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Am J Physiol Endocrinol Metab 290: E1313-E1320, 2006. First published January 24, 2006; doi:10.1152/ajpendo.00071.2005
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Decreased nuclear hormone receptor expression in the livers of mice in late pregnancy

Trevor R. Sweeney, Arthur H. Moser, Judy K. Shigenaga, Carl Grunfeld, and Kenneth R. Feingold

Department of Medicine, University of California, San Francisco, and Metabolism Section, Department of Veterans Affairs Medical Center, San Francisco, California

Submitted 22 February 2005 ; accepted in final form 15 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the third trimester of pregnancy, there is an increase in serum triglyceride and cholesterol levels. The mechanisms accounting for these changes in lipid metabolism during pregnancy are unknown. We hypothesized that, during pregnancy, the expression of nuclear hormone receptors involved in regulating lipid metabolism would decrease. In 19-day pregnant mice, serum triglyceride and non-HDL cholesterol levels were significantly increased, whereas total cholesterol was slightly decreased, because of a decrease in the HDL fraction. Peroxisome proliferator-activated receptor (PPAR){alpha}, PPARbeta/{delta}, and PPAR{gamma}, liver X receptor (LXR){alpha} and LXRbeta, farnesoid X receptor (FXR), and retinoid X receptor (RXR){alpha}, RXRbeta, and RXR{gamma} mRNA levels were significantly decreased in the livers of 19-day pregnant mice. Additionally, the expressions of thyroid receptor (TR){alpha}, pregnane X receptor, sterol regulatory element-binding proteins (SREBP)-1a, SREBP-1c, SREBP-2, and liver receptor homolog 1 were also decreased, whereas the expression of TRbeta, constitutive androstane receptor, and hepatic nuclear factor 4 showed no significant change. mRNA levels of the PPAR target genes carnitine-palmitoyl transferase 1{alpha} and acyl-CoA oxidase, the LXR target genes SREBP1c, ATP-binding cassettes G5 and G8, the FXR target gene SHP, and the TR target genes malic enzyme and Spot14 were all significantly decreased. Finally, the expressions of PPAR{gamma} coactivator (PGC)-1{alpha} and PGC-1beta, known activators of a number of nuclear hormone receptors, were also significantly decreased. The decreases in expression of RXRs, PPARs, LXRs, FXR, TRs, SREBPs, and PGC-1s could contribute to the alterations in lipid metabolism during late pregnancy.

farnesoid X receptor; liver X receptor; peroxisome proliferator-activated receptor; retinoid X receptor; lipid metabolism

IN MANY ANIMALS, INCLUDING HUMANS, there is an increase in serum triglyceride and cholesterol levels during the third trimester of pregnancy because of an increase in circulating VLDL and LDL (16, 17, 46). A number of factors contribute to this increase in circulating lipids (16–18). Adipose tissue lipoprotein lipase activity is reduced (18), leading to a decrease in the clearance of triglyceride-rich lipoproteins. Moreover, triglyceride synthesis is decreased in adipose tissue, whereas lipolysis is enhanced during pregnancy (18), resulting in an increase in serum free fatty acid levels. In the liver, pregnancy leads to a reduction in fatty acid oxidation (15), although fatty acid synthesis is increased (62). The increase in de novo fatty acid synthesis and the enhanced delivery of fatty acids from adipose tissue coupled with the decrease in fatty acid oxidation increase the pool of fatty acids available in the liver, resulting in an increase in triglyceride synthesis (58). The activity of diacylglycerol acetyltransferase, a key enzyme in the synthesis of triglycerides, is also increased in the liver during pregnancy (50). This increase in triglyceride synthesis in the liver leads to an increase in the hepatic production of VLDL (58). Increased hepatic fatty acid and triglyceride synthesis may also contribute to the increased occurrence of fatty liver during pregnancy.

In addition to changes in fatty acid/triglyceride metabolism, pregnancy is associated with changes in cholesterol metabolism (37). Hepatic cholesterol synthesis is increased during pregnancy (4, 13), and the activity of cholesterol 7 {alpha}-hydroxylase (Cyp7a), the first enzyme in the synthesis of bile acids, is decreased (50). These changes would result in the increased availability of cholesterol in the liver for the production of lipoproteins.

The mechanisms accounting for these changes in hepatic triglyceride and cholesterol metabolism during pregnancy are unknown. One potential site of regulation is nuclear hormone receptors, a large family of transcription factors characterized by a central DNA binding domain that targets the receptor to specific response elements in the promoter of genes and a COOH-terminal portion that contains a ligand binding domain (29). This ligand binding domain recognizes specific compounds depending on receptor type. It is now recognized that, in a subgroup of the nuclear hormone receptors, the ligand binding domain binds lipids, and these receptors serve as liposensors (8). These receptors, which include the peroxisome proliferator-activated receptor (PPARs), liver X receptors (LXRs), and farnesoid X receptor (FXR), monitor the lipid content of cells and then regulate the expression of many proteins that play key roles in lipid metabolism (8). The PPARs are activated by polyunsaturated fatty acids, eicosanoids, and synthetic ligands such as fibrates and thiazolidinediones (24, 47).

