Introduction

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Lung maturation and surfactant production are influenced by sex. A male disadvantage in lung development and surfactant production has been found in many species. In humans these differences are reflected in the increased incidence of respiratory distress syndrome (RDS) in male infants (1). Both in vivo and in vitro studies have shown that estrogen enhances fetal lung development, whereas androgens delay it (4).

In rats, in vivo and in vitro administration of estrogen stimulates pulmonary surfactant production by increasing the rate of choline incorporation into phosphatidylcholine (PC) (4, 11, 12). Estrogen also enhances the activity of cholinephosphate cytidylyltransferase, the rate-limiting enzyme in PC synthesis (11, 12). In contrast, androgens inhibit surfactant production and decrease saturated phosphatidylcholine (SPC) and PC to sphingomyelin ratios (6). Although many studies have confirmed this sexual dimorphism in fetal rat lung development and pulmonary surfactant synthesis, the mechanisms by which estrogen and androgens exert their effects have not been clearly delineated.

In the biosynthesis of surfactant, a number of nonlipid precursors, including glucose, are incorporated into phospholipids (13, 14). Glucose is also the predominant source of adenosine triphosphate (ATP) production in most mammalian cells. Thus, glucose is a vital substrate for fetal lung development, providing a source of energy and carbon atoms for surfactant synthesis. The uptake of glucose is governed by glucose transporters (Glut), structurally related proteins that are encoded by a family of genes and expressed in a tissue-specific manner (15). Seven isoforms exist, designated Glut 1-7, referring to the order in which they were cloned (16). Glut 1 is the most ubiquitous of the glucose transporters and is the predominant isoform expressed in fetal tissues. To date, Glut 1 is the only isoform expressed in fetal rat lung (19). Glucose transport is thought to be the rate-limiting step in glucose utilization in rat lung (20).

In previous studies, we found that Glut 1 is expressed in type II pneumocytes and fibroblasts of the fetal rat (21). The mechanisms that regulate Glut 1 function and expression in the fetus are not well understood. Numerous substrates and a variety of hormones have been shown to influence Glut 1 expression (16, 18). Previous studies have reported that estrogen in female rats and dihydrotestosterone (DHT) in male rats modulate glucose uptake in their respective sex organs (22, 23).

We therefore hypothesized that estrogen and androgens differentially regulate glucose transport in fetal lung in both sexes, thus altering availability of the substrate for the synthesis of the surfactant that may ultimately affect lung maturation. In this study, we attempted to define the effects of estradiol and DHT on glucose transport and hexokinase activity in fetal rat lung explants.

	Materials and Methods

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Time-mated Sprague-Dawley pregnant rats were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN) and individually housed under standard conditions. On Day 20 of gestation (term = 21.5 d), dams were killed by pentobarbital overdose. Fetuses were immediately delivered by cesarean section, decapitated, and weighted, and lungs were quickly harvested. Fetal sex was determined by examination of gonads, and lungs from males and females were pooled separately. These studies were approved by the Animal Care Committee of Children's Memorial Institute for Education and Research (Chicago, IL).

Culture of Lung Explants

Our methods have been described previously (21). In brief, fetal lungs were removed and dissected under sterile conditions. The trachea and major airways were removed and the remaining tissue was chopped into 1-mm³ pieces. Explants were cultured for 24 h in serum-free Waymouth's medium (2 ml/dish) at 37°C in a humidified atmosphere of 95% O₂ and 5% CO₂ on a rocking platform.

After 24 h, the culture medium was removed and replaced with serum-free medium containing either estradiol or DHT solutions at doses of 0, 1, 10, or 100 nM. Previous experiments have shown these concentrations to affect surfactant production. Glucose transport experiments were carried out after 3 and 24 h of exposure to the hormone treatments.

Reversal experiments were also performed. After 24 h in medium with either DHT or estradiol at the foregoing concentrations, explants were washed with warm phosphate-buffered solution (PBS) and cultured for an additional 24 h in standard culture medium.

