Downregulated IRS-1 and PPARgamma in obese women with gestational diabetes: relationship to FFA during pregnancy

Patrick M. Catalano1, Steven E. Nizielski3, Jianhua Shao2, Larraine Preston1, Liping Qiao2, and Jacob E. Friedman1,2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 p85alpha 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-gamma (PPARgamma ) 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 PPARgamma 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 p85alpha 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. PPARgamma exists as two isoforms, PPARgamma 1 and PPARgamma 2, which are transcribed from the same gene using alternative promoters (49). PPARgamma 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 PPARgamma , 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 PPARgamma were embryonic lethal; however, heterozygous PPARgamma (+/-) 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). PPARgamma has also been implicated in the regulation of systemic insulin sensitivity (48). This is supported by the finding that PPARgamma 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 PPARgamma are nonobese yet have severe insulin resistance and type 2 diabetes (3). The target genes induced by PPARgamma 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 PPARgamma , along with key insulin-signaling proteins, might underlie the changes in the FFA metabolism that accompany normal human pregnancy and GDM.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta and IRS-1 antibodies were from Transduction Laboratories (Lexington, KY). The IRS-2 and p-85alpha antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The PPARgamma antibody was purchased from Santa Cruz Laboratories (Santa Cruz, CA) and primarily recognizes the PPARgamma 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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Table 1.   Characteristics of subjects in the longitudinal study at term

In addition to these subjects, a group of patients was recruited as outpatient volunteers to undergo adipose tissue biopsies during either scheduled elective cesarean delivery or gynecological surgery. These included eight obese pregnant subjects (Preg-Con), seven obese subjects diagnosed with GDM during the third trimester (GDM), and seven obese nonpregnant subjects (Non-Preg-Con). All subjects had >30% body fat, and basal insulin levels were significantly higher in GDM compared with Preg-Con and Non-Preg-Con subjects (Non-Preg-Con 10.8 ± 2.1; Preg-Con 13.1 ± 2.1; GDM = 32.1 ± 9.7, P < 0.5 vs. Non-Preg and Preg-Con). Pregnant subjects scheduled for cesarean deliveries were classified as normal or GDM after a 100-g OGTT (7). The OGTT was administered between 26 and 28 wk of gestation. Subjects diagnosed with GDM were treated with diet therapy before delivery. None of the obese pregnant subjects had a family history (1st or 2nd degree relative) of diabetes mellitus. None of the Non-Preg-Con subjects had, on the basis of a 75-g OGTT, diabetes mellitus. Subcutaneous adipose tissue was obtained from the abdomen at the time of elective cesarean or gynecological surgery under a continuous lumbar epidural infusion of local anesthetic. In nonpregnant patients, general anesthesia was induced with a nitrous oxide-oxygen mixture. Only saline was given intravenously before the biopsy.

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 · m-2 · 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.

Insulin sensitivity was estimated as the glucose infusion rate required for maintaining plasma glucose concentrations constant at 90 mg/dl during the clamp. FFA concentrations were measured during the basal period as the mean of serum samples obtained at 90, 100, 110, and 120 min. FFA samples were also measured at 10-min intervals during the last 40 min of the clamp during steady-state insulin infusion (minutes 210, 220, 230, 240) to estimate the percent suppression from basal during insulin infusion and change in suppression over time and between groups.

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-beta , IRS-1, IRS-2, p85alpha , or PPARgamma (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 PPARgamma 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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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Fig. 1.   Longitudinal changes in basal plasma free fatty acids (FFA; A), and insulin suppression of FFA (B) in pregnant-control (Preg-Con) and gestational diabetes mellitus (GDM) subjects. The data were analyzed by ANOVA and Fisher's protected least significant difference testing for post hoc analysis between groups. Data are means ± SD for Preg-Con (n = 4) and GDM (n = 5) subjects.

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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Table 2.   Glucose, insulin, FFA, and FFA-to-insulin ratio values at basal and low- and high-dose insulin clamp over time

During the high-dose insulin clamp, we analyzed the change in FFA concentrations, and there was a significant decrease in the ability of insulin to suppress FFA concentrations over time (P = 0.005), which is reflected by the data in Table 2 as a higher residual FFA concentration in the GDM and control subjects. Although there was no significant difference between groups in terms of the actual FFA concentrations, because the GDM subjects tended to have higher starting basal plasma FFA as shown in Fig. 1A (P = 0.055), we tried to account for this by calculating the percent suppression as shown by Fig. 1B. This showed a significant time-group interaction (P = 0.049), which upon further analysis showed that the GDM subjects had a significant decrease (P = 0.025) in percent suppression between early and late pregnancy. Hence, the difference in actual FFA concentrations between basal and clamp conditions was not different between groups; however, when the percent suppression is looked at, the GDM subjects had a significant decrease in percent suppression over time (Fig. 1B). There were no significant differences in insulin suppression of FFA either between groups or across time during insulin infusion at 20 mU · kg-1 · min-1. However, during 40 mU · kg-1 · min-1 insulin infusion, there was a significant decrease in the ability of insulin to decrease FFA in Preg-Con and GDM subjects from early to late gestation (0.026, Fig. 1B). Insulin's ability to suppress plasma FFA was significantly lower over time (P = 0.025) in the GDM subjects compared with Preg-Con subjects. Because the insulin levels tended to be higher during the clamp across time, we expressed the FFA data divided by the insulin levels to try to account for possible differences in insulin levels (Table 2). During the insulin clamp, the data show that there was a significant increase in the FFA-to-insulin ratio over time in the GDM subjects (P = 0.0021). This suggests that, after differences in insulin levels in late pregnancy during the clamp were accounted for, there was less suppression of FFA levels compared with early pregnancy. These results are consistent with the percent suppression data presented in Fig. 1B and point to a progressive resistance of insulin to suppressing lipolysis despite higher insulin concentrations in late pregnancy.