PPAR{alpha} predominately regulates fatty acid oxidation in the liver (24, 47). PPAR{gamma} plays a key role in adipocyte differentiation, increasing triglyceride formation and storage (44). The role of PPAR{delta} is less well characterized, but it appears to be important in lipid metabolism (57). LXR{alpha} and LXRbeta are both activated by oxysterols and regulate genes involved in reverse cholesterol transport and bile acid formation (52, 54). FXR is activated by bile acids and regulates bile acid synthesis and triglyceride metabolism (9, 11). The PPARs, LXRs, and FXR all form heterodimers with retinoid X receptor (RXR), which facilitates the binding to DNA response elements (28). RXR{alpha} is the isoform expressed most abundantly in the liver, but RXRbeta and RXR{gamma} are also present (27, 30). In addition to these liposensors, the thyroid hormone receptor (TR) also forms heterodimers with RXR and is well known to regulate the expression of a variety of proteins important in lipid metabolism (1, 28, 29).

Inflammation is characterized by a large number of changes in lipid metabolism (20), which are similar to the changes in lipid metabolism that are observed during the last trimester of pregnancy. In previous studies, we have shown that inflammation results in the decreased expression and activity of RXRs, PPARs, LXRs, FXR, and TR in the liver (2, 3, 22). Furthermore, pregnancy is a low-grade inflammatory state characterized by increases in proinflammatory cytokines (55), the mediators of inflammation, and the serum levels of marker proteins of inflammation, such as C-reactive protein, are increased (45). We therefore hypothesized that pregnancy would result in changes in the hepatic levels of nuclear hormone receptors that regulate lipid metabolism.

In the present study, we demonstrate that during the third trimester of pregnancy there is a decrease in the nuclear hormone receptor liposensors and this decrease is accompanied by a decrease in the expression of their target genes. The decreases in expression of RXRs, PPARs, LXRs, FXR, and TRs could be one mechanism that leads to the alterations in lipid metabolism that characterize the third trimester of pregnancy.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Female C57Bl/6 mice were maintained in a normal-light-cycle room and were provided with rodent chow and water ad libitum. Mice were timed pregnant from the day that the vaginal plug was observed. On day 19 of pregnancy, the pregnant mice were killed along with age-matched controls. The animals were fed ad libitum until they were killed. Anesthesia was induced with halothane, and blood was drawn. Liver tissue was harvested and snap frozen in liquid nitrogen. The tissue was stored at –80°C.

RNA isolation and Northern blot analysis. Total RNA from mouse was isolated from 300 mg of snap-frozen whole liver tissue with Tri Reagent (Sigma). Poly(A)+ RNA was subsequently purified with oligo(dT) cellulose. RNA was quantified by measuring absorption at 260 nm; 10 µg of poly(A)+ RNA was denatured and electrophoresed on a 1% agarose-formaldehyde gel. mRNA was then electrotransferred to Nytran membrane (Schleicher & Schuell). Prehybridization, hybridization, and washing procedures were performed as described previously (33). Membranes were probed with [{alpha}-32P]dCTP-labeled cDNAs with the use of the random priming technique (Amersham Biosciences). mRNA levels were detected by exposure to x-ray film and measured by densitometry or by exposure to a phosphor screen and measured on a Bio-Rad Personal Molecular Imager FX phosphorimager. The uniformity of sample applications was checked by ultraviolet visualization of the ethidium bromide-stained gels before transfer to Nytran membranes.

We and others have found that pregnancy decreases mRNA levels of genes that are widely used for normalizing data: cyclophilin, GAPDH, and actin in rodent and bovine liver (43). However, the differing direction of the changes in mRNA levels (≥50% decrease vs. >2-fold increase) for specific proteins in late pregnancy, the magnitude of the alterations, and the relatively small standard errors of the mean make it unlikely that the changes observed are from unequal loading of mRNA or nonspecific effects. In addition, because the expression of certain genes (for example HNF4) did not change in the liver of mice during late pregnancy, we were able to normalize our data using HNF4 to control for loading and transfer.

Human RXR{alpha} cDNA was a gift from Dr. D. Bikle (University of California, San Francisco, CA). Human LXR{alpha} and LXRbeta cDNAs and mouse RXRbeta and RXR{gamma} cDNA probes were a gift from Dr. D. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). PPAR{alpha}, PPARbeta, and PPAR{gamma} cDNAs were gifts from Dr. A. Bass (University of California, San Francisco, CA). Mouse pregnane X receptor (PXR) cDNA was provided by Dr. T. Willson (Glaxo Smith Kline, Research Triangle Park, NC). The following cDNA probes were generated by RT-PCR with the primer sequences listed in Table 1, starting from total RNA from mouse liver [ATP-binding cassettes (ABC)G5 and ABCG8, acyl-CoA oxidase (ACO), constitutive androstane receptor (CAR), carnitine palmitoyltransferase I{alpha} (CPT I{alpha}), FXR, hepatocyte nuclear factor (HNF)4, liver receptor homolog (LRH)-1, malic enzyme (ME), PPAR{gamma} coactivator (PGC)-1{alpha}, PGC1beta, small heterodimer partner (SHP), Spot 14 (S14), and TRbeta1 and heart TR{alpha}.