2-Deoxyglucose Uptake

2-Deoxyglucose (2-DG) uptake studies were performed in lung explants of Day 20 fetal rats. These methods have been described previously (21). Lung explants were washed with 5 ml 37°C PBS, then [³H]2-DG (0.25 mM) was added to the incubation medium (1,900 µl PBS, 100 µl [³H]2-DG: 1 µCi/ml). Explants were incubated for 3, 30, and 60 min. Under these experimental conditions, 2-DG uptake was linear. Uptake was maximal at 60 min. Uptake was stopped by the addition of ice-cold PBS. Explants were washed and sonicated in water. Aliquots were used for the determination of radioactivity and DNA content per well. Glucose uptake rates were calculated per microgram of DNA or milligram of protein. DNA content was determined by fluorometric assay and protein by Lowry's method (24, 25). To correct for extracellular trapping of 2-DG, [³H]insulin was used to determine extracellular water space and was factored into the calculations of specific uptake (26).

Hexokinase Activity

Hexokinase activity was measured in homogenate, and soluble and particulate fractions by the method of Kuwajima and colleagues (27). Briefly, tissue was homogenized at 0°C in five volumes of 50 mM triethanolamine, 0.3 M sucrose, and 1 mM ethylenediaminetetraacetic acid (EDTA). Glucose phosphorylation capacity was measured at 37°C using 5 U of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Reactions rates were linear over the time period studied.

Immunohistocytochemistry

Uncultured lung tissue was fixed in 10% formalin and then embedded in paraffin. Sections were deparaffinized in Hemo-De (Fisher, Pittsburgh, PA), and then washed in ethanol. Endogenous peroxidases were blocked by incubating in 1% H₂O₂ in absolute methanol for 30 min. Sections were hydrated through descending grades of ethanol and rinsed in H₂O and PBS. The slides were blocked by incubating in 1.5% normal goat serum and then incubated with the primary antibodies, Glut 1 (1:1,000). Following incubation, the slides were washed in PBS, and then sections were incubated with the secondary antibody, biotinylated antirabbit immunoglobin G. This incubation was followed by washing in PBS. The slides were then incubated with the avidin and biotinylated peroxidase complex (VECTOR, Burlingame, CA), washed in PBS, and incubated in 0.05% 3,5-diaminobenzine and 0.01% H₂O₂ in PBS to visualize the bound anti-Glut antibodies. After the appearance of brown reaction product, slides were washed, counterstained with hematoxylin, dehydrated in graded alcohols, cleared in Hemo-De, and mounted. As immunohistochemical controls, sections were incubated without the primary antibody or with preimmune serum. To check further the specificity of the staining, sections were incubated with the anti-Glut antibody in the presence of excess Glut peptide (5 µg/ml).

Western Blot Analysis

Membrane proteins for Western blot analysis were prepared from lung explants as previously described (21). In brief, tissues were washed, placed in ice-cold buffer (250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and 20 mM Hepes), and homogenized. The homogenates were centrifuged at 13,500 rpm for 20 min at 4°C, and the supernatant was centrifuged at 200,000 × g for 90 min at 4°C. The samples were then deglycosylated. The pellets were suspended in cold buffer by several passes through a 22-gauge needle. Protein was determined by the modified method of Lowry and colleagues (25). Fifty micrograms of membrane protein were loaded onto a 12% polyacrylamide gel and proteins were separated by the method of Laemmli (28). Separated proteins were electrophoretically transferred to Bio-Rad polyvinyldifluoride membranes using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Richmond, CA). One membrane representing half the gel was stained with Coomasie R-250 (BRL, Gaithersburg, MD), and the other membrane half was used for chemiluminescence detection (ECL Western blotting analysis; Amersham, Buckinghamshire, UK) of Glut 1 using a double antibody system (Glut 1: affinity purified; Alpha Diagnostics, San Antonio, TX). Sample loading corrections were made on the basis of densitometry data from the Coomassie-stained membrane. Gels were stained to check for completeness of transfer. After transfer, membranes were incubated overnight in 5% milk proteins (Carnation dried milk; Carnation Co., Los Angeles, CA). The resulting signals were quantified by linear densitometry.

