Departments of 1 Reproductive Biology and 2 Nutrition, Case Western Reserve University School of Medicine, and MetroHealth Medical Center, Cleveland, Ohio 44106; and 3 Department of Nutrition, Texas A & M University, College Station, Texas 77843
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ABSTRACT |
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Gestational diabetes
mellitus (GDM) is associated with elevated postprandial free fatty
acids (FFA) and insulin resistance; however, little is known about the
cellular mechanisms underlying insulin resistance to suppress lipolysis
during gestation. We evaluated the longitudinal changes in insulin
suppression of FFA before pregnancy and in early (12-14 wk) and
late (34-36 wk) gestation in obese subjects with normal glucose
tolerance and in obese GDM subjects. Abdominal subcutaneous adipose
tissue biopsies were also obtained during cesarean delivery from normal
obese pregnant (Preg-Con), GDM, and nonpregnant obese control
(Non-Preg-Con) subjects during gynecological surgery. GDM subjects had
higher basal plasma FFA before pregnancy (P = 0.055).
Insulin's ability to suppress FFA levels declined from early to late
gestation in both GDM and Preg-Con subjects and was significantly less
in GDM subjects compared with Preg-Con subjects over time
(P = 0.025). Adipose tissue insulin receptor substrate
(IRS)-1 protein levels were 43% lower (P = 0.02) and
p85 subunit of phosphatidylinositol 3-kinase was twofold higher
(P = 0.03) in GDM compared with Preg-Con subjects. The
levels of peroxisome proliferator-activated receptor-
(PPAR
) mRNA
and protein were lower by 38% in Preg-Con (P = 0.006) and by 48% in GDM subjects (P = 0.005) compared with
Non-Preg controls. Lipoprotein lipase and fatty acid-binding protein-2 mRNA levels were 73 and 52% lower in GDM compared with Preg-Con subjects (P < 0.002). Thus GDM women have decreased
IRS-1, which may contribute to reduced insulin suppression of lipolysis
with advancing gestation. Decreased PPAR
and its target genes may be
part of the molecular mechanism to accelerate fat catabolism to meet
fetal nutrient demand in late gestation.
insulin signaling; lipid metabolism; body weight; adipogenesis
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INTRODUCTION |
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HUMAN PREGNANCY IS CHARACTERIZED by a series of metabolic changes that promote adipose tissue accretion in early gestation and later onset of insulin resistance. In early pregnancy, insulin secretion increases (32), whereas insulin sensitivity is unchanged or decreased or may even increase (9, 10). However, in late gestation, maternal adipose tissue depots decline (44), whereas postprandial free fatty acid (FFA) levels increase and insulin sensitivity worsens by some 40-60% compared with prepregnancy (9, 10). Insulin's ability to suppress whole body lipolysis is reduced during late pregnancy (42) and more so in subjects with gestational diabetes mellitus (GDM) (46), contributing to greater postprandial increases in FFA in GDM subjects. Insulin suppression of plasma FFA has not been evaluated longitudinally in GDM subjects and could contribute to increased hepatic glucose production, decreased insulin secretion, and greater insulin resistance found in GDM subjects (9, 19, 32, 46).
The cellular mechanisms that trigger the transition from lipid storage
to increased lipolysis during pregnancy are unknown. In rodents the
activity and mRNA for lipoprotein lipase (LPL), which regulates lipid
storage, is greatly reduced in adipose tissue during late gestation,
whereas hormone-sensitive lipase (HSL), which catalyzes the
rate-limiting step in adipose tissue lipolysis, increases in late
pregnancy (29). Likewise, the ability of insulin to
suppress cAMP-mediated lipolysis in isolated adipose tissue explants is
reduced during late pregnancy compared with nonpregnant controls
(22). The intracellular mechanisms responsible for insulin
suppression of lipolysis involve activation of the insulin receptor
protein tyrosine kinase, which phosphorylates the intracellular insulin
receptor substrates, mainly insulin receptor substrate (IRS)-1 and
IRS-2 (13, 43). Phosphorylated IRS-1 binds to the p85
regulatory subunit of phosphatidylinositol (PI) 3-kinase, which in turn
activates the p110 catalytic subunit (16). The antilipolytic effect of insulin requires PI 3-kinase activity (33), which mediates serine phosphorylation of
cAMP-phosphodiesterase (PDE3B), associated with the insulin receptor
(37). In obese patients and in type 2 diabetes mellitus,
IRS-1 expression is markedly reduced in adipocytes, resulting in
decreased IRS-1-associated PI 3-kinase activity, and is presumably
responsible for the failure of insulin to suppress lipolysis as well as
reduced insulin-stimulated glucose transport (38).