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  		Table 1. Primer sequences used to make cDNA probes for Northern blot

 
Quantitative real-time PCR. First-strand cDNA was synthesized from 1 µg of total RNA with random hexamer primers using Advantage RT-for-PCR kit (BD Biosciences Clontech). The real-time PCR contained, in a final volume of 20 µl, 20 ng of reverse-transcribed total RNA, 300 nM forward and reverse primers, and 10 µl of 2x SYBR Green PCR Master Mix (Stratagene, La Jolla, CA). PCR was carried out in 96-well plates with Mx3000P real-time PCR system (Stratagene). We calculated the relative amount of all mRNAs by use of the comparative CT method. 36B4 mRNA was used as the invariant control for all experiments. Primer sequences used for quantitative PCR analysis are listed in Table 2.


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  		Table 2. Primer sequences used for qualitative PCR analysis

 
Measurement of serum triglyceride and cholesterol. Total triglyceride levels were measured in the serum of nonpregnant and day 19 pregnant mice as per Sigma diagnostics procedure 337. Total cholesterol and HDL cholesterol were measured as per Sigma cholesterol procedure 352. Separation of HDL cholesterol from non-HDL cholesterol was performed with the ThermoDMA HDL precipitating reagent procedure.

Preparation of nuclear extracts. Fresh liver (1.5–2 g) was homogenized in 10 mM HEPES (pH 7.9), 25 mM KCl, 0.15 mM spermine, 1 mM EDTA, 2 M sucrose, 10% glycerol, 50 mM NaF, 2 mM sodium metavanadate, 0.5 mM dithiothreitol, and 1% protease inhibitor mixture (Sigma). Immediately after homogenization, nuclear proteins were extracted as described by Neish et al. (34), except that 1 mM NaF, 0.1 mM metavanadate, and 1% protease inhibitor mixture (Sigma) were added to all buffers. Nuclear protein content was determined by the Bradford assay (Bio-Rad), and yields were similar in control and LPS-treated groups.

Western blot analysis. Denatured nuclear protein (20 µg) was loaded onto 10% polyacrylamide precast gels (Bio-Rad) and subjected to electrophoresis. After electrotransfer onto polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), blots were blocked with PBS containing 0.10% Tween 20 and 5% dry milk for 1 h at room temperature and incubated for 1 h at room temperature with anti-RXR antibody (Santa Cruz Biotechnology) at a dilution of 1:5,000. Immune complexes were detected using horseradish peroxidase-linked donkey anti-rabbit IgG (dilution 1:20,000) according to the ECL Plus Western blotting kit (Amersham Pharmacia Biotech). Immunoreactive bands obtained by autoradiography were quantified by densitometry.

Measurement of ME activity. The assay method of Thorne et al. (53) was used. Briefly, 100 mg of liver were homogenized in nine parts of ice-cold homogenization buffer consisting of 50 mM Tris (pH 8.0), 0.25 M sucrose, and Sigma protease inhibitor cocktail. Homogenates were spun at 700 g at 4°C for 10 min. The supernate was then spun at 15,900 g for 10 min. The supernate of this spin was then centrifuged at 100,000 g at 4°C for 60 min. The assay was run at 25°C with the supernatant of the 100,000-g spin for enzyme activity. The reaction mixture contained 940 µl of sodium glycine buffer (90 mM), pH 10.0, 30 µl of sodium L-malate (1.0 M), 20 µl NAD (12.3 mM), and 10 µl of liver enzyme. The reaction was started with the addition of enzyme. Readings of optical density at 340 nm were made against a blank containing all components except NAD, at intervals of 15 s for 3 min. The rate of NADH formed per minute was used to determine enzyme activity. The amount of activity was then corrected for the amount of protein in the sample.

Statistical analysis. Data are expressed as means ± SE, and differences between experimental and control groups were analyzed with the unpaired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lipid levels were measured in the serum of day 19 pregnant mice. As shown in Fig. 1A, total triglyceride was increased twofold (control 52 ± 2.9 mg/dl vs. pregnant 119 ± 6.9 mg/dl). Non-HDL cholesterol (VLDL and LDL) was significantly increased by 21% (control 15.8 ± 1.2 mg/dl vs. pregnant 21.0 ± 0.7 mg/dl; Fig. 1B). Total cholesterol was slightly decreased in the pregnant mice to 83% of control (control 57.7 ± 6.2 mg/dl vs. pregnant 46.3 ± 1.9 mg/dl) (Fig. 1C), which was because of a 35% decrease in HDL cholesterol (control 41.9 ± 5.3 mg/dl vs. pregnant 25.2 ± 1.4 mg/dl; Fig. 1D).


Figure 1
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  		Fig. 1. Serum lipid levels in day 19 pregnant mice. Serum triglyceride and cholesterol levels were measured as described in MATERIALS AND METHODS. A: total serum triglyceride. B: serum non-HDL cholesterol (total cholesterol – HDL cholesterol). C: total serum cholesterol. D: serum HDL cholesterol. Data (means ± SE, n = 5) are representative of >1 experiment. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

 
The effect of pregnancy on nuclear hormone receptor mRNA in the liver is shown in Fig. 2. The mRNA level of RXR{alpha}, the most abundant isoform in the liver, was significantly reduced to 62% of control levels. In addition, RXR{alpha} protein levels were reduced 26% in the pregnant animals (Fig. 3). Similarly, RXRbeta and RXR{gamma} mRNA levels were also significantly reduced to 78% and 82% of controls, respectively (Fig. 2). Thus the expressions of all three isoforms of RXR, the obligate heterodimer partner for a number of nuclear hormone receptors, were reduced in the liver of third trimester pregnant mice.