Northern Blot Analysis

Total RNA was extracted from control and treated tissues using RNAzol (guanidinium thiocyanate, phenol, 2-mercaptoethanol; Cinna/Biotex, Friendswood, TX). Total RNA (20 µg/lane) was separated by 1.1% agarose-4.9% formaldehyde gels and transferred to Nytran membranes. The gel was stained with ethidium bromide and visualized under ultraviolet light. RNA was fixed to the membrane by baking for 30 to 60 min at 80°C. Membranes were prehybridized for 2 to 3 h at 42°C in 50% deionized formamide, 5× saline sodium citrate (SSC) (3 M NaCl and 3 M Na₃ citrate-2H₂O), 5× Denhardt's solution (0.02% each of Ficoll 400, polyvinylpyrrolidone, and bovine serum albumin), 100 µg herring sperm DNA, and 1,000 µg yeast tRNA. The blots were probed using radiolabeled [³²P]deoxy-CTP Glut 1 cDNA (gift of Dr. Graeme Bell, University of Chicago, Chicago, IL) overnight at 42°C. After hybridization, the blots were washed four times for 15 min each time at 42° to 50°C in 0.1% SSC and 0.1% sodium dodecyl sulfate (SDS) and then once for 30 min at 65°C with 0.4% SSC and 1.0% SDS. The blots were autoradiographed for 48 to 72 h at 70°C. The relative amount of Glut 1 mRNA was quantified by densitometric analysis. Glut mRNA levels were normalized to 28S ribosomal RNA by dividing the quantity (densitometric determination) of Glut mRNA by the quantity of 28S ribosomal RNA.

Statistical Analysis

Arcsin transformation of percentages was used when appropriate. Statistical analyses were performed using analysis of variance (Fisher's exact test) (29).

	Results

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At 24 h, basal glucose uptake, measured in the absence of sex hormones, was 37% higher (P < 0.05) in lung explants from female fetal rats than in explants from males (Figure 1). After 24 h of culture, estradiol at 10 nM and 100 nM (no significant effect at 1 nM) enhanced glucose uptake in both female (53% and 31%, respectively) and male (32% and 26%, respectively) lung explants above their respective control values (P < 0.05) (Figure 1). Treatment with DHT (24 h) significantly decreased glucose uptake in female and male lung explants at doses of 10 and 100 nM. In females, treatment with DHT at 10 and 100 nM reduced glucose uptake to 74% and 54% of controls, respectively (P < 0.05). DHT at doses of 10 and 100 nM also significantly decreased glucose uptake in lung explants of male fetuses (67% and 73% of controls, respectively) (P < 0.05) (Figure 2). Short-term exposure (3 h) to hormone treatment did not significantly alter glucose uptake in female or male lung explants (data not shown).

View larger version (42K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   Figure 1.   2-Deoxyglucose uptake in female and male fetal rat lung explants treated for 24 h with 0, 1, 10, or 100 nM of estradiol (top panel) or DHT (bottom panel), as described in MATERIALS AND METHODS. Tissue was pooled from four to six litters (approximately 12 pups per litter) for each experiment (n = 4 experiments, five to seven replicates per experiment). The results are means ± SEM, *P < 0.05 versus 0 (control). Glucose uptake was significantly different in males versus females, P < 0.05.

View larger version (100K): [in this window] [in a new window]   Figure 2.   Immunohistochemical localization of Glut 1 in uncultured Day 20 fetal lung. Control for immunostaining is shown in the left panel (A). Glut 1 antibody preabsorbed with Glut 1 peptide shows no significant immunoreactivity, a finding that demonstrates staining is specific. Glut 1 (B) is localized to alveolar epithelial cells (arrow) and mesenchymal cells (double arrow).