The insulin sensitivity index (ISI), measured during the insulin clamp by quantifying steady-state glucose disposal, was reported previously in these subjects (9) and declined significantly across time (P = 0.03) and was lower in the GDM group compared with the Preg-Con group (P = 0.03) (data not shown). During late pregnancy, there was a significant correlation between fasting plasma FFA and ISI as measured during high-dose insulin infusion (r = 0.88, P < 0.0001, Fig. 2).


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Fig. 2.   Correlation between basal plasma FFA and insulin sensitivity as measured during high-dose insulin infusion (40 mU · kg-1 · min-1) in Preg-Con and GDM subjects.

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 (beta -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 p85alpha subunit of PI 3-kinase was increased twofold in GDM subjects compared with Preg-Con subjects (P = 0.03).


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Fig. 3.   Expression of insulin-signaling proteins in human adipose tissue from nonpregnant control (NP-Con, Non-Preg-Con), Preg-Con, and GDM subjects. Adipose tissue biopsies from the subcutaneous abdominal compartment were obtained at the time of scheduled cesarean delivery or during gynecological surgery. Samples were analyzed by SDS-PAGE as described in METHODS. The images show an example autoradiogram of proteins detected with anti-insulin receptor (COOH terminal) antibody, anti-insulin receptor substrate (IRS)-1, anti-IRS-2, and anti-phosphatidylinositol (PI) 3-kinase (p85 alpha  subunit) detection. Bar graphs depict quantification of the autoradiograms from multiple Western blots analyzed by scanning densitometry. Data are means ± SE, expressed as arbitrary units relative to the values for nonpregnant controls.

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 PPARgamma , 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 PPARgamma 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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Fig. 4.   Representative Northern blot analysis of peroxisome proliferator-activated receptor (PPAR)gamma , leptin, uncoupling protein (UCP)-2, adipose-specific intracellular fatty acid-binding protein (aP2), and lipoprotein lipase (LPL) in subcutaneous adipose tissue biopsies from Non-Preg-Con, Preg-Con, and GDM subjects. Total cellular RNA was isolated from adipose tissue biopsies obtained under anesthesia as outlined in METHODS. RNA was size-fractionated, transferred, and hybridized with specific cDNA probes as outlined in METHODS.



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Fig. 5.   Altered mRNA expression levels of PPARgamma (A), leptin (B), UCP-2 (C), aP2 (D), and LPL (E) in subcutaneous adipose tissue biopsies from Non-Preg-Con, Preg-Con, and GDM subjects. Relative levels of mRNA expression were determined by densitometry and expressed as a percentage of mRNA hybridization (arbitrary units) from Non-Preg-Con samples detected on the same Northern blot. All values were corrected for 18S ribosomal mRNA to account for loading differences. Results are means ± SE of 7-8 patients/group.

To confirm that changes in steady-state PPARgamma mRNA levels were present at the protein level, Western blots were performed in a limited number of subjects (Fig. 6). PPARgamma protein levels were significantly reduced in GDM subjects by 37% (P = 0.05) and by 25% (P = 0.06) in Preg-Con compared with Non-Preg-Con subjects.


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Fig. 6.   Expression of PPARgamma protein in human adipose tissue from Non-Preg-Con, Preg-Con, and GDM subjects. Adipose tissue biopsies from the subcutaneous abdominal compartment were obtained at the time of scheduled cesarean delivery or during gynecological surgery as outlined in METHODS. Samples were analyzed by SDS-PAGE as described. The images show an example autoradiogram detected with anti-PPARgamma antibody. Bar graphs depict quantification of the autoradiograms analyzed by scanning densitometry. Data are means ± SE expressed as arbitrary units relative to the values for Non-Preg-Con subjects.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 p85alpha levels are both associated with reduced PI 3-kinase activity. Thus, although decreased IRS-1 and increased p85alpha 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 p85alpha 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 p85alpha 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 p85alpha 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 p85alpha 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 p85alpha , 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)-alpha 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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Table 3.   Plasma hormone levels in obese control and GDM subjects during gestation

The transcription factor PPARgamma is required for adipocyte differentiation and expression of genes important for lipid metabolism in adipose tissue. We observed a decrease in steady-state PPARgamma 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 PPARgamma mRNA and protein levels in adipose tissue (45). The decrease in PPARgamma 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-alpha induces adipose de-differentiation via downregulation of PPARgamma (47). We previously reported that plasma levels of TNF-alpha 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-alpha mRNA in adipose tissue samples from these subjects by means of conventional Northern blot analysis. However, others have found that TNF-alpha mRNA and protein are present in human placenta at early and late gestation (14), suggesting that increased circulating TNF-alpha during pregnancy may be placenta derived. Increased TNF-alpha could downregulate PPARgamma expression, thereby triggering insulin resistance in adipose tissue as well as reduced insulin signaling in skeletal muscle. Whether decreased PPARgamma 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. PPARgamma 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 PPARgamma 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-alpha (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 PPARgamma 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 PPARgamma 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 p85alpha protein levels in adipose tissue, may contribute to impaired suppression of FFA by insulin in GDM. Decreased PPARgamma 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.


    ACKNOWLEDGEMENTS

We thank the subjects who participated in these studies and gratefully acknowledge Dr. Saeid B. Amini, CWRU, for his assistance in statistical analysis.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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