The peroxisome proliferator-activated receptors (PPARs) are a family of
fatty acid sensors (receptors), which transduce stimuli from fatty
acids into changes in gene expression. PPAR exists as two isoforms,
PPAR
1 and PPAR
2, which are transcribed
from the same gene using alternative promoters (49).
PPAR
1 is highly expressed in adipose tissue and plays an
essential role in fat cell differentiation and lipid storage by binding
in the nucleus to response elements in several adipose-specific genes,
and as a central regulator of the adipogenic transcriptional cascade (5, 39). A great deal of attention has recently focused on PPAR
, since it has been shown to coregulate, along with the retinoid X receptor (RXR), the adipocyte apoptosis pathway
(12). Mice with a targeted disruption of the gene for
PPAR
were embryonic lethal; however, heterozygous PPAR
(+/
)
mice accumulate less lipid and show lower expression of fat-specific
markers relative to wild-type mice when placed on a high-fat diet
(27). PPAR
has also been implicated in the regulation
of systemic insulin sensitivity (48). This is supported by
the finding that PPAR
agonists currently used for treatment of type
2 diabetes (thiazolidinediones) increase sensitivity and fat mass
(34) and by the recent finding that patients with dominant
negative mutations within PPAR
are nonobese yet have severe insulin
resistance and type 2 diabetes (3). The target genes
induced by PPAR
include several important genes involved in fatty
acid metabolism, including LPL (40), the adipose-specific
intracellular fatty acid-binding protein aP2 (28), and the
mitochondrial uncoupling protein UCP-2 (4, 31). The aP2
protein plays a key role in the regulation of fat cell insulin
sensitivity, as demonstrated by aP2 knockout mice that are resistant to
diet-induced insulin resistance (25). The goal of the
present study, therefore, was to determine whether changes in the
expression pattern of PPAR
, along with key insulin-signaling proteins, might underlie the changes in the FFA metabolism that accompany normal human pregnancy and GDM.
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METHODS |
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Materials.
Chemiluminescence reagents (ECL kit) and horseradish peroxidase were
obtained from Amersham Life Science (Arlington, IL). Nitrocellulose
membrane, Western blotting reagents, and protein assay kit were from
Bio-Rad (Hercules, CA) and Sigma (St. Louis, MO). Leupeptin and
pepstatin were from Calbiochem (La Jolla, CA), and phenylmethylsulfonyl
fluoride (PMSF) was obtained from Sigma. Anti-insulin receptor- and
IRS-1 antibodies were from Transduction Laboratories (Lexington, KY).
The IRS-2 and p-85
antibodies were purchased from Upstate
Biotechnology (Lake Placid, NY). The PPAR
antibody was purchased
from Santa Cruz Laboratories (Santa Cruz, CA) and primarily recognizes
the PPAR
1 isoform.
Subject recruitment.
This study was conducted in the General Clinical Research Center
(GCRC), MetroHealth Medical Center, Case Western Reserve University.
The protocol was approved by the institutional review board and
scientific review committee of the GCRC. Written informed consent was
obtained from each study subject before enrollment. For the
longitudinal studies, we recruited four obese women with no history of
gestational diabetes (Preg-Con) and five obese women with a history of
GDM in a previous pregnancy (GDM) preceding a planned pregnancy. We
defined obesity as pregravid body fat >25%. None of the subjects had
diabetes mellitus before conception according to oral glucose tolerance
tests (OGTT). Other than GDM, all of these pregnancies were
uncomplicated. None of the subjects was breast feeding, using hormonal
contraception, taking other medicines that might affect glucose/lipid
metabolism, or using tobacco products. All subjects planned to conceive
after baseline pregravid studies were completed. The subjects with
normal glucose tolerance in a previous pregnancy all had normal glucose
tolerance before and throughout pregnancy. Women with a previous
history of GDM all had 2 abnormal values on an oral glucose tolerance test during pregnancy and comprised the GDM group, according to Carpenter and Coustan (7). The demographics of these
subjects are given in Table 1.