Figure 2
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  		Fig. 2. Expression of RXR, PPAR, LXR isoforms, and FXR mRNAs in livers of day 19 pregnant mice. RNA was isolated, and Northern Blot analysis was performed as described in MATERIALS AND METHODS. Data (means ± SE, n = 10–20) are expressed as a percentage of controls. RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; LXR, liver X receptor; FXR, farnesoid X receptor. *P < 0.05 and **P < 0.01 vs. control.

 

Figure 3
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  		Fig. 3. Protein levels of RXR{alpha} in livers of day 19 pregnant mice. Hepatic nuclear extracts were prepared, and Western blot analysis was carried out as described in MATERIALS AND METHODS. Data (means ± SE, n = 4) are expressed as a percentage of controls. **P < 0.01 vs. control.

 
We next determined the effect of pregnancy on the levels of nuclear hormone receptor liposensors. As shown in Fig. 2, hepatic PPAR{alpha}, PPAR{delta}, and PPAR{gamma} mRNA levels were significantly reduced in the pregnant mice to 62, 73, and 41% of control levels, respectively. Similarly, LXR{alpha} and LXRbeta mRNA levels were significantly reduced to 70 and 63% of control levels, respectively, in the livers of pregnant mice. Lastly, FXR mRNA levels were significantly reduced by 25% in the livers of pregnant mice. Thus, not only is RXR expression decreased during the third trimester of pregnancy, but there is also a decrease in the expression of nuclear hormone receptor liposensors.

Given the decrease in RXR isoforms and nuclear hormone receptor liposensor expression during pregnancy, we next examined the expression of other nuclear hormone receptors that play a role in lipid metabolism. As shown in Fig. 4, TR{alpha}, LRH-1, and PXR mRNA levels were significantly reduced to 34, 77, and 54% of control, respectively, in the livers of pregnant mice. In contrast, hepatic TRbeta, HNF4, and CAR mRNA levels were not significantly altered by pregnancy. Thus the expressions of many, but not all, nuclear hormone receptors were decreased during the third trimester of pregnancy.


Figure 4
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  		Fig. 4. mRNA expressions of TR{alpha}, LRH-1, PXR, TRbeta, HNF, and CAR in livers of day 19 pregnant mice. TR, thyroid receptor; LRH, liver receptor homolog; PXR, pregnane X receptor; HNF, hepatocyte nuclear factor; CAR, constitutive androstane receptor. RNA was isolated, and Northern Blot analysis was performed as described in MATERIALS AND METHODS. Data (means ± SE, n = 10) are expressed as a percentage of controls. *P < 0.05 and **P < 0.01 vs. control.

 
In Figs. 2 and 4, the mRNA levels were not normalized because we found that the levels of the typical "housekeeping" genes (actin, cyclophilin, and GAPDH) were all altered in the liver during late pregnancy. However, not all mRNA levels are altered; as shown in Fig. 4, the mRNA levels of HNF4 are the same in the livers of control and pregnant mice. Therefore, to ensure that our results were not from loading or transfer problems, we normalized our data by using HNF4 (Table 3) and found that the same genes were decreased.


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  		Table 3. Comparison of gene expression changes in livers of day 19 pregnant mice when not normalized or normalized to HNF4

 
We next examined the expression of target genes of PPAR, LXR, FXR, and TR in the liver of pregnant animals. As shown in Fig. 5 and Table 3, the mRNA levels of PPAR target genes ACO and CPT1{alpha}, LXR target genes ABCG5 and ABCG8, FXR target gene SHP, and thyroid target genes S14 and ME were all significantly decreased in the liver of pregnant mice. Moreover, the activity of malic enzyme was reduced by 50% in the liver of pregnant mice (Fig. 6). Thus, not only does the expression of the nuclear hormone receptors decrease during the third trimester of pregnancy but the expression of target genes is also decreased.


Figure 5
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  		Fig. 5. Expression of nuclear hormone receptor-regulated genes in livers of day 19 pregnant mice. ACO, acyl-CoA oxidase; CPT, carnitine palmitoyltransferase; ABC, ATP cassette-binding protein; SHP, small heterodimer partner; ME, malic enzyme; S14, spot 14. RNA was isolated, and Northern Blot analysis was performed as described in MATERIALS AND METHODS. Data (means ± SE, n = 10–20) are expressed as a percentage of controls. *P < 0.05 and **P < 0.01 vs. control.

 

Figure 6
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  		Fig. 6. Malic enzyme activity in livers of day 19 pregnant mice. Hepatic proteins extracts were prepared, and malic enzyme activity was measured as described in MATERIALS AND METHODS. Data (means ± SE, n = 5) are expressed as a percentage of controls. *P < 0.05 vs. control.

 
Recent studies have demonstrated the great importance of the coactivator PGC-1 in regulation of the activity of several nuclear hormone receptor liposensors (26, 38, 51). Therefore, we next determined the mRNA levels of PGC1{alpha} and PGC1beta in the livers of pregnant mice. As shown in Fig. 7 and Table 3, the levels of PGC-1{alpha} and PGC-1beta are significantly decreased by 46 and 37%, respectively. Thus there is not only a decrease in the expression of nuclear hormone receptor liposensors but also a decrease in the key coactivator PGC-1.