Decreased glucose uptake was not due to cell death because morphology (light microscopy) of the explants was not altered by any of the treatments. In addition, DNA, RNA, and protein content of the explants were not affected by estrogen or DHT treatment (data not shown).

Hexokinase activity was quite low in fetal lung in the soluble (14 to 19 nM/min/mg protein) and particulate (2 to 4 nM/min/mg protein) fractions, and did not differ between sexes. Estrogen and DHT treatment at any dose did not alter activity in explants (Table 1).

The photomicrograph in Figure 2 shows the distribution of Glut 1 protein in uncultured fetal lung on Day 20 of gestation. Glut 1 protein was localized to alveolar epithelial cells, mesencymal cells, and bronchial columnar epithelium. In sections treated with either preimmune serum or Glut 1 antibody preabsorbed with Glut 1 peptide, there was no significant immunoreactivity, a finding that demonstrates that staining was specific for Glut 1.

Glut 1 protein, measured by Western blot analyses, was identified as a single band of 43,000 molecular weight. Densitometric analyses (n = 3 experiments) of autoradiographs demonstrated that Glut 1 protein levels were significantly elevated after a 24-h treatment with estradiol at 10 and 100 nM in both female and male fetal lung explants (Figure 3). However, as was the case for glucose uptake, male lung explants were not as responsive to estradiol as female explants. Estradiol increased Glut 1 protein levels approximately 2-fold in females as compared with a 1.5-fold increase in males (P < 0.05). Treatment with DHT at doses of 10 and 100 nM resulted in a substantial decrease in Glut 1 protein levels in male and female explants (P < 0.05) (Figure 4). There was no difference in response to DHT between females and males.

View larger version (33K): [in this window] [in a new window]   Figure 3.   Western immunoblot and densitometric analysis of Glut 1 protein in male and female fetal lung explants treated for 24 h with 0, 1, 10, or 100 nM estradiol. Fifty micrograms of protein were loaded into each lane. Immunoreactive proteins were visualized with autoradiography. Glut 1 was identified as a single band of 43,000 mol wt. Data represent the means ± SEM of three experiments. *P < 0.05 versus 0 nM (control).

View larger version (38K): [in this window] [in a new window]   Figure 4.   Western immunoblot and densitometric analysis of Glut 1 protein in fetal lung explants treated with DHT 0, 1, 10, or 100 nM for 24 h. Fifty micrograms of protein were loaded into each lane. Immunoreactive proteins were visualized with autoradiography. Glut 1 was identified as a single band of 43,000 mol wt. Data represent the means ± SEM of three experiments. *P < 0.05 versus 0 nM (control).

In Northern blots, Glut 1 mRNA was identified as a single band of approximately 2.8 kb. Analysis of densitometry data (n = 5 experiments) showed that Glut 1 mRNA levels were significantly increased in female and male explants cultured for 24 h in 10 and 100 nM estradiol (Figure 5). Again, females were more responsive to estradiol treatment than males. Glut 1 mRNA levels were enhanced 2 to 3 times in females and only 1.5 to 2 times in males. In comparison with estradiol, DHT treatment for 24 h led to a substantial decline in Glut 1 mRNA at 10 and 100 nM in both females and males (P < 0.05) (Figure 6). There was no difference in response to DHT treatment between the sexes.

View larger version (36K): [in this window] [in a new window]   Figure 5.   Analysis of Northern blot densitometry data of Glut 1 mRNA in fetal lung explants treated for 24 h with 0, 1, 10, or 100 nM of estradiol. Twenty micrograms of total RNA were loaded into each lane. Glut 1 was visualized with autoradiography and was identified as a single band of 2.8 kb. Data represent the means ± SEM of three experiments. *P < 0.05 versus 0 nM (control).

View larger version (42K): [in this window] [in a new window]   Figure 6.   Northern blot and densitometric analysis of Glut 1 mRNA in fetal lung explants treated with different concentrations of DHT for 24 h. Twenty micrograms of total RNA were loaded into each lane. Glut 1 was visualized with autoradiography and identified as a single band of 2.8 kb. Data represent the means ± SEM of three experiments. *P < 0.05 versus 0 nM (control).