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Longitudinal protocol. A series of experiments was performed before pregnancy and repeated in early (12-14 wk) and late (34-36 wk) gestation. Nonpregnant subjects were evaluated in the follicular phase of the menstrual cycle by use of both history and serum progesterone concentrations. Each subject was instructed in a standard dietary regimen 2 wk before each study period by the GCRC research nutritionist. The dietary regimen was designed to standardize nutritional intake for each subject, to maintain weight before conception, and to allow appropriate increases in weight during pregnancy. The regimen was identical to that used in the treatment of GDM at our institution. At the time of diagnosis of GDM, all subjects were asked to perform self-monitoring of blood glucose levels. Target fasting and preprandial glucose values were <100 mg/dl, and target postprandial values were <120 mg/dl. The following sets of studies were performed on 3 separate days: 1) OGTT and routine laboratory studies; 2) hydrodensitometry with correction of residual lung volume and total body water using H218O; and 3) a two-step hyperinsulinemic-euglycemic clamp to estimate insulin sensitivity and suppression of serum FFAs.
OGTT. Before conception, all subjects were administered a 75-g OGTT as defined by the National Diabetes Data Group. During pregnancy, all subjects were given a 100-g OGTT. Normal glucose tolerance was defined according to the criteria of Carpenter and Coustan (7). The venous plasma glucose was measured using the glucose oxidase method with a Yellow Springs glucose analyzer (Yellow Springs Instrument, Yellow Springs, OH).
Body composition analysis. Each subject's body composition was estimated by underwater weighing with adjustment for residual lung volume using nitrogen dilution. Total body water was measured using isotope dilution of 18O. The details of these methodologies have previously been published (11). Because all subjects had estimates of both total body water and density, a two-compartment model was estimated using the Siri model.
Hyperinsulinemic euglycemic clamp and measurement of FFA.
The hyperinsulinemic euglycemic clamp was performed to estimate
peripheral insulin sensitivity after an 11-h fast as originally described by DeFronzo et al. (17) and as used by us
previously (9). The first 2-h infusion was at 20 mU · m2 · min
1 to achieve a
plasma insulin concentration of ~50 µU/ml, and the second 2-h
infusion was at 40 mU · m
2 · min
1 to achieve a
plasma insulin concentration of ~100 µU/ml. Plasma glucose
concentrations were maintained at 90 mg/dl for the entire 4-h insulin
infusion by sampling the plasma glucose level every 5 min and varying
the infusion of the 20% glucose according to a computer program.
Plasma was collected for measurement of insulin concentrations every 10 min during the last 40 min of the 2-h insulin infusion at each
concentration. The amount of glucose infused was calculated for each
10-min interval and averaged for the last 40 min of each 2-h infusion.
This value was used to estimate insulin sensitivity in peripheral
tissue under steady-state conditions. The primary purpose of the
two-dose insulin infusion clamp was to provide a range of insulin
concentrations to estimate the changes in insulin sensitivity and to
examine suppression of FFA concentrations before and during pregnancy.
Statistical analysis. Data were expressed as means ± SD. Statistical analysis was performed using two-way analysis of variance with repeated measures. These analyses allowed us to evaluate the longitudinal changes over time and differences between groups. Statistical analyses were performed with the Statview II statistical package (Abacus Concepts, Berkeley CA). P < 0.05 was considered significant.
Western blot analysis.