Figure 7
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  		Fig. 7. Nuclear hormone receptor coactivator expression in livers of day 19 pregnant mice. PGC, PPAR coactivator. RNA was isolated, and Northern Blot analysis was performed as described in MATERIALS AND METHODS. Data (means ± SE, n = 10) are expressed as a percentage of controls. **P < 0.05 vs. control.

 
Finally, sterol element binding proteins (SREBPs) are transcription factors that are important regulators of hepatic lipid metabolism (5, 48). As shown in Fig. 8, the mRNA levels of SREBP-1a, SREBP-1c, and SREBP-2 are decreased in the liver of pregnant mice.


Figure 8
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  		Fig. 8. Expression of sterol regulatory element-binding proteins (SREBPs) in livers of day 19 pregnant mice. RNA was isolated, and 1 µg of total RNA was reverse transcribed to cDNA. SREBP-1a, -1c, and -2 expressions were measured by quantitative real-time PCR as described in MATERIALS AND METHODS. Data (means ± SE, n = 4) are expressed as a percentage of controls. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During the third trimester of pregnancy, elevations in serum lipid levels are commonly observed. The specific lipoproteins that increase depend on the species studied; however, in humans, VLDL and LDL levels increase, resulting in an elevation in both serum cholesterol and triglyceride levels (17, 37). In the present study, we observed an increase in serum triglycerides and non-HDL (VLDL + LDL) cholesterol in mice. However, total serum cholesterol levels were slightly decreased because of a marked decrease in HDL cholesterol, the predominant cholesterol carrying lipoprotein in mice. It is postulated that this increase in specific maternal serum lipoprotein levels provides a source of lipid for the fetus, allowing for normal growth and development (59).

The regulation of lipid metabolism is very complex. Recently, it was recognized that members of the nuclear hormone receptor family of transcription factors are activated by lipid ligands, and these nuclear hormone receptors regulate lipid metabolic pathways (8). These nuclear hormone receptors have been referred to as liposensors and include PPARs, LXRs, and FXR. To activate gene transcription, these liposensors form heterodimers with RXR; hence, the three RXR isoforms can be considered key components of the nuclear hormone receptor liposensor group. In the third trimester of pregnancy, we observed in our study that the hepatic mRNA levels of the liposensors RXR{alpha}, RXRbeta, RXR{gamma}, PPAR{alpha}, PPARbeta/{delta}, PPAR{gamma}, LXR{alpha}, LXRbeta, and FXR were all decreased. This reduction in liposensor expression in the liver could contribute to a number of the alterations in lipid metabolism that are observed during the third trimester of pregnancy.

In the liver, PPAR/RXR is a key regulator of fatty acid oxidation, stimulating both mitochondrial and peroxisomal fatty acid oxidation (10, 47). In the present study, we observed that the expression of two key genes required for fatty acid oxidation and regulated by PPAR/RXR (ACO and CPT1{alpha}) were decreased in the liver of third trimester pregnant mice. ACO catalyzes the first step of oxidation in peroxisomes (14), and CPT1{alpha} regulates the transport of fatty acids into mitochondria (31), an essential rate-limiting step in mitochondrial fatty acid oxidation. Previous studies by other investigators have reported a decrease in fatty acid oxidation in the livers of mice in late pregnancy (15). In addition, the studies of Wasfi et al. (58) have shown that the livers of pregnant rats preferentially esterify fatty acids rather than channeling them into the oxidative pathways. This esterification will increase the formation of triglycerides, which could provide substrate for the increased synthesis of VLDL. This increase in triglyceride synthesis could also contribute to the high risk of developing fatty liver during pregnancy. Thus the reduction of PPAR/RXR activity could contribute to the alterations in hepatic fatty acid metabolism that characterize the last trimester of pregnancy.

LXR/RXR regulates many genes involved in reverse cholesterol transport (52). In the present study, we demonstrate that the levels of ABCG5 and ABCG8 are decreased in the livers of third trimester pregnant mice. These transporters play a key role in the excretion of cholesterol into the bile (40, 60). In rodents, the excretion of cholesterol in the bile is decreased during late pregnancy (39, 42). In mice, LXR/RXR activity is also well recognized to regulate the expression of Cyp7a, the rate-limiting enzyme in the conversion of cholesterol to bile acids (36). Studies by other investigators have shown that Cyp7a activity decreased during pregnancy (50). Hence, a decrease in LXR/RXR activity could result in a reduction in the excretion of cholesterol by two major pathways, direct excretion into bile and conversion of cholesterol to bile acids. A decrease in cholesterol excretion would result in the increased availability of cholesterol for the formation of lipoproteins (25, 41). Finally, LXR activation is known to increase SREBP1c expression. We show that SREBP1c mRNA levels are decreased in the liver, which could be due to the reduction in LXR.

The effect of FXR/RXR on bile acid metabolism is complex. Activation of FXR/RXR inhibits bile acid synthesis (49) and decreases serum triglyceride levels (11, 19). FXR activation increases the transcription of SHP, a nuclear hormone receptor that lacks a DNA binding domain. SHP in turn binds to and inactivates LRH-1, which is required for the expression of Cyp7a in the liver (9). During the third trimester of pregnancy, in our study, we observed a decrease in expression of both SHP and LRH-1. Because of the decrease in LRH-1 (and LXR), one would predict that the decreased activation of FXR and the resulting reduction in SHP expression would not lead to an increased expression of Cyp7a.