In contrast to the lack of effect of short-term treatment on glucose uptake, 3-h treatment with 10 and 100 nM of estradiol increased Glut 1 mRNA levels in females (247% and 132%, respectively) and in males (182% and 52%, respectively) above their respective control values (P < 0.05) (data not shown). In agreement with previous data, female explants were more responsive to estradiol treatment than males. Unlike estradiol, short-term treatment with DHT did not significantly affect Glut 1 mRNA levels in either male or female explants (data not shown).

To determine if the effects of estradiol and DHT on glucose transport were reversible, treated explants were washed after 24 h and subsequently cultured for an additional 24 h in standard culture medium. 2-DG uptake, Glut 1 protein, and mRNA returned to levels seen in control rats, indicating that the effects of estradiol and DHT on glucose transport were reversible and not permanent (Figure 7).

View larger version (33K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   Figure 7.   Reversal experiments show that the addition of estradiol (top panel) or DHT (bottom panel) for 24 h is not permanent in lung explants. Data represent the means ± SEM of five different experiments. Glucose uptake was significantly different in males versus females, P < 0.05.

	Discussion

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Lung maturation in male fetuses lags 1 to 2 wk behind the process in females (30). Because of delayed surfactant production, premature male infants are at increased risk for RDS (1). It is not know whether the sexual dimorphism observed in fetal lung development is primarily due to beneficial effects of estrogen or detrimental effects of androgens. Fetal levels of unconjugated 17B-estradiol are similar in male and female fetuses (31), whereas testosterone levels are significantly higher in males than in females in late gestation (32). Androgens, endogenous and exogenous, delay lung maturation structurally and functionally (6). Synthesis of SPC by type II cells in vitro is inhibited by DHT in doses similar to those used in our study (33, 34). Although these studies suggest that androgens play a greater role in retarding lung development, other studies indicate that estrogen plays an equally important role by enhancing lung maturation. Estrogen has been found by many investigators to stimulate surfactant production and secretion in vivo and in vitro (4, 5, 10, 11). Estradiol in vitro increases PC content, the most abundant phospholipid in surfactant, as indicated by the rate of choline incorporated into PC.

Our results showing differences in glucose uptake between the sexes and the effects of sex hormones on glucose transport suggest that this process may also be involved in the sexual dimorphism in lung development. Glucose is an important precursor for lipid biosynthesis in the lung (13, 14). Glucose uptake into the type II pneumocyte is controlled by Glut 1 (21, 35). Our study demonstrates that in male and female lung explants, 24-h treatment with estrogen significantly increases glucose uptake, Glut 1 protein, and Glut 1 mRNA levels. In contrast, 24-h treatment with DHT significantly decreases 2-DG uptake, Glut 1 protein, and Glut 1 mRNA levels in all three explant types. Although we did not measure phospholipid synthesis in these experiments, several studies have shown that the doses used in our experiments significantly alter phospholipid synthesis by the fetal lung (4).

In these studies we have demonstrated that Glut 1 localizes to type II pneumocytes in situ. This finding complements our previous findings that showed that Glut 1 is expressed in type II pneumocytes in culture (21). In contrast, in situ immunohistochemical studies done in other laboratories have observed that Glut 1 is expressed in perineural sheaths in fetal mouse lung (36). They were unable to demonstrate the presence of Glut 1 in alveolar epithelial cells. This same group also failed to show localization of Glut 1 in alveolar epithelial cells in human fetal lung and newborn rat lung. These discrepant findings may be explained by species differences (human versus rat) and by age differences (fetal versus juvenile). In prior studies we have shown that Glut 1 expression in the lung rapidly declines after birth (37).

