different degree in the various fat depots
(27). In this context, we have
shown in a previous study (37)
that there is a 30% decrease in subcutaneous adipose tissue weight from
days 15 to 20 of gestation. Consequently, the overall
decrease in maternal fat in late pregnancy should be the result of the
contribution of the different adipose stores. Thus the results of the present
study, which focuses specifically on lumbar adipose tissue, do not exclude the
possibility that, in late pregnant rats, other fat depots, such as
subcutaneous adipose tissue, exhibit an enhanced fat breakdown. A study
performed in rabbits reveals that the 3- and
2-adrenoceptor components are enhanced in late pregnancy
(5). These results are not in
disagreement with ours, since they show an increase in the
2/
ratio, and this corresponds to a higher
antilipolytic component in adipocytes from late pregnant rabbits
(5). Furthermore, because the
-adrenoceptor activity is impaired when nonpregnant animals become
hyperinsulinemic by means of the euglycemic hyperinsulinemic clamp
(23), we can speculate that
the lower catecholamine-stimulated lipolysis in late gestation could be a
consequence of decreased adrenoceptor activity secondary to maternal
hyperinsulinemia. The antilipolytic effect of insulin is considered the most
sensitive of its metabolic actions
(56), so it requires a much
lower insulin concentration (1 nM in our study) than other response sites like
the stimulation of glucose transport (>10 nM for glucose uptake and
lipogenesis). Hence, even in insulin resistance states, in which glucose
transport is impaired, sensitivity to insulin's antilipolytic effect is
relatively preserved. This is in agreement with the hypothesis that insulin
resistance is not a general phenomenon but is confined to specific effector
systems (33). This could
explain that, although insulin resistance of glucose transport is a
characteristic of late pregnancy (insulin stimulation of glucose uptake is
reduced by 50%) (36),
hyperinsulinemia accounts for the decreased catecholamine-stimulated lipolysis
found in late pregnant rats (insulin inhibition of lipolysis is impaired by
only 25% in 20-day pregnant rats), resulting in the maintenance or expansion
of adipose stores. In support of this hypothesis, it has been found that
adipose tissue from late pregnant women retains its sensitivity to the
antilipolytic effect of insulin
(9). Thus, similar to the
results obtained in pregnant rats fed a different fat diet
(23), hyperinsulinemia
occurring in late pregnancy might be responsible for decreasing (2-fold) both
lipolytic activity and
3-adrenoceptor protein in adipose
cells.
Together with the changes observed in lipolysis, it was found here that basal lipid synthesis from glucose is significantly increased in the 7-day pregnant rat. In midpregnancy (day 14), the values returned to those found in the nonpregnant condition, whereas in late pregnancy there was a tendency for the values to decrease. The tendency of adipose tissue lipogenesis to decrease in late pregnancy is in agreement with the sharp decline observed in Wistar rats on day 21 of gestation (45). In this context, it should be mentioned that, in late pregnancy, the mother has hypoglycemia, so the "in vivo" availability of glucose for lipid synthesis is even lower. These results, together with a decreased hydrolysis and uptake of circulating triglycerides resulting from an impaired lipoprotein lipase activity (38), reflect the net breakdown reduction of the various adipose tissue depots seen around delivery (37).
It could be suggested that plasma insulin per se is responsible for the modulation of lipid synthesis from glucose during pregnancy. However, in our study, the highest lipogenic activity of adipose tissue was detected in early pregnancy, when rats are normoinsulinemic and the lowest in the 20-day pregnant rats despite these animals being hyperinsulinemic. Furthermore, the dose-response relationship between insulin and activation of lipogenesis reveals that insulin responsiveness is drastically enhanced in the 7-day pregnant rats. Thus modulation of lipogenesis during pregnancy can be better explained on the basis of insulin responsiveness. Fat accumulation in early gestation is caused by enhanced adipose tissue lipogenesis (17, 32), which is related to maternal hyperphagia because it does not happen in food-restricted rats (41). Our results support this hypothesis because after a meal, when insulin secretion and glucose availability are enhanced, the higher insulin responsiveness of the mother favors accumulation of glucose into lipids, contributing to the hypertrophy of adipose tissue and increased maternal fat depots.
Alternatively, given its participation in the regulation of energy
homeostasis, leptin might be involved in fat accretion during pregnancy.
Biological effects of leptin include inhibition of food intake and impairment
of metabolic action of insulin, including stimulation of glucose uptake,
lipogenesis, and inhibition of isoproterenol-stimulated lipolysis
(42). However, the present
study does not support this interaction in pregnancy, as no relationship
between leptin concentration and lipogenesis, lipolysis, or insulin
responsiveness was observed. Data from our present study are in agreement with
observations indicating that both adipose tissue and plasma leptin levels peak
1219 days of gestation and subsequently display a pronounced
decline to values similar to those of nonpregnant animals by day 21
of gestation (29).
Furthermore, in a previous study from our group
(25), we have already reported
that plasma leptin levels parallel changes in fat mass during pregnancy and
lactation. These data suggest that the maternal fat depots contribute,
together with placenta (35,
39), to the change in maternal
leptin levels during pregnancy.
Modulation of insulin sensitivity in pregnancy might be due to changes in
gestational hormones. Specifically, a decline in progesterone, an anti-insulin
hormone, during early pregnancy has been proposed to be responsible for lower
insulin requirements (28,
31,
32). In addition, it has been
suggested that low concentrations of 17-estradiol, similar to early
pregnancy, could be responsible for the enhanced insulin sensitivity in muscle
and adipose tissue by increasing the number of insulin receptors
(21). On the other hand, high
17
-estradiol concentrations, as found in late pregnancy, could favor
insulin resistance by decreasing the insulin receptor number in peripheral
tissues (21). Nevertheless,
the hormonal and molecular environmental factors responsible for such changes
remain to be established.
In conclusion, to our knowledge, this is the first study providing evidence that, at early gestation, an enhanced insulin sensitivity accounts for increased activation of adipose tissue lipid synthesis and inhibition of lipolysis. Normoinsulinemia in the presence of augmented insulin responsiveness, therefore, may drive the anabolic tendencies of the mother during the first two-thirds of gestation, and this accounts for increased fat deposition. This mechanism should have special relevance after a meal, when insulin secretion is enhanced and there is enough glucose availability. With this metabolic adaptation, the mother ensures that circulating glucose is actively taken up by adipose tissue and converted into triglycerides, which are not actively hydrolyzed. In this way, the mother develops a net fat depot that is known to have a major impact on the appropriate availability of substrates that warrant the normal development of the fetus at late pregnancy.
![]() |
DISCLOSURE |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|