Frozen samples were homogenized in 10 volumes of solubilization
buffer A (in mM: 50 HEPES, pH 7.5, 137 NaCl, 1 MgCl2, 1 CaCl2, 2 Na3VO4, 10 Na2P2O7, 10 NaF, and 2 EDTA plus
1% NP-40, 10% glycerol, 2 µg/ml aprotinin, 10 µg/ml antipain, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 1.5 mg/ml benzamidine, 34 µg/ml PMSF) using a Polytron PTA 20S generator at maximum speed for
30 s. The homogenate was then centrifuged at 65,000 rpm at 4°C
in a model 70 Ti rotor (Beckman, Fullerton CA) for 60 min to remove
insoluble material, and the supernatant was used for analysis. The
pellet was resuspended in solubilization buffer, and 10-40 µg of
protein (Bradford procedure, Bio-Rad Biochemical) were treated with
Laemmli sample buffer, boiled for 5 min, and resolved on 8% SDS-PAGE
gel using a Bio-Rad miniprotein gel apparatus at 100 V for 1 h. To
ensure that the proteins were in a linear range of detection, each
sample was run in an average of three distinct assays on separate
minigels with the use of a concentration between 10 and 40 µg of
protein. Each gel contained an internal standard of adipose tissue
protein (20-µg aliquot) prepared similarly to the adipose samples.
Proteins were electrotransferred from the gel to nitrocellulose
membrane at 90 V (constant) for 1 h using a minitransfer
apparatus. Nonspecific protein binding to the filter was blocked using
5% milk, 10 mM Tris, 150 mM NaCl and 0.02% Tween 20. The filter was
incubated with antibodies to IR-, IRS-1, IRS-2, p85
, or PPAR
(1.5 µg/ml) diluted in blocking buffer for 4 h at 22°C,
followed by extensive washing with Tris-buffered saline (150 mM NaCl,
10 mM Tris + Tween 20). The blots were incubated with secondary
antibody linked to horseradish peroxidase in 10 ml of blocking buffer
for 1 h at 22°C and washed again before the membranes were
exposed to the ECL reagent according to the manufacturer's
instructions (Amersham). Autoradiography was carried out using Kodak
XAR X-ray film, with exposure time varied from 30 s to 3 min, and
the average specific band intensities from each exposure were
quantified by optical density using a Digiscan scanner (US Biochemical,
Cleveland, OH) for integrating the autoradiographic signals. The
results were expressed as arbitrary units relative to an internal
standard sample run together with each blot.
RNA extraction and Northern blot analysis.
Total RNA was extracted from frozen adipose tissue using the guanidine
thiocyanate procedure. Solutions were made in diethyl pyrocarbonate-treated water, and materials were soaked overnight in
diethyl pyrocarbonate-treated water. RNA concentration was determined
by absorbance at 260 nm, and purity was checked by A260/280 ratio and
by minigel electrophoresis. Twenty micrograms of total RNA per sample
were loaded onto a 1.4% agarose/0.66 M formaldehyde gel in 1× MOPS
buffer and size-fractionated by electrophoresis. RNA was transferred
overnight to a GeneScreen Plus membrane (NEN) and ultraviolet
cross-linked in a Stratalinker (Stratagene). Prehybridization was done
at 65°C for 2 h in Church buffer. Random-primed labeling (kit
from GIBCO-BRL) was used to generate 32P-labeled cDNA
(106 dpm/µg). Probes used were leptin, 1.1-kb fragment;
UCP-2, a 1.7-kb EcoR1-EcoR1 fragment of the 3'
end of the rat cDNA (provided by Dr. Bradford Lowell, Harvard Medical
School); and aP2, a 1.1-kb Xba1-PstI fragment of
the human cDNA (provided by Dr.Gokan Hotamisligil, Harvard University).
The LPL cDNA probe was a 1.5-kb fragment obtained from American Type
Culture Collection (no. 63159). The cDNA for PPAR was a 1.5-kb
fragment obtained from Dr. Bruce Spiegelman (Howard Hughes Medical
Center, Boston, MA). The membrane was prehybridized for 30 min in
ExpressHyb (Clontech) and hybridized for 68°C for 2 h. The
membrane was washed in 0.1× standard sodium citrate (SSC) sodium
chloride-sodium citrate-0.1% SDS at 50°C for 2 × 10-20 min. For reprobing, the blots were stripped in 0.1× SSC-2% SDS for
3 × 10 min at 85°C. The blots were then washed with 0.1× SSC, 3 × 10 min at 42°C or until no counts remained. For
quantitative analysis, the hybridized lots were imaged, and the counts
per minute of each hybridized band were determined using an
InstantImager (Packard). Values were normalized to hybridization to
ribosomal RNA (28S) to account for loading differences. Each RNA sample was run an average of three times on different Northern blots and
specific hybridization verified by molecular size markers. Statistical
comparisons between groups were made using Student's t-test
or analysis of variance.