In addition to forming heterodimers with PPARs, LXR, and FXR, RXR also forms heterodimers with other class 2 nuclear hormone receptors, including TRs, PXR, and CAR. Because of the marked changes in expression of the liposensors in the present study, we also determined the effect of pregnancy on the hepatic expression of TR{alpha}, TRbeta, CAR, and PXR. Interestingly, TR{alpha} but not TRbeta mRNA levels were decreased in the liver in the third trimester of pregnancy. Thyroid hormone influences all major metabolic pathways, including lipid metabolism in the liver. In the present study, we show that the hepatic mRNA levels of two genes, ME and S14, well recognized to be regulated by TR/RXR (3), are decreased during the third trimester of pregnancy. PXR regulates the expression of a large number of genes involved in the detoxification and removal of toxic compounds (23). The impact of the decrease in PXR expression remains to be elucidated, but studies by other investigators have shown that the expression of genes regulated by PXR, such as organic anion-transporting protein (Oatp)2, are decreased during late pregnancy (6, 7).

In addition to decreased expression of many class 2 nuclear hormone receptors, the present study also demonstrates a marked reduction in the mRNA levels of PGC-1{alpha} and PGC-1beta. These nuclear hormone receptor coactivators are well recognized to play important roles in regulating glucose and lipid metabolism by interacting with a variety of nuclear hormone receptors, including the PPARs, LXRs, FXR, and TRs (32, 35, 56, 61). A decrease in the PGC-1 coactivators would be expected to potentiate the decrease in the nuclear hormone receptors. Recent studies in liver cells have shown that ectopic expression of PGC-1{alpha} reduces triglyceride secretion by a process that is dependent on FXR; in intact mice, fasting induces an increase in PGC-1{alpha}, leading to a reduction in serum triglyceride levels in wild-type but not FXR-deficient mice (61). Hence, one could speculate that the decrease in PGC-1 observed in the liver of third trimester pregnant mice could contribute to the increase in serum triglyceride levels by affecting the activity of FXR and perhaps other nuclear hormone receptors.

Finally, we also demonstrate that the mRNA levels of SREBP-1a, SREBP-1c, and SREBP-2 are decreased in the livers of pregnant mice. It is well recognized that SREBPs are transcription factors that are key regulators of both cholesterol and fatty acid synthesis with increased levels of SREBPs stimulating the expression of many of the enzymes in these biosynthetic pathways (5). Thus it is somewhat paradoxical that SREBP mRNA levels are decreased in the livers of pregnant animals, a situation where lipid synthesis has been reported by numerous investigators to be increased (4, 13, 62). However, the molecular mechanisms for the increase in cholesterol and fatty acid synthesis in the livers of pregnant animals is not yet understood, but our results would suggest that an increase in SREBP expression is not a major contributor. The increase in cholesterol synthesis during inflammation is also not accompanied by increases in many of the genes regulated by SREBPs (12).