Glut 1 also localizes to lung fibroblasts (21). Fetal rat lung fibroblasts store neutral lipids as the lung matures and triglycerides of fibroblast origin can be utilized as substrate for surfactant synthesis by type II epithelial cells (38). Recent studies have shown that DHT inhibits neutral lipid trafficking, thus depriving type II cells of substrate for surfactant synthesis (38). Lipid trafficking is an energy-dependent process and interference with fuel provision by inhibiting glucose transport may be one mechanism by which androgens decrease triglyceride uptake by lung fibroblasts.

Although the present study shows that glucose uptake is strongly affected by both sex hormones, the molecular mechanism by which these hormones act remains unknown. Sex hormones may act directly on Glut 1 at the transcriptional level by inducing or repressing the gene, or indirectly through other mediators. Steroid hormones belong to a gene superfamily whose members modulate the initiation of transcription of several target genes (39). Although receptors for estrogen and androgens have been identified in fetal lung tissue, it is not known if they are on type II pneumocytes (43). The activated hormone receptor complex interacts with specific DNA sequences in the promoter region (hormone response elements). Hormone response elements may either induce or repress gene transcription. Estrogen response elements have been identified in several genes in both humans and rats (39, 40); however, it is not known if Glut 1 contains a traditional estrogen response element. To date, androgen response elements have not been identified in any gene but there is evidence that androgens act through other hormone response elements (42). Alternatively, sex hormones may mediate their effect through changes in mRNA stability. The molecular mechanisms remain to be determined.

Estrogen and androgens may also act on other cells in the rat lung that produce factors known to influence glucose uptake by the type II pneumocyte. For example, androgens inhibit the production of epidermal growth factor (EGF) and EGF is an important stimulus of glucose uptake (43). Androgens also reduce cyclic adenosine monophosphate (cAMP) production by lung fibroblast. Cyclic AMP increases glucose uptake in many cell types, including type II pneumocytes (48). Estradiol increases the production of insulinlike growth factor-1 and EGF, two growth factors that stimulate glucose uptake in fetal lung (49).

In our study, the magnitude of change in glucose uptake after 24-h treatment with doses of estradiol or DHT paralleled the change in Glut 1 protein and mRNA levels. These findings indicate that changes in glucose uptake result from increased synthesis of the transporter. Although it is likely that the increase in Glut 1 mRNA was due to an increase in transcription of the gene, estradiol and DHT could also affect the stability of mRNA. Alterations in glucose uptake induced by treatment with estradiol or DHT could also be due to changes in the intrinsic activity or translocation of the transporter from inside the cell to the plasma membrane. A majority of Glut 1 normally resides on the plasma membrane in the lung; therefore, it is unlikely that enhanced translocation is responsible for the observed increase in glucose uptake. In addition, the lack of change observed in glucose uptake after short-term treatment suggests that increased biosynthesis, rather than translocation of Glut-1, is responsible for the changes in glucose transporter activity.

In addition to exhibiting higher basal glucose uptake, female rat lung was also more responsive than male lung to estradiol treatment (3 h and 24 h). Differences between the sexes may be related to dissimilar regulation at the transcriptional, post-transcriptional, or receptor level. For example, there may be a difference in absolute number of estrogen receptors between the sexes, or females may have increased receptor activity. Male rat lung may also lack factors that make it less responsive than the female lung. Fibroblasts from male fetal rat lung lack 11-oxireductase activity and are thus unable to increase production of fibroblast pneumocyte factor in response to cortisone treatment (50).

For analyzing hormonal effects on lung tissue, organ culture is a practical system and has several advantages over cell monolayer culture systems. Biochemical and morphologic maturation, including surfactant production and synthesis of Glut 1, occurs in a manner similar to that observed in vivo (51, 52). The explant system has the advantage of maintaining standard cell-architectural relationships that permit normal cell-to-cell interaction. Because explants are cultured in serum-free medium, this system also allows for fine regulation of the hormonal environment.

In conclusion, there are many factors that contribute to the sexual dimorphism in lung maturation and surfactant synthesis. We propose that the regulation of substrate supply via glucose transporters by estrogen and androgens is an important additional mechanism for controlling surfactant production and lung development.