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RESULTS |
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Subject characteristics.
There were no significant differences in age, parity, or fasting
glucose between the Preg-Con and GDM subjects (Table 1). Fasting plasma
insulin levels were significantly greater in GDM compared with the
Preg-Con groups (P < 0.05). There was no difference in
percent fat or body mass index between the obese control and GDM groups
before or during pregnancy. Fasting plasma FFA levels were measured
longitudinally throughout gestation as shown in Fig.
1A. Basal plasma FFA levels
were higher in GDM subjects during prepregnancy (P = 0.055) and remained constant throughout gestation. Plasma FFAs tended
to increase with advancing gestation in Preg-Con subjects, but this
difference did not achieve statistical significance (P = 0.13).
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Hyperinsulinemic euglycemic clamp studies.
Plasma insulin, glucose, and FFA are shown during low- and high-dose
insulin clamp in Table 2. There were no
significant differences in plasma glucose during the clamps. Although
the insulin concentrations tended to be higher in the GDM group,
analysis of the data in Table 2 demonstrated no significant difference (P = 0.09) between the pregnant control and GDM
subjects. There was, however, a significant difference
(P = 0.004) over time in the insulin concentrations in
both groups. On the basis of our previous publication (9),
we attributed the increase in insulin during the clamps between early
and late gestation to the reduced metabolic clearance rate (MCR) of
insulin with advancing gestation in both control and GDM subjects.
There is a further increase in the MCR of insulin in the pregnant
control compared with GDM (P = 0.046). Thus, although
there is a higher insulin concentration over time, this may be related
to a decrease in the MCR of insulin under the experimental conditions,
possibly due to hepatic insulin resistance. The insulin levels were not
statistically different between control and GDM subjects, however,
during the high-dose insulin clamps.
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Western blot analysis.
Western blot analysis was performed on abdominal subcutaneous adipose
tissue biopsies and the relative levels of insulin signaling proteins
quantified by densitometry as shown in Fig.
3, A-D. There were no
significant differences in insulin receptor protein levels (-subunit) in adipose tissue among the three groups
(P = 0.54). Adipose tissue IRS-1 protein levels were
43% lower in the GDM compared with the Preg-Con groups
(P = 0.02). In contrast, IRS-2 protein content was
increased 1.7-fold in GDM subjects and 1.5-fold in Preg-Con compared
with Non-Preg-Con subjects. The level of p85
subunit of PI 3-kinase
was increased twofold in GDM subjects compared with Preg-Con subjects
(P = 0.03).
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Northern blot analysis.
To further explore the potential molecular mechanisms underlying the
decrease in FFA storage during late pregnancy, we measured the
steady-state mRNA expression pattern of PPAR, leptin, UCP-2, aP2,
and LPL, as shown in representative blots in Fig.
4 and quantified by scanning densitometry
in Fig. 5. The levels of PPAR
mRNA
were significantly decreased by 48% in GDM (P = 0.005)
and in Preg-Con subjects by 38% (P = 0.006) compared
with Non-Preg-Con subjects. The expression pattern for leptin mRNA
tended to be lower in GMD and Preg-Con subjects but was not
statistically significant compared with Non-Preg-Con subjects. The
levels of UCP-2 mRNA were decreased in GDM subjects by 39%
(P = 0.03) and by 45% (P = 0.006) in
Preg-Con subjects compared with Non-Preg-Con subjects. The level of aP2 mRNA was decreased in GDM subjects by 52% compared with Preg-Con subjects (P = 0.04) and by 74% (P = 0.02) compared with Non-Preg-Con subjects. Similarly, GDM subjects
demonstrated a ~73% reduction in LPL mRNA levels compared with
Preg-Con (P = 0.001) and a 47% reduction compared with
Non-Preg-Con subjects (P = 0.007). Interestingly, LPL
mRNA levels were higher in Preg-Con subjects compared with Non-Preg-Con
subjects by 32% (P = 0.02).