In summary, the present study demonstrates that, during late pregnancy, there are marked decreases in the expression of nuclear hormone receptors and many of the genes that these transcription factors characteristically regulate. It is possible that these decreases in nuclear hormone receptors have profound effects on the metabolism of the pregnant animal.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Feingold, SFVAMC-Metabolism Section, 4150 Clement St. 111F, San Francisco, CA 94121 (e-mail: kfngld{at}itsa.ucsf.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baxter JD, Webb P, Grover G, and Scanlan TS. Selective activation of thyroid hormone signaling pathways by GC-1: a new approach to controlling cholesterol and body weight. Trends Endocrinol Metab 15: 154–157, 2004.[CrossRef][Web of Science][Medline]
  2. Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR. The acute phase response is associated with retinoid X receptor repression in rodent liver. J Biol Chem 275: 16390–16399, 2000.[Abstract/Free Full Text]
  3. Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR. Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver. Am J Physiol Endocrinol Metab 284: E228–E236, 2003.[Abstract/Free Full Text]
  4. Belknap WM and Dietschy JM. Sterol synthesis and low density lipoprotein clearance in vivo in the pregnant rat, placenta, and fetus. Sources for tissue cholesterol during fetal development. J Clin Invest 82: 2077–2085, 1988.[Web of Science][Medline]
  5. Brown MS and Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: 331–340, 1997.[CrossRef][Web of Science][Medline]
  6. Cao J, Huang L, Liu Y, Hoffman T, Stieger B, Meier PJ, and Vore M. Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin. Hepatology 33: 140–147, 2001.[CrossRef][Web of Science]
  7. Cao J, Stieger B, Meier PJ, and Vore M. Expression of rat hepatic multidrug resistance-associated proteins and organic anion transporters in pregnancy. Am J Physiol Gastrointest Liver Physiol 283: G757–G766, 2002.[Abstract/Free Full Text]
  8. Chawla A, Repa JJ, Evans RM, and Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science 294: 1866–1870, 2001.[Abstract/Free Full Text]
  9. Davis RA, Miyake JH, Hui TY, and Spann NJ. Regulation of cholesterol-7{alpha}-hydroxylase: BAREly missing a SHP. J Lipid Res 43: 533–543, 2002.[Abstract/Free Full Text]
  10. Desvergne B and Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20: 649–688, 1999.[Abstract/Free Full Text]
  11. Edwards PA, Kast HR, and Anisfeld AM. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis. J Lipid Res 43: 2–12, 2002.[Abstract/Free Full Text]
  12. Feingold KR, Pollock AS, Moser AH, Shigenaga JK, and Grunfeld C. Discordant regulation of proteins of cholesterol metabolism during the acute phase response. J Lipid Res 36: 1474–1482, 1995.[Abstract]
  13. Feingold KR, Wiley T, Moser AH, Lear SR, and Wiley MH. De novo cholesterogenesis in pregnancy. J Lab Clin Med 101: 256–263, 1983.[Web of Science][Medline]
  14. Ghisla S. Beta-oxidation of fatty acids. A century of discovery. Eur J Biochem 271: 459–461, 2004.[Web of Science][Medline]
  15. Grimbert S, Fromenty B, Fisch C, Letteron P, Berson A, Durand-Schneider AM, Feldmann G, and Pessayre D. Decreased mitochondrial oxidation of fatty acids in pregnant mice: possible relevance to development of acute fatty liver of pregnancy. Hepatology 17: 628–637, 1993.[CrossRef][Web of Science]
  16. Herrera E. Lipid metabolism in pregnancy and its consequences in the fetus and newborn. Endocr J 19: 43–55, 2002.
  17. Herrera E. Metabolic adaptations in pregnancy and their implications for the availability of substrates to the fetus. Eur J Clin Nutr 54, Suppl 1: S47–51, 2000.[Web of Science][Medline]
  18. Herrera E, Lasuncion MA, Palacin M, Zorzano A, and Bonet B. Intermediary metabolism in pregnancy. First theme of the Freinkel era. Diabetes 40, Suppl 2: 83–88, 1991.[Web of Science][Medline]
  19. Kast HR, Nguyen CM, Sinal CJ, Jones SA, Laffitte BA, Reue K, Gonzalez FJ, Willson TM, and Edwards PA. Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids. Mol Endocrinol 15: 1720–1728, 2001.[Abstract/Free Full Text]
  20. Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, and Grunfeld C. Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host. J Lipid Res 45: 1169–1196, 2004.[Abstract/Free Full Text]
  21. Khovidhunkit W, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR. Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages: differential role of LXR. J Lipid Res 44: 1728–1736, 2003.[Abstract/Free Full Text]
  22. Kim MS, Shigenaga J, Moser A, Feingold K, and Grunfeld C. Repression of farnesoid X receptor during the acute phase response. J Biol Chem 278: 8988–8995, 2003.[Abstract/Free Full Text]
  23. Kliewer SA, Goodwin B, and Willson TM. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev 23: 687–702, 2002.[Abstract/Free Full Text]
  24. Lee CH, Olson P, and Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 144: 2201–2207, 2003.[Abstract/Free Full Text]
  25. Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, and Brown MS. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 277: 9520–9528, 2002.[Abstract/Free Full Text]
  26. Lin J, Yang R, Tarr PT, Wu PH, Handschin C, Li S, Yang W, Pei L, Uldry M, Tontonoz P, Newgard CB, and Spiegelman BM. Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP. Cell 120: 261–273, 2005.[CrossRef][Web of Science][Medline]
  27. Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro AE, Kakizuka A, and Evans RM. Characterization of three RXR genes that mediate the action of 9-cis retinoic acid. Genes Dev 6: 329–344, 1992.[Abstract/Free Full Text]
  28. Mangelsdorf DJ and Evans RM. The RXR heterodimers and orphan receptors. Cell 83: 841–850, 1995.[CrossRef][Web of Science][Medline]
  29. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, and Evans RM. The nuclear receptor superfamily: the second decade. Cell 83: 835–839, 1995.[CrossRef][Web of Science][Medline]
  30. Mano H, Ozawa T, Takeyama K, Yoshizawa Y, Kojima R, Kato S, and Masushige S. Thyroid hormone affects the gene expression of retinoid X receptors in the adult rat. Biochem Biophys Res Commun 191: 943–949, 1993.[CrossRef][Web of Science][Medline]