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DISCUSSION |
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Plasma FFAs are important regulators of insulin resistance, and circulating FFA concentrations tend to be higher in women with GDM. Although there was a tendency for circulating FFA concentrations to be higher in GDM subjects before conception, our longitudinal results indicate that most of the changes in plasma FFA occur in late pregnancy during physiological hyperinsulinemia. When the FFA levels are expressed relative to insulin, the ratio increased significantly over time in GDM subjects (Table 2), especially during the hyperinsulinemic clamp. Although there were few subjects in this study, the data suggest a gradual loss of insulin suppression of lipolysis during late pregnancy. These results are similar to those of Xiang et al. (46), who reported no change in basal FFA but impaired insulin suppression of FFA in a large series of Latina subjects with GDM studied during the third trimester compared with normal pregnant controls. Sivan et al. (42) showed that, in normal pregnant subjects, the basal rates of FFA oxidation, reesterification, and lipolysis were similar; however, the inhibitory effect of insulin on the rate of lipolysis declined by 30% from the second to the third trimester in normal pregnancy. FFA concentrations between basal and clamp conditions were not different between groups; however, when the percent suppression by insulin was considered, the GDM subjects had a significant decrease in percent suppression over time (Fig. 1B). We interpret this to mean that slightly higher basal FFA levels and less insulin suppression (despite higher insulin levels) suggest that GDM subjects are more resistant in the basal state and tend to remain that way during late gestation. This is very similar to data reported previously for glucose disposal in these subjects (9). Taken together, these data indicate that normal pregnancy is associated with insulin resistance to suppress lipolysis with advancing gestation, whereas GDM is associated with insulin resistance to suppress lipolysis before gestation, which is exacerbated during pregnancy, especially during late gestation.
Although the mechanism by which insulin suppresses lipolysis in human
adipocytes is not completely understood, studies strongly suggest that
a key antilipolytic action of insulin involves the level of
IRS-1-associated PI 3-kinase activity. Zierath et al. (50)
reported that IRS-1 protein expression and tyrosine phosphorylation were reduced by 50% in human omental compared with subcutaneous fat
cells, and this corresponded with a 50% reduction in PI 3-kinase activity and the inability of insulin to suppress lipolysis. Similarly, incubation with wortmannin, a PI 3-kinase inhibitor, inhibited the
normal ability of insulin to block FFA release in normal human fat
cells. Rondinone et al. (38) recently reported that, in adipocytes from non-insulin-dependent diabetes mellitus subjects, IRS-1
protein and tyrosine phosphorylation were severely reduced, whereas
IRS-2 levels remained unchanged; however, maximal IRS-2-associated PI
3-kinase activity was significantly decreased compared with nondiabetic
subjects. Our results in normal pregnancy did not show any significant
decrease in protein expression in adipose tissue compared with
nonpregnant controls, indicating that declining insulin regulation of
lipolysis over time could be due to a change in protein activity rather
than a change in protein levels. Existing data in the literature
suggest that decreased IRS-1-associated PI 3-kinase activity is the
best early effector of reduced suppression of plasma FFA by insulin,
and decreased IRS-1 expression and increased p85 levels are both
associated with reduced PI 3-kinase activity. Thus, although decreased
IRS-1 and increased p85
in GDM subjects do not imply causality, it
is tempting to speculate that the significant changes in GDM vs. normal
pregnant control subjects may inhibit insulin signaling to PI 3-kinase,
which might be responsible for the greater insulin resistance to
suppression of lipolysis in GDM. Although we did not measure PI
3-kinase activity, there is a close relationship between decreased
IRS-1 protein and the inability of insulin to stimulate PI 3-kinase
activity in humans with insulin resistance (21, 38, 50).
Studies are currently underway in our laboratory to identify whether
decreased PI 3-kinase activity in response to insulin in isolated
adipocytes is causally related to reduced insulin inhibition of
lipolysis in pregnancy relative to GDM.
The physiological significance of increased p85 subunit of PI
3-kinase remains uncertain. However, our results are consistent with
recent in vivo animal data that both proteins are important in the
insulin resistance of GDM. We recently found in an animal model of GDM
that pregnancy triggers a novel functional redistribution of the
insulin-signaling environment in vivo. This environment preferentially
increases p85
expression and activity associated with the insulin
receptor away from IRS-1. Despite an increase in p85 and total PI
3-kinase activity, IRS-1-associated PI 3-kinase was severely impaired
and IRS-1 protein levels were decreased. In conjunction with the
redistribution of PI 3-kinase to the insulin receptor, there is a
selective activation of serine kinases downstream from PI 3-kinase.
Consistent with this result, IRS-1 is degraded, insulin-stimulated
coupling between IRS-1 and p85
is inhibited, and GLUT-4
translocation was markedly decreased (40a). Unfortunately, we
did not have the opportunity to be able to test these specific mechanisms in humans in the present study. However, our current thinking is that both increased p85
and decreased IRS-1 together could be important mechanisms underlying impaired insulin signaling and
reduced suppression of lipolysis in the adipocyte during GDM.
Alternatively, we found an increase in IRS-2 levels in GDM subjects compared with nonpregnant controls, suggesting that increased IRS-2 could compensate for the lack of IRS-1 expression. However, the levels of IRS-2 were increased in both Preg-Con and GDM subjects compared with nonpregnant controls, suggesting that increased IRS-2 was unable to compensate for the lack of IRS-1 in GDM subjects. This is similar to transgenic mice lacking IRS-1, in which there is an upregulation of IRS-2 but reduced insulin-stimulated glucose transport (1). Other possibilities for impaired insulin action to suppress FFA levels in GDM could include the inability to activate phosphodiesterases, as well as other metabolic events, such as failure to reesterify FFA.
Although our results need to be confirmed in isolated adipocytes from
GDM subjects, impaired suppression of FFA may have important implications for the mechanisms of whole body insulin resistance in
these subjects. Women with GDM are more insulin resistant in the
nonpregnant state compared with obese subjects with normal glucose
tolerance (9) and are at high risk for subsequent
development of type 2 diabetes (6). Thus a reduction in
adipose tissue IRS-1 or an increase in p85, if retained postpartum,
could be an important factor underlying the tendency for higher fasting plasma FFA in former GDM women. Relative to possible hormone
differences that could account for increased insulin resistance to
suppression of FFA in GDM vs. normal pregnancy, we performed an
analysis of progesterone, estradiol, human chorionic gonadotropin,
human placental lactogen, cortisol, prolactin, leptin, and tumor
necrosis factor (TNF)-
in these subjects as shown in Table
3. As expected, the levels of all of
these hormones increased significantly throughout gestation.
However, at no time was there a significant difference between normal
pregnant and GDM subjects. These data underscore the observation that
the greater insulin resistance in GDM is due mostly to intrinsic or
genetic insulin resistance that is maintained throughout pregnancy,
perhaps combined with a change in sensitivity to these hormones, rather
than a change in a specific hormone in pregnancy.
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The transcription factor PPAR is required for adipocyte
differentiation and expression of genes important for lipid metabolism in adipose tissue. We observed a decrease in steady-state PPAR
mRNA
and protein concentration in normal and GDM subjects during late
gestation, at a time when adipose tissue stores are being mobilized to
provide nutrients for maternal and fetal metabolism (44).
In humans, fasting is associated with a fall in PPAR
mRNA and
protein levels in adipose tissue (45). The decrease in
PPAR
in pregnancy was beyond that in normal, nonpregnant obese subjects, suggesting that PPAR downregulation may be part of the molecular adaptation to pregnancy termed "accelerated starvation," in which fasting accelerates fat catabolism to meet fetal nutrient demand in late gestation (18). In adipocyte cell lines,
the cytokine TNF-
induces adipose de-differentiation via
downregulation of PPAR
(47). We previously reported
that plasma levels of TNF-
increase significantly between early and
late pregnancy and correlate well with the extent of insulin resistance
measured by hyperinsulinemic clamps (8). In the present
study, we were unable to detect TNF-
mRNA in adipose tissue samples
from these subjects by means of conventional Northern blot analysis.