  31. McGarry JD and Brown NF. The mitochondrial carnitine palmitoyltransferase system: from concept to molecular analysis. Eur J Biochem 244: 1–14, 1997.[Web of Science][Medline]
  32. Meirhaeghe A, Crowley V, Lenaghan C, Lelliott C, Green K, Stewart A, Hart K, Schinner S, Sethi JK, Yeo G, Brand MD, Cortright RN, O'Rahilly S, Montague C, and Vidal-Puig AJ. Characterization of the human, mouse and rat PGC1beta (peroxisome-proliferator-activated receptor-{gamma} co-activator 1beta) gene in vitro and in vivo. Biochem J 373: 155–165, 2003.[CrossRef][Web of Science][Medline]
  33. Memon RA, Fuller J, Moser AH, Smith PJ, Feingold KR, and Grunfeld C. In vivo regulation of acyl-CoA synthetase mRNA and activity by endotoxin and cytokines. Am J Physiol Endocrinol Metab 275: E64–E72, 1998.[Abstract/Free Full Text]
  34. Neish AS, Khachigian LM, Park A, Baichwal VR, and Collins T. Sp1 is a component of the cytokine-inducible enhancer in the promoter of vascular cell adhesion molecule-1. J Biol Chem 270: 28903–28909, 1995.[Abstract/Free Full Text]
  35. Oberkofler H, Schraml E, Krempler F, and Patsch W. Potentiation of liver X receptor transcriptional activity by peroxisome-proliferator-activated receptor gamma co-activator 1{alpha}. Biochem J 371: 89–96, 2003.[CrossRef][Web of Science][Medline]
  36. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE, and Mangelsdorf DJ. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR-{alpha}. Cell 93: 693–704, 1998.[CrossRef][Web of Science][Medline]
  37. Potter JM and Nestel PJ. The hyperlipidemia of pregnancy in normal and complicated pregnancies. Am J Obstet Gynecol 133: 165–170, 1979.[Web of Science][Medline]
  38. Puigserver P and Spiegelman BM. Peroxisome proliferator-activated receptor-gamma coactivator 1 {alpha} (PGC-1{alpha}): transcriptional coactivator and metabolic regulator. Endocr Rev 24: 78–90, 2003.[Abstract/Free Full Text]
  39. Ravi Subbiah MT and Buascaglia MD. Studies concerning the hypercholesterolemia of pregnancy: cholesterol catabolism and excretion during late pregnancy in the rat. Res Commun Chem Pathol Pharmacol 13: 529–539, 1976.[Web of Science][Medline]
  40. Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, and Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors {alpha} and beta. J Biol Chem 277: 18793–18800, 2002.[Abstract/Free Full Text]
  41. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, Shan B, Brown MS, Goldstein JL, and Mangelsdorf DJ. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXR{alpha} and LXRbeta. Genes Dev 14: 2819–2830, 2000.[Abstract/Free Full Text]
  42. Reyes H and Kern F Jr. Effect of pregnancy on bile flow and biliary lipids in the hamster. Gastroenterology 76: 144–150, 1979.[Web of Science][Medline]
  43. Rhoads RP, McManaman C, Ingvartsen KL, and Boisclair YR. The housekeeping genes GAPDH and cyclophilin are regulated by metabolic state in the liver of dairy cows. J Dairy Sci 86: 3423–3429, 2003.[Abstract/Free Full Text]
  44. Rosen ED and Spiegelman BM. PPAR{gamma}: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 276: 37731–37734, 2001.[Free Full Text]
  45. Sacks GP, Seyani L, Lavery S, and Trew G. Maternal C-reactive protein levels are raised at 4 weeks gestation. Hum Reprod 19: 1025–1030, 2004.[Abstract/Free Full Text]
  46. Salameh WA and Mastrogiannis DS. Maternal hyperlipidemia in pregnancy. Clin Obstet Gynecol 37: 66–77, 1994.[CrossRef][Web of Science][Medline]
  47. Schoonjans K, Staels B, and Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res 37: 907–925, 1996.[Abstract]
  48. Shimomura I, Bashmakov Y, Shimano H, Horton JD, Goldstein JL, and Brown MS. Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver. Proc Natl Acad Sci USA 94: 12354–12359, 1997.[Abstract/Free Full Text]
  49. Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, and Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 102: 731–744, 2000.[CrossRef][Web of Science][Medline]
  50. Smith JL, Lear SR, Forte TM, Ko W, Massimi M, and Erickson SK. Effect of pregnancy and lactation on lipoprotein and cholesterol metabolism in the rat. J Lipid Res 39: 2237–2249, 1998.[Abstract/Free Full Text]
  51. Spiegelman BM and Heinrich R. Biological control through regulated transcriptional coactivators. Cell 119: 157–167, 2004.[CrossRef][Web of Science][Medline]
  52. Steffensen KR and Gustafsson JA. Putative metabolic effects of the liver X receptor (LXR). Diabetes 53, Suppl 1: S36–S42, 2004.[Abstract/Free Full Text]
  53. Thorne CJ, Grossman LI, and Kaplan NO. Starch-gel electrophoresis of malate dehydrogenase. Biochim Biophys Acta 73: 193–203, 1963.[Medline]
  54. Tontonoz P and Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol 17: 985–993, 2003.[Abstract/Free Full Text]
  55. Veenstra van Nieuwenhoven AL, Heineman MJ, and Faas MM. The immunology of successful pregnancy. Hum Reprod 9: 347–357, 2003.
  56. Vega RB, Huss JM, and Kelly DP. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor {alpha} in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 20: 1868–1876, 2000.[Abstract/Free Full Text]
  57. Wang YX, Lee CH, Tiep S, Yu RT, Ham J, Kang H, and Evans RM. Peroxisome-proliferator-activated receptor-{delta} activates fat metabolism to prevent obesity. Cell 113: 159–170, 2003.[CrossRef][Web of Science][Medline]
  58. Wasfi I, Weinstein I, and Heimberg M. Hepatic metabolism of [1–14C]oleate in pregnancy. Biochim Biophys Acta 619: 471–481, 1980.[Medline]
  59. Woollett LA. Origin of cholesterol in the fetal golden Syrian hamster: contribution of de novo sterol synthesis and maternal-derived lipoprotein cholesterol. J Lipid Res 37: 1246–1257, 1996.[Abstract]
  60. Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, and Hobbs HH. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci USA 99: 16237–16242, 2002.[Abstract/Free Full Text]
  61. Zhang Y, Castellani LW, Sinal CJ, Gonzalez FJ, and Edwards PA. Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) regulates triglyceride metabolism by activation of the nuclear receptor FXR. Genes Dev 18: 157–169, 2004.[Abstract/Free Full Text]
  62. Zimmermann T and Hummel L. Studies on the fatty acid synthesis in maternal and fetal rats. Acta Biol Med Ger 37: 223–227, 1978.[Web of Science][Medline]



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