However, others have found that TNF-
mRNA and protein are present in
human placenta at early and late gestation (14),
suggesting that increased circulating TNF-
during pregnancy may be
placenta derived. Increased TNF-
could downregulate PPAR
expression, thereby triggering insulin resistance in adipose tissue as
well as reduced insulin signaling in skeletal muscle. Whether decreased
PPAR
expression plays a role in increased lipolysis, or adipose cell
de-differentiation in late pregnancy, remains to be investigated.
The levels of UCP-2 mRNA were also significantly decreased in GDM and
normal pregnant subjects. PPAR modulates UCP2 gene expression
indirectly by altering the activity or expression of other
transcription factors that bind to the UCP-2 promoter
(31). The role of UCP-2 in adipose tissue metabolism is
currently under investigation. Although UCP-2 has uncoupling activity
(36), recent studies in UCP-2 knockout mice suggest that
body composition is normal in these mice (2). Possible
roles for UCP-2 may include the regulation of ATP synthesis or control
of reactive oxygen species production by mitochondria (4,
35). The later functions suggest that UCP2 expression might be
involved in regulation of adipose tissue apoptosis.
The levels of steady-state LPL mRNA were significantly reduced in GDM
subjects; however, LPL mRNA levels did not correspond to reduced
PPAR and insulin resistance in normal pregnant subjects. A number of
studies have shown that translational modification is an important
mechanism for the downregulation of LPL activity, including decreased
activity induced by TNF-
(26). In humans with type 2 diabetes, adipose tissue LPL activity is reduced, but this is not
accompanied by a decrease in LPL mRNA (41). Our results
suggest that other factors besides the level of PPAR
may be involved
in the regulation of steady-state LPL mRNA levels in normal pregnancy.
We also did not detect a significant change in steady-state leptin mRNA levels in pregnant or GDM subjects compared with nonpregnant controls. Plasma leptin levels increase during early gestation (23), either from placenta (30), maternal adipose tissue stores (24), or both. Our data suggest that, during late pregnancy, maternal adipose tissue leptin synthesis does not contribute to increased plasma leptin levels, suggesting that the placenta may be an important source for leptin production or that leptin synthesis is downregulated in adipose tissue during late pregnancy.
Finally, the levels of aP2 mRNA were significantly lower in GDM
subjects compared with both Preg-Con and Non-Preg-Con groups. Because
aP2 binds to cytosolic fatty acids, it may modulate their intracellular
concentrations, availability, or subcellular trafficking (15). This could have effects on the activity of nuclear
hormone receptors that are regulated by fatty acid ligands. Thus
PPAR activity may be altered in GDM subjects in addition to
decreased concentration of the receptor.
In summary, we have shown that normal pregnancy is associated with a
decline in insulin suppression of FFA throughout gestation, whereas in
GDM subjects the insulin suppression of FFA is more severe in late
pregnancy. Because of the strong association between IRS-1, p85, and
insulin signaling, our data suggest the decreased IRS-1 protein, as
well as increased p85 protein levels in adipose tissue, may
contribute to impaired suppression of FFA by insulin in GDM. Decreased
PPAR
mRNA and protein levels in adipose tissue are consistent with
insulin resistance and could contribute to the declining adipose tissue
mass and increasing plasma FFA that accompany late gestation.
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ACKNOWLEDGEMENTS |
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We thank the subjects who participated in these studies and gratefully acknowledge Dr. Saeid B. Amini, CWRU, for his assistance in statistical analysis.
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FOOTNOTES |
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This research was supported by National Institute of Child Health and Human Development Grant R01-HD-22965 (P. M. Catalano) and General Clinical Research Center Grant M01-RR-80. J. E. Friedman was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50272 and by Perinatal Emphasis Research Center Grant NIH-NICHD-11089.
Present address of S. Nizielski: Biomedical and Health Sciences, 327 Padnos Hall, Grand Valley State University, Allendale, MI 49401.
Address for reprint requests and other correspondence: J. Friedman, Univ. of Colorado Health Sciences Center, Section of Neonatology-B195, RM 3402, 4200 E. Ninth Ave, Denver, CO 80262 (E-mail: Jed.Friedman{at}UCHSC.edu).
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.
10.1152/ajpendo.00124.2001
Received 15 March 2001; accepted in final form 7 November 2001.
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